Group C and G Streptococci, their role in acute pharyngitis

Group C and group G Streptococci: Their Role in acutepharyngitis

By

Alaa H. Al-CharrakhPh.D Microbial Biotechnology

Babylon University, Iraq

Jawad K. Al-Khafaji Rana Hadi Al-Rubaye Ph.D Microbiology M.Sc Microbiology

Dedication

The authors would like to dedicate this works to their families,friends, and students with love.

Authors

Acknowledgements

Praise to the Almighty Allah, the glorious creator of the universe, forhis kindness and mercy, and blessing upon Mohammad the prophet andupon his family and followers. The authors would like to thank Dr. Nabeel Al-A'raji, President ofBabylon University, for his support, advice, and encouragement. Theauthors also grateful to department of Microbiology, College ofMedicine, Babylon University for providing all the needed facilities,which were essential for successful completion of the present work. I would like to thank the Dean of College of Medicine University ofBabylon and Head of Microbiology Department in the College ofmedicine University of Babylon for their in achieving my research. Special thanks also go to staff of Hilla teaching hospital in Babylonprovince, for their help and distinguished assistance. Also we would liketo thank staff of the Microbiology Lab in Microbiology Department inCollege of Medicine for their unlimited support and encouragement.

Authors

Contents_________________________________________________________________

I

List of Contents

No. Subject Page

List of Contents I

List of Tables V

List of Figures VI

List of Abbreviations VII

Chapter One: Introduction and Literature Review

1.1. Introduction 1

1.2. Literatures review 2

1.2.1. History and Taxonomy of Group C Streptococci 2

1.2.2. Habitats of group C streptococci 6

1.2.3. Characteristics and Identification of group C streptococci 7

1.2.3.1. Morphological and Physiological Characteristics 7

1.2.3.2. Antigenic and Serological Characteristics 8

1.2.3.3 Identification of group C streptococci 9

1.2.4. Hemolytic Reactions of group C streptococci 14

1.2.5. Virulence Factors of group C streptococci 15

1.2.5.1. Gliding motility 15

1.2.5.2. Hemolysin and Cytotoxins 16

1.2.5.3. Protein antigens 17

1.2.5.4. IgG Fc( ) receptors 17

1.2.5.5 Streptokinase 18

1.2.5.6. Hyaluronidase 19

1.2.5.7. Capsule production 19

1.2.5.8. Extracellular products 20

1.2.5.9. Platelet aggregation 20

1.2.5.10 Co-colonization 21

1.2.6. Medical importance of group C streptococci 21

1.2.7. Susceptibility to Antimicrobial Agents 25

Chapter two : Materials and Methods

2.1. Materials 29

Contents_________________________________________________________________

II

2.1.1. Patients 29

2.1.2. Laboratory equipments 29

2.1.3. Chemical materials 30

2.1.4. Culture Media 30

2.1.5. Diagnostic kits 31

2.1.6. Diagnostic Disks 31

2.1.7.1. Antibiotic powders 31

2.1.7.2. Antibiotic Disks 32

2.1.8. Bacterial Isolates 32

2.2. Methods 33

2.2.1. Specimen collection 33

2.2.2. Preparation of Reagents and Solutions 33

2.2.2.1. Catalase Reagent 33

2.2.2.2. Phosphate buffer solution (PBS) 33

2.2.2.3. Coppric sulfate solution (20%) 33

2.2.2.4. Crystal violet solution (1%) 33

2.2.2.5. Tannic acid solution (1%) 34

2.2.6. D-mannose solution (0.1 M) 34

2.2.2.7. Normal Saline solution (0.15 M) 34

2.2.2.8. Trichloroacidic acid (TCA) solution (5%) 34

2.2.2.9. -Lactamase detection solutions 34

2.2.2.10.

McFarland tube standard (0.50) 35

2.2.3. Preparation of culture media 35

2.2.3.1. Blood agar medium 35

2.2.3.2. Nutrient agar medium 35

2.2.3.3. Nutrient Broth 36

2.2.3.4. Müller- Hinton agar 36

2.2.3.5. M9 medium 36

2.2.3.6. Brain heart infusion(BHI) glycerol broth medium 36

2.2.3.7. Brain heart infusion-glycerol agar medium 36

2.2.3.8. Egg yolk agar medium 36

Contents_________________________________________________________________

III

2.2.3.9. Tryptic Soy Agar medium 37

2.2.3.10. Columbia Blood Agar 37

2.2.3.11. Chocolate Agar 37

2.2.3.12. Sugar fermentation medium 37

2.2.4. Stains 38

2.2.5. Isolation and Identification of bacterial isolates 38

2.2.5.1. Colonial morphology& Microscopic Examination 38

2.2.6. Physiological and Biochemical Tests 38

2.2.6.1 Catalase test 38

2.2.6.2. Optochin Susceptibility Test 39

2.2.6.3. Bacitracin sensitivity test 39

2.2.6.4. CAMP Test 39

2.2.6.5. Sugar fermentation test 39

2.2.7. Commercial API 20 test system 40

2.2.8.. Serological test: Streptex agglutination test for Lancefield

grouping

40

2.2.9. Virulence factors tests 41

2.2.9.1. Capsule stain test (Hiss s Method) 41

2.2.9.2. Hemolysin production test 41

2.2.9.3. Extracellular protease production test 41

2.2.9.4. Detection of Colonization Factor antigen (CFA) I and III 41

2.2.9.5. Bacteriocin production test 42

2.2.9.6. Lipase production test 43

2.2.9.7. Gliding motility test 43

2.2.10. Antimicrobial susceptibility test 44

2.2.10.1.

Disk Diffusion test 44

2.2.10.2 Detection of -Lactamase Production 44

2.210.3. Determination of Minimum Inhibitory concentration MICs 45

2.2.10.3.1.

HiComb MIC Test 45

2.2.10.3.2.

Determination of MICs by agar dilution method 45

Contents_________________________________________________________________

IV

2.2.11. Preservation of bacterial isolates 47

Chapter three: Results and discussion

3.1. Isolation and Identification of Bacterial Isolates 48

3.2. Biochemical and Physiological tests 49

3.3. Serological Identification 53

3.4. Virulence factors of the bacterial isolates 54

3.4.1. Capsule production 55

3.4.2. Hemolytic reaction 56

3.4.3. Extracellular protease production 56

3.4.4. Lipase production 57

3.4.5. Colonization Factor Antigen(CFA) 57

3.4.6. Bacteriocin production 58

3.4.7. Gliding motility 58

3.5. Detection of Antibiotic Resistance 59

3.5.1 Disk diffusion method 59

3.5.2 Detection of -Lactamase Production 63

3.5.3 MICs Determination of anginosus group isolates 64

Conclusions and Recommendations 69

References 70

Contents_________________________________________________________________

V

List of Tables

Table No. Title Page No.

1-1 Key characteristics for laboratory identification of Strept.

anginosus group

11

2-1 Laboratory instruments and equipments 29

2-2 Chemical materials 30

2-3 Culture media 30

2-4 Diagnostic kits 31

2-5 Diagnostic Disks 31

2-6 Antibiotic powders 31

2-7 Antibiotic Disks 32

2-8 Standard Strains and Local Isolates 32

3-1 Distribution of -hemolytic Streptococci belonged toanginosus group isolated from throat swab samples

49

3-2 Biochemical and physiological tests used for identification

of Streptococcus anginosus group from other Streptococci

50

3-3 Additional tests (other than that in API 20strep system)used for Identification of Streptococcus anginosus group

50

3-4 Lancefield grouping of anginosus group by streptex

agglutination test

53

3-5 Virulence factors detected in isolates of anginosus group

streptococci

55

3-6 Antibiotic resistance of anginosus group streptococci by disk

diffusion method

61

3-7 MICs of a number of antibiotics against Strep.anginosus

group isolates.

66

Contents_________________________________________________________________

VI

List of Figures

Figure No. TitlePage No.

3-1 API 20 strep system test used for identification of

Streptococcus anginosus group isolates.

52

3-2 Percentages of group C and F antigens in the -hemolyticstreptococci recovered in this study.

53

3-3 Gliding motility test 59

3-4 Antibiotic resistance of anginosus group streptococci byusing disk diffusion method.

62

3-5 HiComb MIC test used for determination of MIC ofantibiotics against Streptococcus anginosus group isolates.

65

List of Abbreviations

Abbreviation Key

Am Ampicillin

AZM Azithromycin

BHI Brain heart infusion

BHS Beta-hemolytic streptococci

CAMP Christie, Atkins, and Munch-Petersce (authrs)

CTX Cefotaxim

CTR Ceftriaxon

CDC Center of disease control

CFA Colonization Factor Antigen

CFA/I Colonization Factor Antigen-I

CFA/II Colonization Factor Antigen-II

CFA/III Colonization Factor Antigen-III

CFU Colony forming unit

CRL Clarythromycin

CIP Ciprofloxacin

C Chloramphinicol

CLSI Clinical and Laboratory Standards Institute

DA Clindamycin

DNA Deoxyribonucleic Acid

Contents_________________________________________________________________

VII

emm Protein antigen gene

erm (B) Erythromycin resistance gene

erm TR Erythromycin resistance gene

erm erythromycin resistance methylaseE Erythromycin

FEP Cefepime

gm gram

GCS Group C streptococci

GGC Group G streptococci

GCGS Group C and G streptococci

GBPs Glucan binding proteins

IgG Immunoglobulin G

IgA Immunoglobulin A

KDa Kilo dalton

M.W. Molecular weight

MIC Minimum inhibitory concentration

MBC Minimum bactericidal concentration

MLSB Macrolide-lincosamide-streptomycin B

M Molar

c MLSBConstitutive Macrolide-lincosamide-

streptomycin B

i MLSB Inducible Macrolide-lincosamide-streptomycin

B

mefA macrolide efflux gene A

mefE macrolide efflux gene E

NCCLS National committee for clinical laboratory

standards

OF Serum opacity factor

ONPG O-nitrophenyl galactoside

OFX Ofloxacin

RT-PCR Real-time polymerase chain reaction

PBS Phosphate buffer solution

P Penicillin G

PAM Plasmin and/or plasminogen An Emm-like protein

Contents_________________________________________________________________

VIII

PBPs Penicillin-binding proteins

rpm. Round per minute

SAG Streptococcus anginosus group

SDS-

PAGE

Dodecylsulfate-polyacrylamide gel

electrophoresis

SDI Samarah Drug Industries

SDSE Streptococcus dysgalactiae subsp. Equisimilis

SMG Streptococcus milleri group

TCA Trichloroacidic acid

TE Tetracycline

TSA Trypticase soy agar

U unitVA Vancomycin

VGS Viridans group streptococci

VP Voges Proskauer

WHO World health organization

g Microgram

Chapter One Introduction & Literature review

1

1.1. Introduction:

The Streptococcus milleri group (SMG) is a highly diverse group which includes

three species: Streptococcus anginosus, Streptococcus intermedius, and

Streptococcus constellatus. The group also includes hemolytic streptococci

belonging to Lancefield group A, C, F or G as well as non-groupable and non-

hemolytic streptococci (Whiley and Beighton, 1991).

The nomenclature, identification and classification of Streptococcus milleri have

been confusing. In 1989, it was proposed in the United States to rename this

different group under one species name, Streptococcus anginosus, and while in

Greate Britain the designation Strep. milleri was preferred (Belko et al., 2002).

Whiley performed DNA relatedness studies on strains classified as Streptococcus

anginosus, and observed that three DNA homology groups could be identified that

corresponds to three ditinict strains: Streptococcus anginosus, Streptococcus

intermedius and Streptococcus constellatus (Whiley and Hardie, 1989).

The Streptococcus milleri group (SMG) is commensal organisms commonly

isolated from mouth, oropharynx, gastrointestinal tract and vagina, but they can

cause a variety of human and animal infections (Ruoff, 1988).

The roles of group C streptococci in causing endemic pharyngitis are still

controversial (Turner et al., 1990), although Lancefield group C streptococci are

implicated in the outbreaks of pharyngitis and associated disorders (Bradley et al.,

1991). It is well known that group C streptococci are often isolated from clinical

specimens. Strep. anginosus is the most common beta-hemolytic group C

streptococcus isolated from the human throat (Lebrun et al., 1986).

Unlike other viridians streptococci, SMG species are often associated with

bacteremia and abscess formation. However, the pathogenic mechanism of SMG

is not yet completely understood. The frequent presence of polysaccharide capsule

may help these pathogens to escape from being phagocytosed before adhering to

the site of tissue damage.


2

The production of extracellular enzymes including hyaluronidase,

deoxyribonuclease, ribonuclase, gelatinase and collagenase by these organisms

may contribute to its pathogenicity by degradation connective tissues. SMG have

also been observed to release extracellular products with immunosuppressive

effects which may allow the organism to survive within an abscess (Arala-Chaves

et al., 1981).

In Iraq, so far little information are available on prevalence of group C and G

streptococci in patients with acute pharyngitis, in addition there is no information

regarding their virulence and antibiotic resistance. So that this wok was conducted

for phenotypic characterization of these groups of pathogenic bacteria in causing

pharyngitis. To achieve these aims, the following objectives were followed:

1. Isolation and identification of group C streptococci from patients suffering

from pharyngitis and detection of Lancefield groups (C, F, G).

2. Detection of some virulence factors such as protease, lipase, and capsule

production.

3. Determination of antibiotic susceptibility (AST) using:

a- Disk diffusion test (DDT).

b- Detection of -lactamase production by using Rapid Iodometric method.

c- MICs against different antibiotics.

1.2. Litratures Review:

1.2.1. History and Taxonomy of Group C Streptococci:

The nomenclature, identification and classification of Streptococcus milleri

have been confusing. It was first described by Guthof who gave the name to a

group of streptococci with similar biochemical and physiological properties which

had been isolated from abscesses around the mouth (Guthof, 1956).

Group C streptococci is a common cause of infection in animals but there is

little information about their overall importance as a cause of human infection.

Group C and G streptococci were first recognized as human pathogens by

Lancefield and Hare (1935).


3

Hutchinson (1946) found that their occurrence was approximately one-sixth of

that of group A streptococci and suggested that their isolation to be of the same

significance as that organism. Since then it has become clear that streptococci

possessing the group C polysaccharide antigen are taxonomically divers, with the

number of species, as well as their nomenclature, still unclear.

The species name was chosen to honor the oral microbiologist W.D.Miller.

The definition of Strep.milleri as a species was further clarified by cell wall

analysis, numerical classification, and DNA transformation (Colman and

Williams, 1965; Colman, 1968; Colman, 1969) to link taxonomically Guthof,s

Strep. mlleri and non-hemolytic streptococci of serological groups A,C,F, and G.

Later, small beta-hemolytic streptococci along with other non-hemolytic

streptococci group F, C or G streptococci collectively referred to as the

Streptococcus milleri group (Colman and Williams, 1972).

There was a unification of these streptococci into a single species

Streptococcus anginosus which was the oldest approved name for these bacteria

and therefore had precedence over the name streptococcus milleri , a puplication

of an emended description of Strep. anginosus by Coykendall and co-workers in

Strep. milleri nomenclature was developed in 1987. The emended description,

contained in a paper examining genetic relationships of Strep. milleri

organisms, establishes Strep. anginosus as the approved name for all biotypes of

organisms unofficially referred to a Strep. milleri (Coykendall et al., 1987).

DNA relatedness studies were performed on strains classified as Streptococcus

anginosus, and observed that three DNA homology groups could be identified

that correspond to three distinct strains: Streptococcus constellatus, Streptococcus

intermedius, and Streptococcus anginosus (Wihley and Hardie, 1989).

Taxonomic studies, the logical foundation for nomenclature, have examined

Strep. Milleri organisms by a variety of methods. Computer-assisted numerical

taxonomy studies by Colman (1968), Lutticken and associates (1978), and Bridge


4

and Sneath (1983) found that streptococci identified as Strep. milleri formed tight

clusters when examined by the more impartial numerical classification methods.

In studies of the cellular fatty acid composition of Strep. milleri strains,

reported homogeneity in fatty acid profiles despite diverse physiological

characteristics of Strep. milleri strains examined (Labbe et al., 1985), while others

were able to correlate biotypes of Strep. milleri strains with various fatty acid

profiles (Druker and Lee, 1981).

A close genetic relation was demonstrated between the type strains of Strep.

anginosus, Strep. constellatus, and Strep. intermedius (Farrow and Collins, 1984).

A close relationship between the type strain of Strep. anginosus (a beta-hemolytic

group G isolate) and minute hemolytic streptococci of group A, C, F, or G or with

no detectable serogroups was demonstrated via hybridization studies (Ezaki et al.,

1986).

Phenotypically, the members of this group are characterized by their

microaerophilic or anaerobic growth requirement, the formation of minute

colonies and the frequent presence of characteristic caramel-like smell when

cultured in agar plate. Later investigators applied molecular taxonomic techniques

other than phenotypic traits for classification of these streptococci (Coykendall et

al., 1987; Whiely and Beighton, 1991).

The most currently accepted molecular taxonomic studies of Whiley,

Beighton and Facklam divided the Strep. milleri group of streptococci into three

species Streptococcus anginosus, Streptococcus constellatus, and Streptococcus

intermedius (Whiley and Beighton, 1991, 2002).

The phylogenetic relatedness of these three species has been confirmed by

16SrRNA sequence analysis (Bentley et al., 1991).

Members of the anginosus group are considered part of the viridans group

streptococci, the majority of which display -hemolytic or non-hemolytic

reactions. However there is general agreement on a division into large colony

pyogenic group and a small colony anginosus group (Killian, 1998).


5

The distribution of the group antigens also shows some association with the

species. Isolates of Strep. intermedius rarely have group antigens, while isolates of

Strep. anginosus and Strep. constellatus often have group F, C, A, and G antigens

. Other investigators have also found similar distributions of the three species

using Whiley s scheme and DNA re-association as reference identification

procedures (Taketoshi et al., 1993).

Evidence of further taxonomic heterogeneity within the group has been

supported by phenotypic and genotypic criteria that include biochemical and

cultural characteristics and pyrolysis mass spectra (Winstanley et al., 1992;

Bergman et al., 1995), long chain fatty acid composition (Cookson et al., 1989),

DNA hybridization (Whiley & Hardie 1989; Whiley et al., 1997), and rRNA-

based studies (Doitt et al., 1994; Whiley et al., 1997).

A distinct 16 rRNA population described within the anginosus group , this

rRNA population appeared to be most closely to the Strep. anginosus species

(Bergman et al., 1995).

However the nomenclature and classification have been controversial. In

1989, Streptococcus milleri group (SMG) was named Strep. milleri in Europe,

whereas North American bacteriologists grouped them with Strep. anginosus, and

several adaptations have been made (Clarridge et al., 2001; Belko et al., 2002).

The name anginosus or previously termed Streptococcus milleri species group

used for the small colony-forming -hemolytic strains with Lancefield group A,

C, F, or G antigens, in a study on viridians group streptococci in a tertiary Korean

hospital (Uh et al., 2007).

In study by Silvana et al., (2010) in India, the designation Strep.milleri

(Guthof, 1956) has often been used for streptococci of this group, although it has

never an officially approved name (Ruoff, 1988).


6

1.2.2. Habitats of group C streptococci:

Isolation of Strep. milleri from a variety of body sites suggests that it is a

common commensal organism in humans. Specimens of the oral cavity and

throat (Bannatyne and Randall, 1977; Piscitelli et al., 1992; Shinzato and Saito,

1995;; Whiley et al., 1999), feces (Unsworth, 1980), and vagina (Wort, 1975;

Ruoff, 1988), appendix (Poole and Wilson, 1977) have all yielded Strep. milleri

strains. Recent analysis using real-time polymerase chain reaction (PCR) found

extremely low levels of Strep. anginosus in the saliva (Kumagai et al., 2003;

Sugano et al., 2003).

Most of the vaginal strains, unlike other Strep. Milleri isolates, produced acid

from raffinose and melibiose. This biotype, which corresponds to mannitol

positive Strep. intermedius in Facklam s nomenclature (Facklam, 1984), is

isolated frequently from the urine cultures of female patients and ferments

mannitol in addition to raffinose and melibiose (Ruoff et al., 1983).

Srept. intermedius, Srept. constellatus, Srept. anginosus form part of normal

flora of mouth, oral pharynx, gastrointestinal tract, and genitourinary tract

(Gossling ,1988; Whitworth, 1990; Piscitelli et al., 1992).

However, each Srept. milleri group species has a predilection for specific

anatomic sites (Whiley et al., 1992). Streptococcus anginosus is more commonly

isolated from gastrointestinal and genitourinary tract infections. Streptococcus

constellatus has a propensity for the respiratory and gastrointestinal tract.

Streptococcus intermedius is responsible for most head and neck infections, such

a supportive otitis media, pyogenic sinusitis, and intracranial abscesses (G mez-

Garcés et al., 1994; Jacobs et al., 1995).

Streptococcus milleri was initially described as an oral cavity organisms

causing periodontal abscess. Streptococcus anginosus, one of the oral viridians

streptococci, is a normal flora preferentially found in dental plaque (Hamada and

Slade, 1980).


7

Group C streptococci are found as normal pharyngeal flora in up to 8% of

healthy adults (Bradley et al., 1991). They were also colonies the skin, nose,

rectum, and in the umbilicus of newborns, (Mohr et al., 1979).

1.2.3. Characteristics and Identification of group C streptococci:

1.2.3.1. Morphological and Physiological Characteristics:

Streptococci are Gram positive organism, spherical or ovoid, and no larger

than 2 m in diameter. Cell division occurs in one plane by the formation of an

equatorial plate, which results in the formation of chains of varied length, usually

6 to 12 cocci but fewer for encapsulated strains, chain length diminishes under

optimal growth conditions (Stollerman, 1996).

They are catalase negative, do not reduce nitrates and are not soluble in bile

salts. If carbohydrate is fermented by streptococci, large amounts of lactic acid are

produced without gas (Frobisher, 1973).

Like physiological characteristics, colony morphology of Strep. milleri

isolates is also variable. On blood agar group C streptococcal strains fall into two

morphological categories large and small colony types. These colony types are

distantly related genetically (Schleifer and Kilpper-Balz, 1987).

smooth-and rough-colony variants were noted in a strain of group F beta-

hemolytic Sterp. milleri and these colony types can be also observed with no

hemolytic isolates (Liu, 1954).

Regardless of nomenclature, organisms referred to as Strep. milleri share a

core of physiological traits. The majority of isolates produces acetione from

glucose (Bucher and Graeventiz, 1984; Piscitelli, 1992) ferment lactose, trehalose,

Salicin, and sucrose (Facklam, 2002) and hydrolyze esculine and arginine

(Colman and Williams, 1972; Facklam, 1977; Ball and parker, 1979).

Some authors (Bannatyne and Randall, 1977; Ingham and Sisson, 1977; Sisson

et al., 1978) have remarked on a characteristic, caramel like odor produced by

cultures of Strep.milleri but this is not a feature of all strains, caramel smell is


8

produced in precetible quantities by some strains, but has never been fully

evaluated for screening identification of Strep.milleri group (Ruoff, 1988).

Carbon dioxide stimulates growth or is required for growth by some strains of

Strep.milleri (Ball and Parker, 1979). The requirement of CO2 , which could also

be satisfied by oleic acid, was first noted in group F and G beta-hemolytic St.

milleri or minute streptococci (Deibel and Niven, 1955;Liu, 1954). Later studies

revealed no hemolytic Strep.milleri isolates with similar properties. Sisson and co-

workers (1978) and Ball and Parker (1979) have observed that Strep. milleri

strains which require CO2 for growth may be mistakenly referred to as anaerobic

streptococci, since they grow well in an anaerobic environment but not in air

without increased CO2.

In a study of more than 300 Strep. milleri isolates, two minor groups were

identified whose characteristics differed slightly from these of the majority of

strains. Isolates of one of the minor groups lacked some of the biochemical

abilities of the major group and were more often beta-hemolytic and groupable

with Lancefield group A, C, For G antiserum. The second minor group was found

to have extended biochemical activities, fermenting raffinose and melibiose or

mannitol. These organisms were usually no hemolytic and non-groupable (Ball

and Parker, 1979)

1.2.3.2. Antigenic and Serological Characteristics:

Strep.milleri strains are known to show a wide serological variation, on the basis

of their cell surface carbohydrate antigens, distinct from Lancefield group

antigens.

Ottens and his colleagues first demonstrated the occurrence of five

carbohydrate antigens I-V (designated Ottens I-V) shared among indifferent

streptococci, including those belonging to Lancefield groups and also non-

groupable strains (Willers et al., 1964; Jablon et al., 1965).

Most strains do not have Lancefield group specific carbohydrate, and those

that do usually belong to Lancefield group A, C, F or G (Poole, Wilson 1976).


9

Further more, it is reported that Strep. intermedius has five carbohydrate

antigens I-V (Osano I-V) (Osano et al., 1990). It is noted that many species of

viridans streptococci elaborate A, C, F or G or other Lancefield antigens

(Facklam, 1977).

Both beta-hemolytic and no hemolytic Strep.milleri strains with group A, C,

F, G or no detectable Lancefield antigen have been observed (Facklam, 2002).

However, it does appeared that, as a general rule, the majority of minute-

colony-forming beta-hemolytic streptococci with group A, C, F or G antigen are

physiologically identical to Strep. milleri (Lawrence et al., 1985).

Protein antigens described, which were present in a majority of 99

Srept.milleri isolates from pyogenic infections, and hypothesized that these

proteins contribute to the pathogenicity of Strep.milleri. Protein analysis might

further subdivide Strep. milleri group and help in assessment of pathogenicity

(Luttichen et al., 1978).

Other studies also have demonstrated the presence of 11 serotypes (a-k)

among Strep. milleri isolates from the mouth and various systemic infections

(Yakushiji et al., 1988; Kitada et al., 1992), and the distribution of each serotype

antigen is generally restricted to strains of a species in the Strep. milleri group

(Taketoshi et al., 1993a; Taketoshi et al., 1993b).

1.2.3.3. Identification of group C streptococci:

Studies referred that, clinically significant streptococci should be first

identified by a combination of colonial appearance and cell morphology,

hemolysis on blood agar, Gram stain, catalase reaction, Lancefield grouping,

Optochin, bacitracin and sulphonamide sensitivity. However, routine

sulphonamide testing is useful. Together with enterococci, Strep. milleri is

uniformly resistant to sulphonamide while other streptococci show variable

sensitivity. This property can be used in the preparation of selective media for

routine clinical use (Tillotson and Ganguli, 1984).


10

Sulphonamide resistant groups A, C, and G streptococci can be differentiated

by bacitracin sensitivity and -D-glucuronide activity. Only Optochin resistant

non-groupable streptococci, which are also sulphonamide resistant, need be

biochemically tested to identify Strep. milleri.

The SMG have been historically grouped based on the formation of

microcolonies, ability to produce acetoine, hydrolyze arginine, inability to

ferment sorbitol, and often a distinctive caramel smell (Ruoff, 1988; Piscitelli et

al., 1992).

Despite their similarities these organisms have tremendous phenotypic

heterogeneity-even within each species-displaying various patterns of hemolysis

( , , and ), Lancefield grouping (A, C, F, G, and non-type able), and ability to

ferment various sugars (Facklam, 2002). Results of these tests are more consistent

and thus useful to identify these organisms. Key characteristics of Strep.

anginosus group organisms are summarized in Table (1-1).

This heterogeneity has historically made laboratory based misidentification of

these organisms commonplace-resulting in a significant underestimation of the

true burden of SMG disease (Ruoff, 1988).

Compounding these difficulties are the fact that: (1)SMG may require 5-10%

CO2 to grow and thus may not be identified unless this is provided; (2) without

stringent standards SMG may be inappropriately identified as anaerobic

streptococci (Ruoff, 1988; Belko et al., 2002); (3) some SMG may have fastidious

nutritional requirements and fail to grow adequately on standard culture media-in

fact up to half of clinical isolates for susceptibility testing fail to grow (Belko et

al., 2002). While the adoption of new standard of laboratory practices has greatly

improved upon SMG isolation, several limiting issues remain.

Two minor groups were identified in a study of more than 300 Strep.milleri

isolates, whose characteristics differed slightly from these of the majority of

strains. Isolates of one of the minor groups lacked some of the biochemical

abilities of the major group and were more often beta-hemolytic and groupable


11

Table (1-1): Key characteristics for lab identification of Strept. anginosus group (Gray, 2005).

Trait Result

Colony characteristics Small, grey, pinpointcolonies on blood agar

Hemolysis Alpha hemolysis*Typical biochemicalreactionsCatalaseVogues-ProskauerMannitolSorbitolArginineEsculinUrease

NegativePositive*Negative*Negative*PositivePositiveNegative

Lancefield group reaction Group F*Other Butterscotch odor of

colony on blood agar*An asterisk indicates that the delineated test result is the most common; however, other results are

possible.

with Lancefield group A, C, F or G antiserum. The second minor group was

found to have extended biochemical activities, fermenting raffinose and melibiose

or mannitol. These organisms were usually no hemolytic and non-groupable (Ball

and Parker, 1979).

The identification of these organisms has been the source of much confusion

(Facklam, 1984; Coykendall et al., 1987).The caramel smell of Strep. milleri in

culture is a distinctive but variable feature making it unsuitable as a screening test

(Ruoff, 1988).

The methods of Whiley et al. (1990) were used to perform the enzyme assays

except that the strains were grown overnight at 35C on Brucella agar plates

containing 5% hoarse blood in an atmosphere containing 5% CO2, and the cell

suspensions used in the assays were adjusted to an optical density equal to that of a

No.1 McFarland standard.

-D-glucuronidase assay provided for rapid differentiation of species within

-hemolytic group C and G streptococci. Both streptococcus equisimilis (group


12

C) and large colony human biotype group G strains were consistently

differentiated from group C and G streptococcus milleri group bacteria by their

ability to hydrolyze the -D-glucuronidase substrate (Cimolai and Mah, 1991).

The ID-32 Strep system (Analytic Products(API) was evaluated for use as a

convenient identification method and reported agreement in identification with

conventional methods for 95.3% of 413 streptococci and related genera , including 32

strains of SMG (Freney et al., 1992). A second commercial system available for

identification of the three species of Strep. anginosus is the Becton Dickinson

Microbiology Crystal Gram-positive system (Von Baum et al., 1998). This

system has -N-fucosidase, -glycosidase, and -glucosidase. Very little

information is available about the utility of this system.

Strains were characterized as belonging to the SMG by means of biochemical

profiling done with the use of the API 20 Strep system test. Strains were assigned

to the Strep. milleri group based on the results of API-20 Strep system tests and were

further spectated by PCR amplification and sequence analysis of a segment of the 16S

rRNA gene (Clarridge et al., 1999).

Many laboratories report the identification of group C a G streptococci till the

group level but not to the species level. However, differentiation to the precise

species among these GCS and GGS should not be ignored, because it has been

reported that large-colony GCS or GGS (referring to Strep. dysgalactiae subsp.

Equisimilis) may have virulence factors similar to those of Strep. pyogenes

(Schnitzler et al., 1995; Igwe et al., 2003) and are more invasive than the

anginosus group among GCS or GGS (Cimolai et al., 1990).

Strains were also tested by using the Fluo-Card Milleri test Kit. The Fluo-Card

Milleri test kit were supplied by the manufacturer and consisted of filter paper strips

with circles containing 4-MU- -D-glucoside. 4-MU- -D-glucoside and 4-MU- -D-

fuciside. Bacterial growth from the same plates used for the conventional assays was

tested with the Fluo-Card Milleri Kit (Limia et al., 2000). Isolates that demonstrated

disagreement between the conventional and the Fluo-Card Milleri Kit identification


13

were first retested by both methods, and if the identification were again discrepant, a

second test with the API 20 Strep strip was performed to confirm that the isolate was

in fact Strep.milleri.

The Streptex Agglutination Kit was used for determination of Lancefield group

antigens A, B, C, D, F and G. Any strain exhibiting multiple group reactions was

retested with diluted extraction enzyme (1:5) (Lancefield, 1933; Birch et al., 1984;

Facklam and Carey, 1991; Collee et al., 1996 ; Levinson and Jawetz, 2001).

The authors state that Lancefield grouping is of little help in recognising

possiple Strep.milleri group cultures (Brogen et al., 1997) . This is because only a

quarter of Strep. milleri isolates are thought to possess Lancefield group antigens

A, C, F or G (Colman, 1990).

However, their study of clinically significant strains indicated that only 30 of

87 (34%) strains a failed to group (Brogen et al., 1997). Hence Lancefield

grouping dose have a useful diagnostic role. These differences in presentation can

make identification of Strep. anginosus group bacteria problematic. One study

looked at the utility of finding Lancefield group F antigen to identify the Strep.

anginosus group and found that F grouping has a specificity of 100% but a

sensitivity of only 47% (Brogan et al., 1997).

Interestingly, when a positive Lancefield group F reaction was combined with

the presence of the butterscotch odor as a means to identify the Strep. anginosus

group, the sensitivity dropped to 19.5% (Gray, 2005).

Molecular technology for specific identification of these species has been

described. This techniques include Pulsed-field gel electrophoresis (Bartie et al.,

2000), sequencing of specific genes (Poyart et al., 1998) and 16S rRNA genes

(Bentley et al., 1991), and species-specific probes (Jacobs et al., 1996) have all

been described. Bacterial identification based on 16S rRNA gene sequence data

have been widely accepted as the most informative basis for phylogenetic and

identification of microorganisms. Likewise, the sequence of the highly variable


14

space region between the 16S and 23S rRNA genes and the groESL genes have

been used for identification of streptococci (Teng et al., 2002; Chen et al., 2005).

It is reported that these methods enable reliable identification of

streptococcus isolates to the species level, but they have not been applied to the

whole spectrum of streptococcus species.

Real-time polymerase chain reaction (RT-PCR) with species-specific primers

can provide a precise and sensitive method for more accurate quantification of

individual species and total bacterial counts (Yano et al., 2002). To differentiate

the anginosus group from other viridians group streptococci, a set of PCR primers

specific for members of anginosus group were designed. Based on a pair of

primers developed initially for differentiating the anginosus group from other

viridians streptococci, the PCR also differentiate between members of the

anginosus group and Strep.dysgalactae subsp.equismilis among beta-hemolytic

group C and G streptococci. Restriction digestion of the amplicon with Xbal and

Bsml further differentiated Strep. anginosus from Strep. constellatus within the

anginosus group.

Identification of the anginosus group is complicated by wide phenotypic and

antigenic diversity, even within 1 species. Although most anginosus group

isolates belong to the non- -hemolytic oral streptococci, -hemolytic strains are

found in all 3 species. Some anginosus group strains carry a type able Lancefield

group antigen, which belongs to group F, C, G or A (Facklam, 2002).

1.2.4. Hemolytic Reactions of group C streptococci:

Although SMG strains are part of the viridans streptococci they may exhibit

all three types of hemolysis ( -, - or -hemolysis) (Joseph et al., 2001).

Streptococcus anginosus is mostly non hemolytic and Strep. constellatus is

usually beta-hemolytic. Facklam (1977), who examined only non-beta-hemolytic

strains, found 56% to be no hemolytic and 44% to produce the alpha reaction. Of

isolates examined by Ball and Parker (1979), 56% were nonreactive, 19% were

alpha reacting, and 25% were beta-hemolytic.Some beta-hemolytic Strep.milleri


15

isolates produce reactions that may be interpreted as greening when surface

colonies are examined. Clear-beta-hemolysis is observed if subsurface growth is

examined (Ruoff and Kunz, 1985).

The type of hemolytic reaction displayed on blood agar has long been used to

classify the Streptococci. -hemolysis is associated with complete lysis of red cells

surrounding the colony, whereas - hemolysis is a partial lysis of red blood cells,

often accompanied by a greenish to brownish discoloration of medium associated

with reduction red cell hemoglobin. Non-hemolytic colonies have been termed

-hemolytic in which no apparent hemolytic activity or discoloration produced by

the colony (Forbisher, 1973; Ross, 1996).

Cytotoxins was identified in a Strep. intermedius strain, which is called

intermedilysin and demonstrated that intermedilysin was strongly hemolytic

against human red blood cells (RBC) but did not lyse RBC from non-primates

(Nagamune et al., 1996).

1.2.5. Virulence Factors of group C streptococci

Very little is known about virulence factors produced by this group of

bacteria.

1.2.5.1. Gliding motility:

A distinct 16S rRNA population within the anginosus group was described.

This rRNA population appeared to be most closely to the Strep. anginosus

species. Phenotypically, the strains belonging to this rRNA population displayed a

gliding type of motility on certain types of chocolate agar, which is the reason

why they were named motile strains. Further notice that the spreading of the

strains was not due to cellular organelles but appeared to be related to a high

production of extracellular glycocalyx. Flagella were not observed when the

motile Strep.milleri strains were examined with the flagellum stain and with an

electron microscope. These negative results do not preclude the presence of

flagella; the flagella may have been destroyed during processing of the bacteria.

From the viewpoint of pathogenicity, a potential advantage of the motile strains


16

for colonization in vivo was suggested, as the production of extracellular

glycocalyx might aid in adherence to mucosal surfaces. The ability to move

across surfaces and the production of extracellular material may aid in

establishing populations of an organism on host tissue (Bergman et al., 1995).

1.2.5.2. Hemolysin and Cytotoxins:

Hemolysin plays a role in blood invasion and in supplying the bacteria with

their requirements of iron (Caufield et al., 1990). It was found that genes code for

hemolysin may be present on conjugant or non conjugant plasmids (Novak et

al.,1994).

Despite their similarities these organisms have tremendous phenotypic

heterogeneity-even within each species-displaying various patterns of hemolysis

( , , and ). Poole and Wilson (1979) noted that a majority of isolates from teeth

were hemolytic, but fecal and vaginal strains tended to be no hemolytic.

Several studies report -hemolytic Strep.milleri strains to be associated with

purulent disease more frequently than non-hemolytic strains (Kambal, 1987;

Spertini et al., 1988).

Also a study of 499 SMG clinical strains consecutively isolated irrespective of

their hemolytic behavior found that non-hemolytic strains were isolated more

frequently from abscess-related specimens than -hemolytic strains (Jacobs et al.,

1995), but this association was not found by others (Van der Auwera, 1985).

The majority of Strep. intermedius strains are reported to be non-hemolytic on

sheep blood agar. These findings apparently dispute the assumed association

between -hemolysis and pathogenicity (whiley et al., 1992). Are strains belong

to the three species of the anginosus group are -hemolytic and there are strains

do not lyse the blood completely (partial or non-hemolysis) also belong to the

three species, it is most predominant than -hemolytic strains (Whiley et al.,

1990; Whiley and Bighton, 1991).

There was a cytotoxins identified in a Strep. intermedius strain, which is

called intermedilysin. Intermedilysin was strongly hemolytic against human red


17

blood cells (RBC) but did not lyse RBC from non primates (Nagamune et al.,

1996).

The authors raised the question as whether intermedilysin analogues were

expressed by the other Strep. milleri group species, but no information on this

subject has been reported to date. Moreover, it is not yet known if all Strep.

intermedius strains produce this enzyme.

1.2.5.3. Protein antigens :

Luttichen and associates described Protein antigens which were present in a

majority of 99 Strep.milleri isolates from pyogenic infections, they hypothesized

that these proteins contribute to the pathogenicity of Strep.milleri (Luttichen et

al., 1978). Streptococcus dysgalactiae subsp. Equisimilis likely owes its virulence

in human to homologs of prominent Strep. pyogenes virulence genes (Cleary et

al., 1991; Davies et al., 2007).

Most SDSE strains isolated from human infections possess emm genes (Bisno

et al., 1987; Schitzler et al., 1995), which code for the potent virulence factor

called M protein (Fischetti, 1989). This surface localized protein contributes

substantially towered the virulence of both Strep. pyogenes and SDSE in human

hosts because it acts as an adhesion, invasion, and antiphagocytic factor (Fischetti,

1989). More than 100 genetically distinct M proteins exist within group C and G

streptococci and form the basis for emm genotyping (Vasi et al., 2000; C. D. C.

P., 2009).

1.2.5.4. IgG Fc( ) receptors:

Some strains of beta-hemolytic streptococci belonging to group A, C, and G

possessed receptors for the Fc fragment of human immunoglobulin G (IgG).

These surface structures on some group A, C, and G strains with a high affinity

for immunoglobulin might contribute to pathogenicity (Lebrun et al., 1982). A

link between the presence of IgG Fc ( ) receptors and virulence was described

(Burova et al., 1980). Furthermore, these authors have shown that the presence of

IgG Fc receptors inhibits phagocytosis of streptococci in classical bactericidal


18

tests, probably through interference with antibody-dependent complement

activation (Burova et al., 1982).

1.2.5.5. Streptokinase:

A secreted plasminogen-binding protein is a 46,000 molecular weight

protein with four compact domains. Two different streptokinase are produced by

group A streptococci. These enzymes are antigenically distinct from group C

streptokinase, the source of the commercial streptokinase used in therapeutic

thrombolysis (Huany et al., 1989).

Streptokinase forms a 1:1 complex with plasminogen activator and catalyzes

the conversion of plasminogen to plasmin. Plasmin in turn digests fibrinogen and

fibrin. It also cleaves the third component of complement into the chemotactic

factor C3a (Lottenbery et al., 1994; Stollerman, 1996).

Streptokinase activity found in different streptococci reflects the host range

and is not active in hosts which it does not normally infect. It has been associated

with the pathogenesis of acute post streptococcal glomerulonephritis

(Gunningham, 2000).

The plasminogen-activating activity of streptokinase may also directly

contribute to streptococcal virulence and invasion of tissues, and it probably

contributes to the thinning of streptococcal pus and with hyaluronidase may

facilitate the rapid spread of streptococci (Kuusela et al., 1992; Stollerman, 1996).

The ability to bind plasmin or its zymogen plasminogen has long been

recognized as a virulence factor in group A, C, and G streptococci. Plasmin and/or

plasminogen are bound by PAM (an Emm-like protein), plr, and a 45-kDa cell

surface protein recently identified as enclose. Streptococcal cell-bound plasmin,

derived from streptokinase-cleavage of plasminogen, influences bacterial

infections by degrading fibrin and breaking down soft-tissue glycoproteins. In

support of this concept, strains causing skin infections show high level binding of

plasmin (Wistedt et al., 1995).


19

1.2.5.6. Hyaluronidase:

Many -hemolytic streptococci, including a number that have hyaluronic acid

capsule, form hyaluronidase. It is formed by groups A, B, C, G and F. The enzyme

formed by group A is immunologically distinct from that of streptococci of group

C and G (Cruickshank et al., 1973; Parker, 1983).

Two enzymes that may be considered virulence factors are -N-

acetylneuramidase (sialidase) and hyaluronidase; Strep. intermedius produces both

of these enzymes, Strep. constellatus produce only hyaluronidase, and Strep.

anginosus produce neither. The production of these enzymes is part of the

identification scheme proposed by (Whiley and Beighton, 1991; Whiley et a.,

1992).

Streptococcal hyaluronidase hydrolyzes hyaluronic acid present in the

streptococcal capsule, and also that found in animal tissues. Known as a spreading

factor in the skin, this enzyme appears to be produced readily in the infected host,

because, like streptokinase, in patients with streptococcal pharyngitis it produces

antibodies with virtually the same frequency as streptolysin O (SLO) and DNase B

(Stollerman, 1996).

1.2.5.7. Capsule production:

The frequent presence of polysaccharide capsule may help these pathogens to

escape from being phagocytosed befor adhering to the site of tissue damage.

Brook and Walker, (1985) show that some Strep. milleri group streptococci

possess a polysaccharide capsule, demonstrated by Hiss,s and ruthenium red

stains.

However, passaging of non-capsulate strains with other capsulate organisms

often restored encapsulation and pathogenicity. Encapsulation may be a possible

virulence character (Lewis et al., 1988). The capsular material produced by

encapsulated strains of SAG also might be a pathogenic factor (NCCLS, 1999).

The nature of such capsuler material is unknown, but the typing antigens

present in group F and related streotococci have often been regarded as


20

microcapsuler structures, capable of preventing phagocytosis (Huis and Willers,

1973). A surface, microcapsular location was proposed for the type antigen,

though this capsule was not demonstrable by negative stainingwith India ink

(Huis in,t Veld and Linssen, 1973).

1.2.5.8. Extracellular products:

The production of extracellular enzymes including hyaluronidase,

deoxyribonuclase, ribonuclase, gelatinase and collagenase by these organisms

may contribute to its pathogenicity by degradation of connective tissues. SMG

have also been observed to release extracellular products with

immunosuppressive effects which may allow the organisms to survive within an

abscess (Arala et al., 1981).

The incubation of crude extracts from all strains of Strep. constellatus and

Strep. anginosus with L-cysteine resulted in the production of a large amount of

hydrogen sulfide. A high level of hydrogen sulfide production, which appears to

be a common feature in both Strep. constellatus and Strep. anginosus, may be

associated with their abscess formation (Yoshida et al., 2008).

1.2.5.9. Platelet aggregation:

In studies to elucidate the virulence factors that play a role in endocardial

infection, cells of clinical Strep. milleri isolates have been examined for their

reactivity with platelets, platelet fibrin or fibrin clots, fibrinogen, albumin,

fibronictin, laminin, 2-macroglobulin and plasminogen (Willcox, 1994; Willcox,

1995).

So far, however, few Srept. milleri strains have been examined for the ability

to induce infective endocarditis and bacteremia in rats or rabbits with

experimentally predisposing heart valve damage (Héra , 1982; Yersin, 1982).

No strong correlation was found between the endocardial infectivity and

platelet-aggregating capacity of strains of Strep. milleri. The platelet aggregating

strains were members of the Lancefield groups F and G or ungroupable but not of

group A or C (Kitada et al., 1997).


21

1.2.5.10. Co-colonization:

There was a consistent correlation of SMG disease and co-colonization with

Pseudomonas aeruginosa leading to speculation of polymicrobial interaction

resulting in enhanced virulence. SMG deserves considerable attention as a

potential pathogen within the airways of patients with cystic fibrosis. However,

SMG disease has rarely been identified in cystic fibrosis (Parkins et al., 2008).

1.2.6. Medical importance of group C streptococci

Group C and G streptococci were first recognized as human pathogens by

Lancefield and Hare in 1935. Since then, awareness about their importance has

greatly increased, especially within recent years (Hill et al., 1969: Broyles et al.,

2009).

The main rought of entry of streptococci into the human body is through the

oral cavity. Some species preferentially colonize not the oral cavity but the

nasopharynx and enter the upper respiratory tract. Streptococcal adhesion to oral

surfaces results primarily from initial binding of cells to deposited salivary

components. These include not only the secretory products of the salivary glands

but also: bacterial products, such as glucan polysaccharides, to which streptococci

can bind via glucan binding proteins (GBPs) (Banas et al., 1990); dietary

components, which may include lectins and other molecules, that interact with

bacterial cell surfaces; serum products that originate as an exudates in gingival

crevicular fluid; and other compounds entering whole saliva from gastric or

respiratory reflux (Gibbons, 1984; Malamud, 1985; Terpenning et al., 1993).

The first description of Strep. milleri by Guthof, deal with strains isolated

from oral infections (Guthof, 1956), and subsequent studies have confirmed the

participation of these organisms in the pathogenesis of infections of the mouth

and teeth (Ottens and Winkler, 1962; Crawford and Russell, 1983; Williams et al.,

1983). .

The role of group C streptococci as a cause of sore throat has been debated at

least since 1947, when a prospective study of over 3000 US army recruits


22

admitted to hospital with respiratory tract infections revealed seven with both

cultural and serological evidence of group C infection (The commission on acute

respiratory disease,1947).

While Poole an Wilson (1976) sugest some evidence for the participation of

beta-hemolytic Strep. miller in pharyngitis, Bucher and Von Grvaeventiz (1984)

argue that further data are required to confirm the pathogenicity of these

organisms in the throat.

They are an unusual cause of pharyngitis, particularly in school children, where

most infections are caused by group A streptococci (Bateman et al., 1993).

Lancefield group C beta-hemolytic streptococci have been implicated as

causative agents in purulent pharyngitis (Benjamin and Perriello, 1976; Cimolia et

al., 1988; Efstratiou et al., 1989; Salata et al., 1989).

Although there are a few studies suggesting a role for group C streptococci in

endemic pharyngitis (Cimolia et al., 1990; Meier et al., 1990; Turner et al., 1990).

However, proof of the etiology of purulent pharyngitis has awaited

comparative studies of isolation rates of the various species of Lancefield group C

streptococci, epidemiologic evidence of clinical illness, and serologic evidence of

infection. Two species of Lancefield group C streptococci have been consistently

isolated from cultures of throat swabs from healthy human adults and those with

pharyngitis. These have been the large-colony Lancefield group C streptococcus

Strep. equisimilis and the small-colony Lancefield group C streptococcus Strep.

anginosus (Bucher and von Graeventiz, 1984; Ruoff et al., 1985; Lebrun et al.,

1986; Vance, 1992).

The role of the -hemolytic non-group A streptococci (group C and G) in

community acquired pharyngitis has been studied extensively (Cimolia et al.,

1988). A series of outbreaks have provided most of the support for these bacteria

as causative agents (Barnham et al., 1983; Martin et al., 1990).

When pathogenic, SMG bacteria commonly form abscesses, have local

extension to surrounding tissues, and have been associated with supportive


23

metastatic complications, although distal spread of the infection was less common

than local extension (Gossling, 1988; Jacobs et al., 1994).

There have been increasing reports of serious infections associated with group

C streptococci, including endocarditis (Korzeniowski et al., 1998; Kurland et al.,

1999; Lefort et al., 2002), meningitis (Barbara et al., 1996), acute epiglottitis

(Richard et al., 1982), cellulitis (Portnoy et al., 1944), pneumonia, septic arthritis,

and puerperal fever (Teare et al., 1989; Bradley et al., 1991).

Many of the patients are elderly, and most have other illnesses or predisposing

factors. Bradley found underlying disease in 27.7% of patients, of which 20.5%

was cardiovascular, and 20.5% was malignant, and described bacteremia in 49

years old woman with rectal carcinoma (Bradley et al., 1991).

Similar to infections with Streptococcus pyogenes, the prime example of a

pyogenic streptococcal pathogens, infections with group C and G streptococci

(GCGS) can develop into life-threatening necrotizing fasciitis, sepsis, and

streptococcal toxic shock-like syndrome. Lancefield groups C and G comprise a

variety of species; one of those species, streptococcus dysgalactiae subsp.

Equisimilis (SDSE), frequently causes human infections. This species can cause

the whole spectrum of infections caused by Strep. pyogenes (Nohlgard et al.,

1992; Baracco and Bisno, 2006).

Infections may occur in more unusual sites, particularly in patients with

impaired immunity. A case of pyomyositis associated with group C streptococci

was described in a 41 years old man with AIDS. In some immunocompromised

patients group C streptococci may be associated with septicemia without

identification of a primary infective focus. (Nitta et al., 1991)

Drucker and Green (1978) provided evidence for the cariogenic potential of

Strep. milleri, although the strains they examined were not cariogenic as

streptococcus mutans.

Strep. anginosus is an opportunistically pathogenic bacterium associated with

purulent dental abscesses. Furthermore, there is increasing evidence to associate


24

Strep. anginosus infection with the incidence of oral cancer and upper respiratory

tract carcinoma (Hooper, 2007).

Streptococci of the anginosus group have a certain proponciy to cause,

bacteremia (Bradley et al., 1991), and serious purulent infections in the deep neck

and soft tissue in internal organs such as the brain, lung, and liver (Moore-Gillon

et al., 1981; whiley et al., 1992; Clarridge et al., 2001; Rashid et al., 2007).

Although they are commensal organisms, they can become pathogenic and

lead to an infection to the surrounding or distant sites after mucosal disruption

caused by trauma (Gossling, 1988). Streptococcus anginosus group (SAG) strains

are known for their association with purulent infections that occur after local

disruption of the mucosal barrier, such as in cases of ulceration, perforation,

inflammation, or surgery These infections often cause significant morbidity and

may require repeat drainage procedures. (Ruoff, 1988; Jacobs et al., 1994).

Among the group C streptococci, streptococcus anginosus group, has been

reported to cause intra-abdominal (Clarridge et al., 2001; Rashid et al., 2007),

pulmonary (Watt and Jack, 1977; Shinzato and Saito, 1995), and CNS (Gossling,

1988) infections. However, it remains an uncommon cause of meningitis (Barbara

et al., 1996).

A case of meningitis due to Strep. Milleri was reported in a 5 year old boy (Padhi

et al., 2004). Recent studies have shown an association between oral bacteria and

several systemic diseases (Williams and Offenbacher, 2000; Beck and

Offenbacher, 2001).

Streptococcus anginosus, one of the oral streptococci, is considered a common

commensal organism found in the human oral cavity, however, this organism has

become known as an important pathogen in respiratory infections and sub acute

bacterial endocarditis (Shinzato and Saito 1995; Sasaki et al., 2001; Allen et al.,

2002).

It is also known that the presence of Strep. anginosus is associated with

carcinogenesis of head and neck (Sasaki et al., 1998; Tateda et al., 2000; Shiga et


25

al., 2001). The quantity of putative pathogen present in clinical samples has

previously been studied by cultivation, immunoassay and DNA hybridization.

These studies have provided useful data implicating several species potential

pathogens (Tappuni and Challacombe 1993; Matto et al., 1998; Darout et al.,

2002).

Strep. anginosus cause sever infections after surgical treatment and infect

implanted material, thereby posing a problem of substantial clinical relevance

(Van der Auwera, 1978; Tresadern et al., 1983; Laine et al, 2005). In recent

study, members of the SAG were identified as the frequent cause of infective

pyogenic streptococcal infections in Canada (Laupland et al., 2006).

1.2.7. Susceptibility to Antimicrobial Agents:

Since the early 1990s, antimicrobial resistance among species of streptococci

has been increasing. Organisms classified as Strep. milleri are usually resistant to

bacitracin and nitrofurazone (Pool and Wilson 1976; Ball and Parker 1979).

Bacitracin resistance is a characteristic which allows separation of Strep. milleri

isolates with the group A Lancefield antigen from bacitracin-susceptible Strep.

pyogenes.

Most strains studied have been found to be susceptible to penicillin,

ampicillin, erythromycin, and tetracycline (Bourgult et al., 1979; Shales et al.,

1981; Tillotson and Ganguli, 1984).

In-vitro bactericidal interactions of penicillin G, cefotaxime, or vancomycin in

combination with gentamicin were studied, which were compared against 20

group G streptococci by the timed kill curve method. Synergy was noted at the

following frequencies: penicillin plus gentamicin, 80%; cefotaxime plus

gentamicin, 85%; vancomycin plus gentamicin 90%. There was no bactericidal

antagonism observed (Lam and Bayer, 1984).

Most members of the Strep. milleri group are susceptible to low

concentrations of penicillin G. Approximately 80-90% of strains have MICs of

0.1 mg/L (G mez-Garcés et al., 1994; Doern et al., 1996).


26

Pencillinase-resistant penicillin (nafcillin and methicillin), cephalothin,

cefamandole, rifampin, vancomycin, clindamycin, and chloramphenicol are also

effective in-vitro against Strep. milleri (Bourgult et al., 1979; Shales et al., 1981).

Penicillin tolerance of human isolates of group C streptococci were tested,

most of the strains showed penicillin tolerance. Synergism was demonstrated with

a combination of penicillin and gentamicin for all strains tested. The rate of

antibiotic killing was measured for a part of these strains by using the combination

of penicillin and gentamicin. All isolates were killed within 5 hours with the

combination, but viable organisms were recovered after 48 hours when either drug

was used alone (Portnoy et al., 1981). This study suggests that penicillin tolerance

with group C streptococci may occur frequently and may account for the poor

outcome of serious group C streptococcal infections tested with penicillin alone.

United States surveillance of viridians group streptococcal bloodstream

isolates in the 1990s reported that 32% to 56% were not susceptible to penicillin

and 38% to 46% were resistant to erythromycin (Doern et al., 1996; Diekema et

al., 2001).

Streptococcus anginosus group SAG is generally considered to be susceptible

to penicillin, other -lactam antibiotics and macrolides, but resistant strains have

been reported (Aracil et al., 1999; Limia et al., 1999; Tracy et al., 2001).

The majority of group C and G streptococci strains demonstrate in vitro

susceptibility to erythromycin, and cephalosporin (Rolston et al., 1982; Bayer and

Lam, 1983).

Many other -lactam antibiotics have in-vitro activity similar to that of

penicillin against the SMG, but the susceptibility to different cephalosporin are

quite variable (Alcaide et al., 1995).

Only a few clinical isolates have been reported to exhibit tolerance of

vancomycin (Noble et al., 1980; Roleston et al., 1984). Tolerance of vancomycin

previously reported among pharyngeal isolates of non-group A -hemolytic

streptococci (mostly GCS and GGS) from children, later investigators chose to


27

investigate further antibiotic susceptibility patterns among GCS and GGS isolated

from patients with invasive infections (bacteremia and meningitis, etc.), for whom

similar findings of tolerance may have clinical implications in treating these

seriously ill patients (Zauotis et al., 1996).

Tolerance of vancomycin, defined as an MBC 32 or more times higher than

the MIC, was exhibited by 18 GGS isolates (54%) (Zaoutis et al., 1999).

The aminoglycosides, gentamicin and netilmicin were much more active

against Strep. milleri than the amikacin and kanamycin, but aminoglycosides were

rarely bactericidal at concentrations safely attainable in serum (Bourgult et al.,

1979). Sulfonamides are ineffective against Strep. milleri (Tillotson and Ganguli,

1984).

The incidence and patterns of antimicrobial resistance among -hemolytic

viridans group streptococci, which is the anginosus group part of it were

determined, isolated from various clinical specimens in a Korean hospital and the

macrolide resistance phenotypes and genotypes of erythromycin-resistance

isolates were clarified and found that the overall resistance rates of -hemolytic

VGS were found to be 47.5% to tetracycline, 3.9% to chloramphenicol, 9.7% to

erythromycin , and 6.8% to clindamycin, whereas all isolates were susceptible to

penicillin G, ceftriaxone, and vancomycin. Among ten erythromycin-resistant

isolates, six isolates expressed a constitutive MLSB (cMLSB) phenotype, and each

of the two isolates expressed the M phenotype, and the inducible MLSB (MLSB)

phenotype. The resistance rates to erythromycin and clindamycin of -hemolytic

VGS seemed to be lower than those of non- -hemolytic VGS in this hospital,

although cMLSB phenotype carrying erm (B) was dominant in -hemolytic VGS

(Uh et al., 2007).

Moreover a significant increase in the antimicrobial resistance of viridance

and -hemolytic streptococci has been noticed in recent decades (Seppala et al.,

2003).


28

Macrolide-resistance genes in group C and G streptococci were studied and

the macrolide-lincosamide-streptomycin B (MLSB) resistance in 47 clinical

isolates of group C beta-hemolytic streptococci and 17 group G streptococci was

investigated. Resistance to erythromycin was found in 31.6%; and 47%

respectively. Resistance to erythromycin was due to the presence of mefA, ermB

and ermTR genes. The ermTR genes were predominant among the group C and G

streptococci (90% of the strains) (Seral et al., 2000).

Different erythromycin resistance mechanisms in group C and group G

streptococci have been demonstrated. The two presently recognized mechanisms

for resistance to macrolide antibiotics in streptococci are target-site modification

and active-drug efflux (Kataja et al., 1998).

A worked on the prevalence of erythromycin and clindamycin resistance

among clinical isolates of the streptococcus anginosus group in Germany were

done by some researchers, they analyzed the antimicrobial susceptibility of 141

clinical SAG isolates to six antimicrobial agents by agar dilution. All isolates

were susceptible to penicillin, cefotaxime and vancomycin. However, 12.8%

displayed increased MIC values (0.12mgl-1) for penicillin. Resistance to

erythromycin was detected in eight (5.7%) isolates. Characterization of the

erythromycin-resistant isolates with the double-disc diffusion test revealed

Macrolide-Lincosamide-Streptomycin B and M-type resistance in six and two

isolates, respectively. Resistance and intermediate resistance to ciprofloxacin

were detected in two and six isolates, respectively. The data show that resistance

to erythromycin, clindamycin and ciprofloxacin has emerged among SAG isolates

in Germany (Asmah et al., 2009).

Chapter Two Materials and methods

29

2.1. Materials

2.1.1. Patients:

The specimens of this study include 177 patients suffering from pharyngitis who

submitted to Hilla teaching hospital during the period of three months (from October

2009 to January 2010). The patient s age ranged from (4-75) years. Throat swab

specimens were collected from them.

2.1.2. Laboratory Instruments and equipment:

Table (2-1) shows the instruments and equipments used in this study:

Table (2-1): Instruments and equipment

Instruments and Equipments Company / origin

Candle Jar BBL/USA

RefrigeratorConcord/Italy

AutoclaveFanem /Brazil

Digital cameraGenx/China

Table Top CentrifugeGemmy/Taiwan

OvenIncubatorShaker water bath

Memmert /Germany

Light microscope Olympus/Japan

Millipore FilterProway/China

MicropipetteSlamid/England

pH-meterWTW/Germany

Distillator


30

2.1.3. Chemical Materials:

Table (2-2) shows the chemical materials used in this study:

Table (2-2): Chemical Materials

Materials Company/Origin

Tannic acid , D-MannoseDi-potassium hydrogen phosphatePotassium di-hydrogen phosphateDi-sodium hydrogen phosphateSodium Chloride, Magnesium sulfate,Calcium Chloride, Coppric sulfate ,

Ammonium chloride, Potassium Iodide

BDH/England

Chloroform, Sulfuric acid, BariumChloride

Fluka/Switzerland

Phenol red, Glucose, StarchSalicin, Iodine, Ethanol 99%Glycerol, Hydrogen peroxide

GCC/England

Crystal violet, EDTA Sigma/USA

Gelatin, Trichloroacidic acid Sigma/Germany

Iodine, Ethanol Syrbio/Switzerland

2.1.4. Culture Media: Table (2-3) shows the culture media used in this study:

Table (2-3): Culture Media

Materials Company/Origin

Agar agar, Blood agar baseBrain heart infusion agarBrain heart infusion brothColumbia agar, Muller Hinton agarNutrient agar, Nutrient brothTryptic soy agar

Himedia/India

Egg yolk agar mediumM9 medium, Chocolate AgarSugar fermentation medium

Prepared in laboratory ofMicrobiology department /Collage ofMedicine/Babylon University


31

2.1.5. Diagnostic kits: Table (2-4) shows the diagnostic kits used in this study:

Table (2-4): Diagnostic kits

Diagnostic kits Company/origin

API 20 strep Biomerieux/FranceHicomb MIC test Himedia/IndiaStreptex Kit Remel/USA

2.1.6. Diagnostic Disks:-

The diagnostic disks used in this study are shown in Table (2-5) below:

Table (2-5): Diagnostic Disks

Diagnostic Disk Potency Company/Origin

Bacitracin 10µgHimedia/IndiaOptochin 5 µg

2.1.7.1. Antibiotic powders

Table (2-6) below shows the antibiotic powders used in this study:

Table (2-6): Antibiotic powders

Antibiotics Company/Origin

Ampicillin

SDI/IraqCeftriaxon

ErythromycinTetracyclinePenicillin G Panpharma/France

Vancomycin Julphar/U.A.E

2.1.7.2. Antibiotic Disks (Bioanalyse /Turkey)

The Antibiotics Disks (as recommended by CLSI, 2007) used in this study are

shown in Table (2-7) below:


32

Table (2-7): Antibiotic Disks

Group Antibiotic Disk potency (µg) Symbol

Penicillins Ampicillin 10 Am

Cephems

(cephalosporins)

Cefepime 30 FEP

Cefotaxime 30 CTX

Ceftriaxon 30 CTR

Glycopeptides Vancomycin 30 VA

Macrolides

Erythromycin 15 E

Clarithromycin 15 CLR

Azithromycin 15 AZM

Tetracyclins Tetracycline 30 TE

Fluoroquinolones Ofloxacin 5 OFX

Phenicols Chloramphenicol 30 C

Lincosamides Clindamycin 2 DA

2.1.8. Bacterial Isolates:-

The standard strains and local isolates used in this study are listed in Table (2-8)

below:

Table (2-8): Standard Strains and Local Isolates

Strain Genotype Origin

Escherichia coli Wild type ATCC 25922

Staphylococcus aureus

Local isolatesDepartment of microbiology/College

of medicine/Babylon University

Streptococcus pyogenes

Enterococcus faecalis


33

2.2. Methods

2.2.1. Specimen collection:

One hundred seventy seven swab specimens are collected from patients who

suffering from pharyngitis. Information about their (sex, age, and antibiotic

administration) is taken into consideration. One specimen for each patient for

bacteriological analysis is described below. These specimens are collected with the

help of physician to avoid any possible contamination. The swabs are inserted into

the pharynx and rotated there before withdrawing them and withdrawn carefully

without contamination from the mouth. Swab for culture were placed in tubes

containing normal saline to maintain the swab wet until taken to laboratory. Each

specimen is immediately inoculated on the blood agar plates. All plates are

incubated anaerobically by Candle Jar at 37 C for 24-48 hrs.

2.2.2. Preparation of Reagents and Solutions:

2.2.2.1. Catalase Reagent (Forbes et al., 2007):

3% H2O2 solution was prepared by using 3ml of H2O2 and 97ml of distilled water and

stored in a dark container. It was used for detection of catalase enzyme production.

2.2.2.2. Phosphate buffer solution (PBS):

Phosphate buffer solution is prepared according to the manufacturer instructions, by

dissolving one tablet of PBS (pH 7.3) in 100 ml of distilled water and then sterilized by

autoclave.

2.2.2.3. Coppric sulfate solution (20%):

Coppric sulfate solution prepared by dissolving 20 gm of CuSo4 in small volume of

distilled water and completed up to 100 ml with distilled water. It was used in capsule

staining (Forbes et al., 2007).

2.2.2.4. Crystal violet solution (1%):

Crystal violet solution is prepared by dissolving 1 gm of the material in 100 ml of

distilled water. It was used in capsule staining (Forbes et al., 2007).


34

2.2.2.5. Tannic acid solution (1%):-

Tannic acid solution is prepared by dissolving 1gm of tannic acid in small volume of

distilled water and completed up to 100 ml of distilled water and then sterilized by

Millipore filter paper. It was used in haemagglutination test for detection of colonization

factor antigen-III (Sambrook and Rusell, 2001).

2.2.2.6. D-mannose solution (0.1 M):

D-mannose solution is prepared by dissolving 1.8 gm of D-mannose in 100 ml of

distilled water and then sterilized by filtration by Millipore filter paper. It was used in

haemagglutination test for detection of colonization factor antigen-I (Sambrook and

Rusell, 2001).

2.2.2.7. Normal Saline solution (0.15 M):

Normal saline solution is prepared by dissolving 0.8 gm in 90 ml of distilled water,

and further completed up to 100 ml with distilled water, and sterilized by autoclaving; it

has been used for preparing bacterial suspension that used in haemagglutination test for

detection of colonization factor antigen I and III (Sambrook and Rusell, 2001).

2.2.2.8. Trichloroacidic acid (TCA) solution (5%):

Trichloroacidic acid solution prepared by dissolving 5 gm of TCA in small volume of

distilled water and completed up to 100 ml of distilled water. It is used in the

extracellular protease production test (Piret et al., 1983).

2.2.2.9. -Lactamase detection solutions (WHO, 1978):

• Penicillin G solution: is prepared by dissolving 0.5693 gm of penicillin G in 100 ml of

phosphate buffer solution (2.2.2.2) and the resulting solution was stored at-20 C after

sterilization by filtration and dispensed in small vials.

• Iodine solution: 2.03gm of iodine and potassium iodide were dissolved in 90 ml of

distilled water and the volume was completed with distilled water to 100 ml, and then stored

in dark bottle at 4 C.

• Starch solution: one gram of soluble starch was dissolved in 100 ml of distilled water and

boiled in water bath for 10 min. The solution was stored in dark bottle at 4 C.


35

2.2.2.10. McFarland tube standard (0.50):

A barium-sulfate turbidity standard solution equivalent to a 0.5 McFarland standard

was prepared as described by NCCLS (2003), as follows:

•A 0.5 ml of aliquot of 0.048 M BaCl2 (1.175% w/v BaCl2 .H2O) was added to 99.5

ml of 0.18 M H2SO4 (1% v/v) with constant stirring to maintain a suspension.

•Correct density of the turbidity standard is verified by using reading the

absorbance at wave 625 nm. The absorbance should be 0.08 to 0.10 for the

McFarland standard.

•Barium-sulfate suspension is distributed in 4 ml aliquots into screw cap tubes,

which were tightly sealed and stored in the dark at room temperature.

•Barium-sulfate turbidity standard is vigorously agitated on a mechanical vortex

before each use and inspected for uniformly turbid appearance.

•Barium-sulfate standard should be replaced or their densities verified monthly.

2.2.3. Preparation of culture media:

The general culture media described below are prepared using the routine methods

and used in appropriate assays:-

2.2.3.1. Blood agar medium:

Blood agar medium was prepared according to manufacturer by dissolving 40 gm of

blood agar base in 1000 ml of distilled water. The medium was autoclaved at 121C for

15 minutes, cold to 50 C and 5% of fresh human blood was added. This medium was

used as enrichment medium for cultivation of the bacterial isolates and to determine their

ability of blood hemolysis.

2.2.3.2. Nutrient agar medium:

Nutrient agar medium was prepared according to the manufacturing company by

dissolving 28 gm of nutrient agar in 1000 ml of distilled water. The medium was

autoclaved at 121C for 15 minutes It is used for general tests, cultivation and activation

of bacterial isolates when it is necessary.


36

2.2.3.3. Nutrient Broth:

Nutrient broth medium was prepared according to the manufacturing company by

dissolving 13 gm of nutrient broth in 1000 ml of distilled water. The medium was

autoclaved at 121C for 15 minutes. It used for general tests, cultivation and activation of

bacterial isolates when it is necessary.

2.2.3.4. Müller-Hinton agar:

Müller-Hinton agar was prepared according to the manufacturing Company by

dissolving 38 gm of Müller-Hinton agar in 1000 ml of distilled water. The medium was

autoclaved at 121C for 15 minutes. It was used in anti-bacterial susceptibility testing.

2.2.3.5. M9 medium:

This medium was prepared according to procedure that described by

Sambrook and Rusell (2001) by dissolving 6 gm of Na2HPO4, 3 gm of KH2PO4, 0.5 gm

of NaCL, and 1 gm of NH4Cl in 950 ml of distilled water, with 2% agar, and then

sterilized by autoclave. After cooling, 2 ml of 1M of MgSO4, 10 ml of 20% glucose and

0.1 ml of 1M of CaCl2 (sterilized separately by filtration) were added, and then the

volume was completed to 1000 ml with distilled water. This medium was used for the

detection of extracellular proteases production.

2.2.3.6. Brain heart infusion-glycerol broth medium:-

This medium was prepared by adding 5 ml of glycerol to 95 ml of

BHI broth, then it was sterilized by autoclave. It was used in Bacteriocin production

detection test (Forbes et al., 2007).

2.2.3.7. Brain heart infusion-glycerol agar medium:-

This medium was prepared by adding 5 ml of glycerol to 95 ml of BHI agar before

autoclaving (Forbes et al., 2007). It was used in Bacteriocin production detection test.

2.2.3.8. Egg- yolk agar medium:

It was prepared by adding 15 ml of egg yolk suspension to 85 ml sterile nutrient agar

after cooling it to 55 Co (Collee et al., 1996). This medium was used to detect the ability

of bacteria to produce lipase enzyme.


37

2.2.3.9. Tryptic Soy Agar medium:

This medium was prepared according to manufacturer by dissolving 40 gm of Tryptic

soy agar in 1000 ml of distilled water. to enhance growth of bacterial isolates, this

medium was modified by adding 5% fresh blood after autoclaving the medium at 121C

for 15 minutes, and colling it to 50 C . This medium was used as enrichment medium for

cultivation of the bacterial isolates and to determine their groups by streptex test.

2.2.3.10. Columbia Blood Agar:

This medium was prepared by dissolving 40 gm of Columbia agar in 1000 ml of

distilled water, after autoclaving the medium at 121C for 15 minutes, and cooling it to

50 C , a 5% fresh blood was added to the medium. This medium was used as enrichment

medium for cultivation of the bacterial isolates and to achieve tests in API 20 strep test.

system.

2.2.3.11. Chocolate Agar:

This medium was prepared by dissolving 40 gm of blood agar in1000 ml of distilled

water, after autoclaving the medium at 121C for 15 minutes, and cooling it in a water-

bath at 80C , a10% of sterile blood was added to the medium and allowed to remain at 80

C , then mixing the blood and agar by gentle agitation from time to time until the blood

becomes chocolate-brown in colour, within about 10 minutes. Then this medium was

poured in plates. This medium was used for gliding motility test (Collee et al., 1996).

2.2.3.12. Sugar fermentation medium:

This medium consists of:

Medium base: It was prepared by dissolving 13 gm of nutrient broth and 0.018 gm of

phenol red in 1000 ml of distilled water. After pH was adjusted to 7.4, contents were

distributed into test tubes. The tubes were then autoclaved.

Sugar solution: 1% of each of the following sugars were used; Mannitol, Raffinose,

inulin and, Salicin (0.5%). All sugar solutions were sterilized using chloroform vapor

(Smith, 1932). 0.1 ml of each sterile sugar solution was added to each tube containing

medium base.


38

2.2.4. Stains:

Gram s Stain:-This stain was prepared according to Collee et al. (1996). It was used

to study cells morphology and their arrangement, to differentiate between Gram-negative

and Gram- positive bacteria.

Capsule stain:- This stain was prepared according to Hiss s Method (2.2.9.1). It

contains two solutions: 1% crystal violet (2.2.2.4) and 20% Coppric sulphate

(2.2.2.3). It was used for detection of bacterial isolates capsules (Balows et al.,

1991).

2.2.5. Isolation and Identification of bacterial isolates:

According to the diagnostic procedures recommended in Collee et al. (1996);

McFadden (2000) and Forbes et al. (2007), the isolation and identification of Strep.

anginosus group bacteria in patient s pharynx associated with pharyngitis were

performed as follows:

2.2.5.1. Colonial morphology & Microscopic Examination

Colonial Morphology: All throat specimens were cultured on enriched blood agar

by swabbing and incubated at 37 C for 1-2 days. A single colony was taken from

each primary positive culture and its identification depended on the morphological

properties such as (Colony size, shape, nature of pigments, translucency, edge,

elevation, and texture).

Microscopic Examination: The morphology of bacterial cells was investigated by

gram-stain to observe a shape, arrangement of cells and type of reaction with gram-

stain. After staining the bacteria by gram stains, specific biochemical tests were

done to reach to the final identification.

2.2.6. Physiological and Biochemical Tests

2.2.6.1. Catalase Test:-

Blood agar base was streaked with the selected bacterial colonies and incubated at

37C for 24 hrs. then transfer the growth by the wooden stick and put it on the surface of


39

a clean slide and add a drop of catalase reagent. Formation of gas bubbles indicates

positive results (Forbes et al., 2007).

2.2.6.2. Optochin Susceptibility Test:

Blood agar plate was streaked with an inoculum from a pure isolates of the

organism to be tested , then an optochin disc was placed in the center of the

inoculums and incubated for 24 hrs at 37 C in a Candle Jar ,this test used to

differentiate between Strep. anginosus and Strep. pneumoniae, if observation of

zones of growth inhibition greater than 14 mm surrounding the disc considered a

positive and it was presumptive indication of Streptococcus pneumoniae (Baron et

al., 1994).

2.2.6.3. Bacitracin sensitivity test:

The blood agar was streaked with bacterial culture and the bacitracin disk was

put in the center of the cultur. The diameter of inhibition zone was equal to or more

than 12 mm, indicating a positive result. This test was used to differentiate between

St. pyogenes and St. anginosus group (McFadden, 2000).

2.2.6.4. CAMP Test:

This test used to differentiate between -hemolytic group B Strep. agalactiae

and Strep. anginosus group, the test done by inoculating a -hemolysin producing

Staphylococcus aureus as a streak across a blood agar plat containing 5% sheep

blood. Then inoculate a single streak of the Streptococcus perpendicular to that of

Staphylococcus, leave 1 cm of space between the two streaks, and then incubate the

plate at 37C for 24h. The positive result appears as an arrowhead-shaped zone of

enhanced hemolysis at the juncture between positive Streptococcus and the

Staphylococcus (Collee, et.al., 1996).

2.2.6.5. Sugar fermentation test:-

Tubes of sugar fermentation medium (2.2.3.12) were inoculated and incubated at

37 C for 1-5 days. The changing in the color of indicator to yellow indicates a

positive reaction with this test.


40

2.2.7. Commercial API 20 system:

These tests were done according to the manufacturer instructions. API 20 system

contains the following tests:

Voges Proskauer test, hydrolysis of hippuric acid, hydrolysis of Esculin, PyrrolidonylArylamidase, alpha-Galactosidase, beta-Glucuronidase, beta-Galactosidase, AlkalinePhosphatase, Leucine Amino peptidase, Arginine dihydrolase, and fermentation ofRibose ,Arabinose, Mannitol, Sorbitol, Lactose, Trehalose, Raffinose, Starch, andglycogen.

• These tests were done by picking a well isolated colony and suspend it into 0.3 mlof normal saline.

• Then flood a Columbia-blood agar plate with this suspension.• Incubate the plate for 24 hours at 37C under 5% CO2 conditions. • Using sterile tube containing 2 ml of normal saline , then harvest heavy

inoculum from previously prepared subculture plate, making a densesuspension with standard turbidity of McFarland (4 tube).

• Then inoculating the cupules of the strip with bacterial suspension, and thenfilling all cupules of the underlined tests with mineral oil.

• Incubating the inoculated strip at 37 C for 4-4.5 hours under aerobicconditions. The results will be recorded to obtain a first reading in this time,and after 24 hours to obtain a second reading if required.

2.2.8. Serological test: Streptex agglutination test for Lancefield grouping:

This test was used to identify streptococci groups (A, B, C, D, F, and G) according to

(Facklam, 1991):

• It was done by dispensing 400 l extraction enzymes into an appropriately labeledtest tube for each culture to be grouped.

• Using a bacteriological loop, to make a light suspension from the culture on trypticsoy blood agar in a sterile tube of the enzyme solution.

• The suspension incubated at 37 C in a water bath for a minimum of 10 minutesor any time up to 1 hour. Shaking the tube after 5 minutes incubation.

• One drop of each latex suspension dispensed by a dropper onto a separate circleon a reaction card.


41

• Using a pipette, one drop of extract was placed in each of the six circles on thereaction card. Mixing the contents in each circle in turn with a mixing stick bya separated stick for each circle. The card was rocked gently for 1-2 minutes,and then observes the agglutination in circles.

2.2.9. Virulence factors tests

2.2.9.1. Capsule stain test (Hiss s Method):

This test was carried out by mixing aloopful of physiological saline suspension

of growth with a drop of normal serum on a glass slide. Allow the smear to air dry,

and heat fix. Flood the smear with crystal violet (1% aqueous solution) (2.2.2.4).

Steam the preparation gently for 1 minute and rinse with copper sulfate (20%

aqueous solution) (2.2.2.3).Capsules appear as faint blue halos around dark blue to

purple cells(Balows et al.,1991).

2.2.9.2. Hemolytic reaction test:

Blood agar medium was streaked with a pure culture of bacterial isolate to be tested

and incubated at 37C for 24-48 hrs. The appearance of a clear zone surrounding the

colony is an indicator of -hemolysis while the greenish zone is an indicator of -

hemolysis (Forbes et al., 2007).

2.2.9.3. Extracellular protease production test:-

This test was carried out by using M9 medium (2.2.2.3.4) supported by 1% gelatin,

after inoculation of this medium with bacterial isolates, and incubation for 24 48 hrs. at

37Co, then 3 ml of Trichloroacidic acid (5%) (2.2.2.8) was added. The positive result was

read by observing a transparent area around the colony (Piret et al., 1983).

2.2.9.4. Detection of Colonization Factor antigen (CFA) I and III:

It was performed to detect the ability of bacterial isolates to produce colonization

factors antigen (CFA). Colonization factor antigen I (CFA-I) production can be

detected as follows:

• RBCs suspension was prepared by placing human blood specimens (group A) in

tube contain EDTA and mixing with phosphate buffer solution (2.2.2.2) in


42

proportion 1:1 and centrifuged at 8000 rpm for 5 minutes. The supernatant was

discarded and the sediment of RBCs was washed three times with PBS and then

RBCs were resuspended up to 3%.

• A bacterial suspension is prepared in normal saline by taking the bacterial

growth for each strain from TSA and mixing it with 1ml of 0.15M NaCL

(2.2.2.7), to determine RBC agglutination test and vasticated colonization

factor antigen type I.

• One volume 25µl of bacterial growth was placed on glass slide and mix with same

volume of D-mannose solution (0.1 M) (2.2.2.6), and same volume of the above

RBCs suspension was added and allowed two minutes to observe the

agglutination, and control tests were done by mix 25µl of bacterial growth with

same volume of RBCs suspension without mannose on the other side of slide as

positive control .Absence of agglutination indicates positive result and vice versa.

• Colonization factor antigen III (CFA/ III) production was detected using same

procedure as described above, except using tannic acid solution (1%) (2.2.2.5)

instead of D-mannose solution (Sambrook and Rusell, 2001).

2.2.9.5. Bacteriocin production test:-

This test was performed using cup assay method as described by Al-Qassab and Al-

Khafaji (1992) as follows:

• All Strep. anginosus isolates were grown in BHI broth with 5% glycerol to enhance

their growth (2.2.2.3.5) at 37C for 18-24 hours.

• The growing Strep. anginosus were heavily streaked on BHI agar (2.2.2.6)

supplemented with 5% glycerol and then incubated at 37C for 18 hours.

• Local isolates were used as an indicator (sensitive isolates) for detection of

bacteriocin production by Strep. anginosus group.

• The indicator isolates was allowed to grow in nutrient broth for 2-3 hours in a

shaker water bath at 37C .


43

• A volume of 0.1 ml of indicator growth was spread on nutrient agar plates and left

to dry.

• Sterile cork borer (5mm diameter) was used to cut agar disks from the cultured agar

layer of tested bacteria (bacteriocin producers).

• Then transfer agar disks of tested bacteria to the agar surface seeded with indicator

isolates and incubated for overnight at 37C .

• When inhibition zone around the agar disk of tested bacteria is observed this

indicates a positive result for producing bacteriocin enzyme.

2.2.9.6. Lipase production test:

Lipase test was carried out in egg-yolk agar medium (2.2.3.7) to determine the ability

of tested bacteria to produce lipase enzyme. After inoculation of the medium agar, the

plates were incubated for overnight at 37C . The appearance of opaque pearly layer

around the colonies indicated for a positive result (Collee et al., 1996).

2.2.9.7. Gliding motility test:

Strains were inoculated into duplicate tubes containing motility

test medium and incubated at either 30 or 35C for up to 72 hours before a negative

reaction was recorded (Facklam and Washington, 1991).

The swarm assay method of Wolfe and Berg (1989) was used to measure spreading

of bacteria over agar surface.

• Bacteria grown on chocolate agar (2.2.3.11) were suspended in saline, and then the

turbidity was adjusted to the turbidity of a no. 0.5 McFarland standard.

• The suspension was diluted 1/100 in saline to produce a bacterial concentration of

approximately 106 CFU/ml.

• Aliquots (5 l) of this suspension were placed in the centers of agar plates.

• For each plate the diameter of the area of growth radiating from the inoculation

point was measured with calipers after 24 and 48 hours of inoculation at 35C in the

presence of 5% CO2 .


44

2.2.10. Antimicrobial susceptibility test:

2.2.10.1. Disk Diffusion test:-

It was performed by using a pure culture of previously identified bacterial isolate.

The most effective antibiotic for each bacterial isolate was determined as recommended

by CLSI (2007).

• The inoculums to be used in this test were prepared by adding 5 isolated colonies

grown on blood agar plate to 5 ml of nutrient broth and incubated at 37Co for 18

hours and compared with (0.5) McFarland standard tube (2.2.2.10).

• A sterile swab was used to obtain an inoculums from the bacterial suspension, this

inoculums was streaked on a Müeller-Hinton agar plate and left to dry.

• The antibiotic discs were placed on the surface of the medium at evenly spaced

intervals with flamed forceps or a disc applicator and incubated for 24 hours at 37Co.

• Inhibition zones were measured using a ruler and compared with the zones of

inhibition determined by the National Committee for Clinical Laboratory Standards

(NCCLS, 2003).

2.2.10.2. Detection of -Lactamase Production:

This test was performed for all isolates that were resistant to -lactam antibiotics,

according to Rapid Iodometric Method (WHO, 1978):

• Overnight bacterial cultures on Blood agar were prepared. Using a sterile loop,

several colonies were transferred to Eppendorf tubes containing 100 µL of

penicillin G (2.2.2.9), and the tubes were incubated at 37 C for 30 min.

• Apportion of 50 µL of starch solution (2.2.2.9) was added and mixed well with

the contents of the tube.

• Portion of 20 µL of iodine solution (2.2.2.9) was added to the tube which cause

the appearance of dark blue color, rapid change of the color to white (within 1-5

min.) indicated a positive result.

The results were compared with negative control (E.coli ATCC 25922).


45

2.2.10.3. Determination of Minimum Inhibitory concentration (MICs):

Two methods were used for determination of MICs of isolates:

2.2.10.3.1. HiComb MIC Test:

This test was done according to procedure of Bryskier and Lorian (2005)

• Müeller-Hinton Agar medium which supplemented with 5% human blood. This

medium was inoculated with a suspension of the standard number of organisms

which made by transferring 4-5 colonies with a sterile loop to 5 ml of Trypton Soya

broth and incubate at 37Co for 4-5 hours until light to moderate turbidity develops,

then compare the inoculums turbidity with that of (0.5) McFarland standard tube

(2.2.2.10).

• After inoculating the medium with the standardized inoculums by using a sterile

swab and streaking the entire surface of the plate, allow the inoculums to dry for 5-

15 minutes.

• Apply the HiComb MIC strip on the surface of Müeller-Hinton with the MIC scale

facing upwnwards. Once applied, do not move the strip. Let it absorb to the surface

of the agar media. Incubate at 37Coand examine after 18-24 hrs.

• Results and interpretation are as follow:

The zone of inhibition will be in the form of an ellipse. MIC value would be the

value at which the zone convenes the comb-like projections of the strips and not at

the handle. If there is no zone of inhibition observed, report the MIC as greater than

the highest concentration on the strip. If the zone of inhibition is below the lowest

concentration then report the MIC as less than the lowest concentration.

2.2.10.3.2. Determination of MICs by agar dilution method:

• The agar dilution susceptibility method was used for determination of

MICs of number of antibiotics.

• The ranges of appropriate dilutions of antibiotics for MIC determinations(µg/ml)

were used as described by Miles and Amyes (1996), which were as follows:


46

Suggested ranges for MICs

Antibiotics Determinations(µg/ml)

Ampicillin 0.25-128

Ceftriaxone 0.25-128

Erythromycin 0.06-8

Tetracycline 0.03-128

Vancomycin 0.12-16

• Appropriate dilutions of antibiotic solutions were prepared according to the report of

international collaborative study by Ericsson and sherries (1971), in which one part

of the antimicrobial solution was added to nine parts of liquid Müeller-Hinton agar.

• The prepared dilutions of antibiotic solutions were added to the molten Müeller-

Hinton agar media that have been allowed to equilibrate in a water bath to 45-50 Co,

and then mixed thoroughly. The mixture was poured into Petri dishes, and allowed

to solidify at room temperature.

• Standardized inoculum for agar dilution method was prepared by growing bacteria

to the turbidity of 0.5 McFarland standards (2.2.2.10). The 0.5 McFarland

suspensions were diluted 1:10 in sterile normal saline.

• The agar plates were marked for the orientation of the inoculums spots .one µL

aliquot of each inoculum was applied to the agar surface with standardized loop.

• Antibiotic free media were used as negative controls and inoculated .The inoculated

plates were allowed to stand at room temperature for no more than 30 minutes until

the moisture in the inoculums spots was absorbed by the agar. The plates were

inverted and incubated at 35 Co for 16 to 20 hrs.

• To determine agar dilution break points, the plates were placed on a dark surface,

and The MIC was recorded as the lowest concentration of the antimicrobial agent

that completely inhibits growth. The MIC values were compared with the break

points recommended by CLSI (2007). Results were compared with the results of

negative control (E.coli ATCC 25922) .


47

2.2.11. Preservation of bacterial isolates:

The bacterial isolates were preserved on blood agar slants at 4 C . The isolates were

maintained monthly during the study by subculturing on new culture media .The

bacterial isolates also were preserved for long time in BHI broth supplemented with

15% glycerol then stored at -20C for 6-8 months (Collee et al., 1996).

Chapter Three____________________________________________ Results and Discussion

48

Chapter Three: Results and Discussion

3.1. Isolation and Identification of Bacterial Isolates:

The present study included collection of 177 throat swab samples from patients

with pharyngitis during the period from October 2009 to January 2010 in Hilla

Teaching Hospital. Morphological and biochemical characterization reveald that 137

isolates belonged to the genus streptococci. Of which 67 (37.8%) -hemolytic

isolates, 5 (2.8%) -hemolytic, and 65 (36.7%) non-hemolytic, While others 40

(22.5%) isolates belonged to other bacterial genera. The results showed that out of

67 -hemolytic streptococcal isolates, 11 (16.4%) isolates belonged to the

Strep.anginosus group. However this percentage reached to about (6.2%) of all

positive cultures of throat swabs collected in this study (Table 3-1). The percentage

of the positive isolates (6.2%) in the present study is higher than that recorded by

other studies. Lewis and Balfour (1999) reported that 65 throat swabs of 1493

received (4.4%) yielded group C streptococci. This result may be due to the

increased infections with pharyngitis caused by group C streptococci in Hilla city,

Iraq, and poor hygien in our country. There was no difference in this study in the

isolation rates of BHS between males and females. Of the 11 isolates which belonged

to the anginosus group, 4 isolates (36.3%) were recovered from males and 7 isolates

(63.6%) form females.

This result is inconsistent with Jacobs et al., (1995 ) who found that this group has a

higher incidence of infections among males. This result may be due to the few

numbers of bacterial isolates of group C streptococci recovered during the isolation

period. Samples were collected from all age groups in this study with a documented

age range of 4 to 75 years. They are less common in neonates or infants (Pool and

Wilson, 1979; Singh et al., 1988).


49

Table (3-1): Distribution of -hemolytic Streptococci belonging to anginosus group and

other bacteria isolated from throat swab samples.

Bacterial isolates (No.) %No. of positive

isolates %

Strep. Spp.-haemolytic Streptococci (67) 37.8 11 (16.4)

-haemolytic Streptococci (5) 2.8

Non-haemolytic Streptococci (65) 36.7

Total No: (137) 77.4

Others:

Staphylococcus spp. (28) 15.8

Neisseria spp. (1) 0.5

Gram negative Cocci (10) 5.6

Total No: (40) 22.5

Total No. of samples: (177) 100 11 (6.2)

3.2. Biochemical and Physiological tests:

Results in tables (3-2) and (3-3) showed that the isolates belonging to the anginosus

group were gram positive cocci, chains ranging from long to short, forming

microcolonies, catalas negative, not sensitive for optochin and bacitracin, all of them

were -hemolytic, negative for CAMP test, possess Lancefield group C and F antigens,

produce acid from salicin, trehalose, raffinose and lactose but not sorbitol and manitol,

most of them do not ferment ribose. This result agreed with the results of Lebrun et

al.(1986); and hydrolyse arginine and esculine; produce acetoin from glucose (vogues-

proskauer posetive), some of the anginosus group isolates can be VP negative, these

results match with the discrition of Ball and Parker (1979); Gossling (1988); Piscitelli et

al., (1992) ; Koneman et al. , ( 1997 ); and Mascini and Holm, (2004).


50

Table (3-2): Biochemical and physiological tests used for differentiation of

Streptococcusanginosus group from other Streptococci

Biochemical and physiological tests

Isolate designation -

Hemolysis

CAMP

test

OptochinBacitraci

n

+---Strep.anginosus T 17+---Strep.anginosus T 46+---Strep.anginosus T 91+---Strep.anginosus T 93+---Strep.anginosus T108

+---Strep.anginosus T113+---Strep.anginosus T114+---Strep.anginosus T116+---Strep.anginosus T137+---Strep.anginosus T147+---Strep.anginosus T151

Catalase test was used to exclude Staphylococcus spp. (positive) from isolates (negative) in

this study. CAMP test was used to differentiate between -hemolytic group B Strep.

agalactie (positive) and Strep. anginosus group. Also the results of bacitracin and optochin

sensitivity test were used to exclude streptococci group A Strep. pyogenes and Strep.

pneumoniae respectively (Collee et.al., 1996).

Table (3-3): Additional tests (other than that in API 20 strep system) used foidentification of Streptococcus anginosus group

Test Result

Colony characteristics Small, grey, and large colonies on bloodagar

Cell morphology Gram positive , CocciHemolysis Beta-hemolyticBiochemical reactionsCatalaseMannitolSalicinRaffinose

NegativeNegativePositivePositive

Lancefield group reaction Group F and C


51

Colonies of the Strep.milleri group appear similar to typical streptococcal

colonies, growing as small, pinpoint, and grey on blood agar and testing catalase

negative (Gray, 2005) while Strep. anginosus group colonies are predictable in their

colony size in culture, several other phenotypic characteristics routinely used to

differentiate and identify streptococci are much more inconsistent in the milleri

group. One important way in which the Strep. anginosus group can be variable in its

presentation is with respect to its Lancefield group reactions. Lancefield grouping

differentiates streptococcal bacteria on the basis of a carbohydrate antigens.

In addition to the usual phenotypic colony characteristics, biochemical tests are

also used to assist in the identification of these bacteria. One simple laboratory test

for the Strep. anginosus group is the Vogues-Proskauer test. This test is positive in

the presence of acetoin, which is produced by Strep. anginosus group bacteria and

not by other beta-hemolytic streptococcal species. Other biochemical tests show that

Strep. anginosus group bacteria are positive for arginine dihydrolase and esculine

hydrolysis and negative for fermentation of mannitol and sorbitol (Mascini and

Holm, 2004).

Results of these tests are more consistent and thus useful to identify these

organisms. Difficulty in identification of these organisms has led to the development

of commercial systems as an aid. In general, these systems include the more

consistent biochemical reactions of Strep. anginosus group bacteria (Gray, 2005).

Hemolysis of organisms growing on blood agar is also used to distinguish

streptococcal group organisms. However, this feature is inconsistent throughout the

group, with strains capable of both alpha- and beta-hemolysis, in addition to gamma

hemolysis (Mascini and Holm, 2004).

API-20 strep systems (Appendix 1) were used for the identification of isolates of

anginosus group (Figure 3-1) in addition to another physiological tests. The use of


52

biochemical profiling tests in API 20 strep system test was not sufficient for final

identification of species of this streptococcal group.

Identification may be difficult, as differentaition from other streptococci is based on

standard biochemical testing (Belko et al., 2002). The Strep. anginosus group is

considered by most sources to be part of the viridans group of streptococci (Antony

and Startton, 2000). Comercial kits (e.g., API 20 Strep system) have revolutionised

the identification of the viridance streptococci to species level, and have been

favourably reviewed for the identification of Strep. milleri isolates (Tillotson , 1982;

French et al., 1989). However, many laboratories reported the identificaion of these

bacteria to the group level but not the species level (Liu et al., 2006).

Figure(3-1): API 20 strep system


53

3.3. Serological Identification:

Streptex agglutination test was used for identification of Lancefield groups A, B, C,

F, and G for testd isolates and the results were shown in (Table 3-4).

The results in (Figure 3-2) showed that the isolates in our study possess Lancefield

group antigens C 2 (18.1%) and F 8 (81.8%) that found in -hemolytic streptococci.

This result is similar to the results of Lawrence et al. (1985) who find that Strep.

milleri represnt 56% of the group C, 100% of group F, and 83% of the nongroupable

beta-hemolytic streptococci isolated in their clinical laboratory, whereas the

incedence of Strep. milleri among group A and G streptococci was estimated to be

low.

Table (3-4): Lancefield grouping of anginosus group by streptex agglutination test

Isolates designation Lancefield group antigens

A B C D F G

Strep.anginosus T 17 - - - - + -



Strep.anginosus T 93 - - + - - -

Strep.anginosus T 108 - - + - - -






Strep.anginosus T 151 - - + - + -


54

Figure (3-2): Percentages of group C and F antigens in the -hemolytic streptococcirecovered in this study.

Although the Strep. anginosus group bacteria are considered to belong to

Lancefield group F, studies have reported that Strep. anginosus group strains

presenting with Lancefield group antigens A, C and G, or even no Lancefield

antigen at all (Antony and Startton, 2000; Facklam et al., 2002).

Streptococcal identification in the clinical laboratory is dichotomous.

Classically, only -hemolytic varieties were considered as noteworthy pathogens,

and Lancefield grouping provided a simple and accurate means for their

identification. Strep. milleri represent a special case, in that its members traverse

traditional hemolytic boundaries, and are known to be capable of possessing C

antigens (A, C, F, or G). Isolates may be classified as Strep. milleri on the basis of

characteristic biochemical tests, or, as with the group F streptococci, on the basis of

Lancefield grouping (Whitworth, 1990).

3.4. Virulence Factors of the bacterial isolates:

The virulence factors of anginosus group streptococcal isolates of the present

study were detected and the results were as fallow (Table 3-5):


55

3.4.1. Capsule production: Structural components of the bacterial cell envelope are

often fundamental to pathogenicity, and structural components may be primary

factors in producing disease.The presence of capsules in Strep. milleri strains has

rarely been reported (Whitworth, 1990). However most isolates (7 of 11) (63.6%) in

the present study were capsulated (Table 3-5).

Some studies showed that some Strep.milleri group streptococci possess a

polysaccharide capsule, demonstrated by Hiss,s and ruthenium red stains (Brook and

Walker, 1985; Leung, 2008). This result may be due to the fact that most isolates

recovered during this study belonge to Lancefield group F in which the typing

antigens in this group (and related streptococci) have often been regarded as

microcapsular structures, capable of preventing phagocytosis (Huis in,t Veld and

Willers, 1973).

Table (3-5): Virulence Factors Detected in isolates of anginosus group streptococci

No.ofisolate

Virulance Factors

Capsule hemolysin Protease LipaseColonization

factors Bacteriocin GlidingmotilityI III

17 - + - + - - - -

46 + + - + - - - -

91 + + - + - + - -

93 + + - + - + - -

108 + + - + - + - -

113 - + - + - + - -

114 - + - + - + - -

116 - + - + - + - -

137 + + - + - + - -

147 + + - + - + - -

151 + + - + - - - +


56

3.4.2. Hemolytic Reaction:

Production of hemolysin is mostly associated with pathogenic bacteria, therefore

it considered as an important factor that participate in their pathogenesis (Madigan et

al., 1997).

All isolates in the present study showed -hemolysis (Table 3-5). This result

matched the results of Ball and Parker (1979) who mentioned that although most

strains of Strep. milleri are non-hemolytic, it is estimated that 25% are beta-

hemolytic and may possess Lancefield group A, C, F, or G antigen, Poole and

Wilson (1976, 1979) who described Strep. milleri as alpha, beta, or nonhemolytic,

Whiley et al. (1990) reported that strains of Strep. constellatus and Strep. anginosus

(13 of 15) which belong to Lancefield serological group F, were beta-hemolytic,

whereas most Strep. intermedius (12 of 15 strains) were nonhemolytic. Joseph et al.

(2001) who studied a total of 98 strains for the three species of Strep. milleri group,

found that they are alpha, beta, or gamma-hemolytic.

This result may be due to that we depend on the type -hemolytic in the

isolation and selection of the isolates and also due to the few number of isolates

recovered during the present study.

The organisms belong to the beta-hemolytic group characterized by their ability

of complete hemolysis of blood when growing on blood agar medium and form a

clear zone around their colonies because they own hemolysin enzyme (Schleifer and

Kilpper-Balz, 1987; Vandamme et al., 1996 ).

3.4.3. Extracellular Protease production:

All isolates in the present study do not produce the enzyme protease when tested

on M9 medium which contain gelatine(Table 3-5). This result was consistent with

Bridge and Sneath (1983) who included only three Strep. milleri isolates in their

study, and they found no gelatinase activity.


57

Also Cole et al.(1994) found a single isolate of Strep. anginosus with other oral

streptococci isolates lacked IgA1 protease, since no Fc and Fc fragments were

generated. An increase in mobility of the 1 chain of the IgA1(k), indicative of

depletion of carbohydrate from the 1 chain, was a characteristic of IgA1 protease-

negative isolates of Strp. mitis, Strep. salivarius, and Strep. anginosus.

However, this finding could not be confirmed when these isolates were retested

with an IgA1( ) paraprotein, possibly because of the difference in the carboydrate

side chains of the heavy chains (Reinholdt et al., 1990) while Hayano and Tanaka

(1967) reported a group F streptococcal strains which produced extracellular

neuraminidase which broke down added bovine submaxillary mucin during growth,

and Beighton et al. (1991) found that Strep. intermedius produce neuraminidase.

This glycosidase is capable of removing terminal sialic acid from oligosaccharide

side chains located at the hing region and elsewhere along the 1 heavy chain.

3.4.4. Lipase production: All isolates in the present study produced the enzyme

lipase after incubation for 48 hrs on egg yolk agar medium (Table 3-5), this result is

consistent with Bridge and Sneath, (1983) who reported that lipase have been

observed in some strains of Strep. milleri group, While Ruoff and Ferraro (1987)

have found that all isolates examined in their study gave negative reaction in a test

for lipase production.

3.4.5. Colonization factor antigenes (CFA):

The term fibrillae has been used to describe fine wisps of M protein on the

surface of Strep. pyogenes (Fischetti et al., 1988). Most isolates in the present study

(8 of 11) (72.7%) possessed the colonization factor antigens III (Table 3-5). This

result is consistent with Handley and Carte r(1979, 1985) who have demonstrated

fine fimbrial structures on the surface of oral streptococci, including Strep. milleri

which may be important in adherence to surfaces, and in inter-species interactions

common in the mixed flora of mucosal surfaces, dental plaque, and purulent lesions,


58

and with Kurl et a.(1989) who reported that some strains of Strep. milleri group

have hemagglutination activities, and with Eifuku-Koreeda et al.(1991);Yamaguchi

and Matsunoshita (2004); Yamaguchi et al.(2009) who described the purification

and some morophological and biochemical properties of the fimbriae from an oral

strain of Strep. intermedius. This type of fimbriae is involved in saliva-induced

cellular aggregation of certain Strep. milleri strains. Little work has been done on

the possible role of such structures in Strep. milleri disease (Whitworth, 1990).

3.4.6. Bacteriocin production:

No isolates in the present study produce bacteriocin when tested with sensitive

Gram positve indicator isolates (Table 3-5). This result disagreed with Druker and

Mckillop, (1982) who described the widespread production of antagonistic

substances in the form of bacteriocin-like activity amongst Strep. milleri strains, and

with Schofield and Tagg (1983) who find that three of 30 human strains of group C

streptococci were inhibitor producer and they find that part of the group C

Streptococci produced inhibitors that had bacteriocin-line properties.

Studies of Beukes et al.(2000); and Beukes and Hastings (2001) on Strep.

milleri strain NMSCC 061 is found to produce a bacteriolytic cell wall hydrolase,

termed millericin B, which hydrolyzes the peptide moiety of susceptible cell wall

peptidoglycan, and Heng et al.(2006) who identifiy stellalysin, a new lytic

bacteriocin produced by Strep. constellatus subsp. constellatus. It is surprising to see

the high frequency of peptide bacteriocin-producing streptococcal isolates, and this

observation is supported by gene annotation, as well as genome mining of

sequenced Streptococcus genomes (Nes et al., 2007).

3.4.7. Gliding motility: In this study only one isolate (Table 3-4), showed a positive

result in gliding motility test (Figure 3-3), this result is consistent with Bergman et

al. (1995) who described isolates belonging to the Strep.milleri species group that

appear to exhibit agliding type of motility, which is expresed as spreading growth on


59

certain types of chocolate agar medium. The Strep.milleri isolates, which they

studied, lacked any observable organelles of motility and give negative results when

tested in conventional motility test medium. However, their findings do provide a

description of unusual characteristic that has not previously been described in

streptococci (Bergman et al., 1995).

A B

Figure (3-3): Gliding motility test; A-negative (Isolate No.17), B-positive (Isolate No. 151).

3.5. Detection of Antibiotic Resistance:

3.5.1. Disk diffusion method:

All 11 Strep. anginosus group isolates were tested for their antibiotic resistance

against a number of antibiotics (Table 2-7) using disk diffusion method. Most

isolates were found to be resistant to at least 4 antibiotic tested, (Table 3-6).

The results of this study revealed that there was highly bacterial resistance to -

lactam antibiotics ampicillin (90%), third generation cephalosporins (cefotaxime and

ceftrixone)(100%), and fourth generation cephalosporin (cefepime) (81.8%) (Figure

3-4). These results are comparable with the data reviewed by G mez-Garcés et al.

(1994) who reported a resistance to penicillin by SMG, and Clermont and Horaud

(1990) who reported that resistance to penicillin G was associated with resistance to


60

the other beta-lactam antibiotics. The result of beta-lactamase detection test in this

study reveald that there was no pencilllinase activity could be detected in strains of

this group.

The mechanism of beta-lactam resistance may be due to altered target (penicillin

binding protein, PBP) in these gram positive bacteria. Penicillin-resistance of

viridans streptococci is not due to beta-lactamase production (hence no benefit from

using agents such as ampicillin-sulbactam) (Melia and Auwaerter, 2009).

Streptococcus anginosus group are generally considered to be susceptible to

penicillin, other -lactam antibiotics, but resistant strains have been reported (Aracil

et al., 1999; Limia et al., 1999; Tracy et al., 2001). Moreover, a significant increase

in the antimicrobial resistance of viridans and -hemolytic streptococci has been

noticed in recent decades (Seppala et al., 2003).

Bacterial resistance to macrolide group, which include erythromycin, azithromycin,

and clarithromycin, has also been investigated (Figure 3-4;Table 3-6).

Most isolates were sensitive to these antibiotics with sensitivity rate (72.7%) for

eythromycin, (72.7%) for azithromycin and (81.8%) for clarithromycin. However,

the erythromycin resistance rate seen in this study was significantly higher than that

of SAG isolates from Netherlands (2.6%) and Spain (17.1%) (Jacobs and

Stobberingh, 1996;Limia et al., 1999).

The two presently recognized mechanisms for resistance to macrolide antibiotics

in streptococci are target-site modification and active-drug efflux (Kataja et al.,

1998).

Target-site modification is mediated by an erythromycin resistance methylase

(erm) that reduces binding of macrolide, lincosamide, and streptogramin B (MLS)

antibiotics to the target site in the 50S ribosomal subunit (Leclercq and Courvalin,

1991).

Tab

le(3

-6)

: A

ntib

ioti

c re

sist

ance

of a

ngin

osus

gro

up s

trep

toco

cci

AM

P=am

pici

llin,

FEP=

cefe

pim

e,C

TX

=ce

fota

xim

,CR

O=c

eftr

iaxo

n,V

A=v

anco

myc

in,E

=ery

thro

myc

in,A

ZM

=azi

thro

myc

in,

CL

R=c

lari

thro

myc

in, T

E=t

etra

cycl

ine,

OFX

=ofl

oxac

in,C

=chl

oram

phen

icco

l,D

A=c

linda

myc

in.

Isol

ate

desi

gnat

ion

AN

TIB

IOT

IC

AM

CT

XC

RO

VA

EA

ZM

CL

RT

EO

F XC

DA

FE

P

Stre

p.an

gino

sus

T17

RR

RS

SS

SR

SS

SS

Stre

p.an

gino

sus

T46

RR

RS

SS

SS

SS

SR

Stre

p.an

gino

sus

T91

RR

RS

SS

SS

SS

SR

Stre

p.an

gino

sus

T93

RR

RS

RR

RR

SS

SR

Stre

p.an

gino

sus

T10

8R

RR

SS

SS

SS

SS

S

Stre

p.an

gino

sus

T11

3R

RR

SS

SS

SS

SS

R

Stre

p.an

gino

sus

T11

4R

RR

SR

RS

SS

RS

R

Stre

p.an

gino

sus

T11

6R

RR

SS

SS

SS

SS

R

Stre

p.an

gino

sus

T13

7R

RR

SS

SS

SS

SS

R

Stre

p.an

gino

sus

T14

7R

RR

SR

RR

RS

RS

R

Stre

p.an

gino

sus

T15

1R

RR

RS

SS

SS

SR

R

% o

f re

sist

ance

9010

010

09.

027

.227

.218

.136

.30

189.

081

.8

61


62

Figure(3-4): Antibiotic resistance of anginosus group streptococci

AMP=ampicillin, FEP=cefepime, CTX=cefotaxim, CRO=ceftriaxon, VA=vancomycin,E=erythromycin, AZM=azithromycin, CLR=clarithromycin, TE=tetracycline, OFX=ofloxacin,C=chlorampheniccol, DA=clindamycin.

In active-drug efflux, the protein encoded by the mef A or mef E (macrolide

efflux) gene causes resistance to 14 and 15 membered macrolide compounds only

(Clancy et al., 1996; Tait-Kamradt et al., 1997).

Because of the high frequency of genetic exchange between different

streptococcal species, it is likely that the increased macrolide resistance among

viridance streptococci has contributed to an increased macrolide resistance among

SAG isolates (Asmah et al., 2009). However, they are effective against Gram

positive bacteria despite their uptake difficulties associated with thick cell wall

(Balowes et al., 1991).

Most isolates were sensitive to tetracycline (Figure 3-4; Table 3-6) with

resistance rate (36.3%). This result is comparable with the Spanish report (G mez-

Garcés et al., 1994) that indicates a resistane rate of (37.1%) for tetracycline.


63

According to CLSI (2007), disk dofusion test for vancomycin sensitivity is

17mm with no intermediate value regarding to Streptococcus spp.

Results of (Figure 3-4; Table 3-6) showed that most isolates (10 of 11) were

highly sensitive to vancomycin clindamycin and chloramphenicol with very low rate

of resistance (9.0%), (18%), (9.0%) respectivly. However one isolate was resistant

to vancomycin, this is the first resistant isolate recorded in Iraq.

The disk diffusion procedure cannot differentiate isolates with reduced

susceptibility to vancomycin from susceptible isolates (MIC range 1 µg/mL) even

when incubated for 24 hours.

Vancomycin was first discovered in 1956 in some soil samples from southeast

Asia, where it was produced by an actinomycete, Streptococcus orientalis. It was

first used clinically in 1958, a mere two years after it had been discovered

(Anderson et al.,1961). It is a glycopeptide antimicrobial agent, it binds to the C-

terminal end of late peptidoglycan precursors, preventing the effective formation of

a bacterial cell wall (Courvalin, 2006).

Resistance to erythromycin may be associated with co-resistance to clindamycin

(Asmah et al., 2009). Some authers have expresed concern that clindamycin

resistance in the Strep. milleri group is increasing (G mez-Garcés et al., 1994;

Jacobs and Stobbering, 1996; Limia et al., 1999).

The figure (3-4) also showed that all isolates (100%) (Table 3-6) were sensitive

to the fluoroquinolone (ofloxacin). Since all isolates were sensitive to ofloxacin, it

might serve as the drug of choice for the treatment of infections caused by this

group.

3.5.2. Detection of -Lactamase Production:

Rapide iodometric method was used for detection of -lactamase production in

-lactam resistant anginosus group isolates. Results of this test were negative for all

tested isolates in this study.


64

This method depends on the detection of penicilloic or cephalospoic acid,

resulted from breackdown of amide bond in -lactam ring for each of penicillins or

cephalosporins (Skyes and Mathew, 1976; Livermore, 1995).

Iodine reacts with starch to form dark blue complex, which stays without

changes in the absence of -lactamase enzymes. In the case of -lactamase-

producing bacteria, the resulting penicilloic or cephalospoic acid will reduce iodine

into iodide; consequently, decolorization of starch-iodine complex occurs (changing

the color directly to white) if an isolate is a -lactamase producer but not if the

enzyme is absent (perret, 1954; Sykes and Mathew, 1976).

This result showed that resistance to -lactama antibiotics in this group is not

due to -lactamase production, but may be due to alterd penicillin binding protien.

This result is in the line of Quinn et al.(1988) report on a fatal case of post-

neurosurgical meningitis due to a penicillin-resistant Strep. milleri strains; resistance

in this strain is seen to be mediated by alteraitions of penicillin-binding proteins.

3.5.3. MICs Determination of anginosus group isolates:

Two methods (2.2.10.3 )were used for determination of the MICs of anginosus

group group isolates against seven antibiotics. The MICs for Cefotaxime and

Ciprofloxacin were determined by the HiComb MIC test (Figure 3-5).

The MICs of other antibiotics (Ampicillin, Ceftriaxone, Vancomycin,

Erythromycin, Tetracycline) were determined by the two-fold agar dilution

susceptibility method. The MIC values were based on break point recommended by

CLSI (2007) for estimation of the response. The break point represents the optimum

concentration of the drug that can reach the serum and provide high level of therapy.

The microorganism was considered sensitive if the estimated MICs were less than

the break point.


65

Figure (3-5): HiComb MIC test used for determination of MIC of antibiotics against

Streptococcus anginosus group isolates.

Results in Table (3-7) showed that all 11 anginosus group isolates were highly

resistant to ampicillin with concentrations one fold of the break point ( 128µg/ml)

and most isolates were highly to moderate resistant to ceftriaxone and cephotaxime

with MIC values ranged from 0.25 to 8µg/ml and from 1 to 30 (µg/ml) respectively.

The -lactam group provides the antibiotic of choice in infections caused by

streptococci, penicillin G being used in a large numbr of cases (Ruoff, 1988). The

SMG organisms are almost uniformly sensitive to penicillin, with a few isolates

being of intermediate susceptibility (MIC 0.25-2.0 mg/l) (Pool and Wilson, 1979;

G mez-Garcés et al., 1994; NCCLS, 2003). Many other -lactam antibiotics have

in vitro activity similar to that of penicillin against the SMG, but the

susceptibilities to different cephalosporins are quite variable (Alcaide et al., 1995).

All isolates in the present study were highly susceptibile to vancomycin and

tetracycline (Table 3-7). However, one isolate was found to be resistant to

vancomycin with MIC reached to (7.68 µg/ml) which is seven times more than the

break point recommended by Clinical and Laboratory Standards Institute ( 1 µg/ml)

(CLSI, 2007) documentations.

Tab

le(3

-7):

MIC

sof

anu

mbe

rof

antib

iotic

sag

ains

tStr

ep.a

ngin

osus

grou

pis

olat

es.

Isol

ates

desi

gnat

ion

MIC

(µg/

ml)

of:

AM

P

(0.

25µ

g/m

l)

CR

O

(0.

5µg/

ml)

CT

X

(1m

cg)

VA

(1µ

g/m

l)

E

(0.

25µ

g/m

l)

TE

(2µ

g/m

l)

CIP

(1m

cg)

Stre

p.an

gino

sus

T17

640.

55

0.48

0.96

0.42

0.1

Stre

p.an

gino

sus

T46

644

300.

480.

240.

035

Stre

p.an

gino

sus

T91

321.

05

0.48

0.96

0.03

0.1

Stre

p.an

gino

sus

T93

640.

510

0.24

0.96

0.12

5

Stre

p.an

gino

sus

T10

864

>0.

50

>0.

12>

0.06

>0.

030

Stre

p.an

gino

sus

T11

364

45

0.24

0.06

>0.

035

Stre

p.an

gino

sus

T11

464

25

0.24

0.96

>0.

035

Stre

p.an

gino

sus

T11

664

21

1.92

0.12

0.24

5

Stre

p.an

gino

sus

T13

764

45

0.24

0.12

>0.

035

Stre

p.an

gino

sus

T14

764

85

0.96

1.92

0.24

5

Stre

p.an

gino

sus

T15

164

0.25

07.

680.

240.

030

*Num

bers

bet

wee

nth

e ba

cket

sre

fer

to b

reak

poi

nts

reco

mm

ende

d by

CL

SI

(200

7).A

MP=

ampi

cilli

n,C

TX

=cef

otax

im,C

RO

=cef

tria

xon,

VA

=van

com

ycin

, E=e

ryth

rom

ycin

, T

E=t

etra

cycl

ine,

CP

I=ce

prof

loxa

cin.

66


67

More variable results are obtained with tetracyclines, where rates of resistance in

the range 0-37% have been described (G mez-Garcés et al., 1994; Doern et al.,

1996).

No vancomycin resistance had been recorded previously locally or worldwide

in this group but it were found in Staphylococcus and Strep. pneumoniae. This group

may be tolerant to vancomycin rather than being resistant. The significance of in

vitro vancomycin tolerance is uncertain. Recent evidence obtained by Novak et al.

(1999) demonstrated a molecular mechanism for vancomycin tolerance in Strep.

pneumoniae.

Only a few clinical isolates have been reported to exhibit tolerance to

vancomycin (Noble et al., 1980; Roleston et al., 1984). Tolerance of vancomycin

have been reported previously among pharyngeal isolates of non-group A -

hemolytic streptococci (mostly GCS and GGS) from children (Zaoutis et al., 1996;

Zaoutis et al., 1999).

Result of the single isolate that was less susceptibile to vancomycin may be

considered as vancomycin tolerant. However, the antimicrobial tolerance, defined as

a minimum bactericidal concentration (MBC) 32 or more times higher than the

MIC, among GCS and GGS has been reported for penicillin and other agents

(Roleston et al., 1984).

The less susceptibility (or resistance) of this isolate to vancomycin may be

recorded only when the reistance genes (VanA, VanB, VanC, etc…) could be

detected by genetic methods (PCR) which is not determined in the present study.

However, resistance to vancomycin in this group not recorded locally or

worldwide and it may be the first recored in Iraq.

Concerns about potential antimicrobial tolerance in group C and G streptococci

and reports of clinical failures in pateints with severe infections have led many

authors to recommend combination therapy for synergy (aminoglycoside plus a cell


68

wall-active agent) in the treatment of these patients (Roleston et al., 1984; Salata et

al., 1989; American Academy of Pediatrics, 1997).

The agent vancomycin may prove valuable as monotherapy or in combination

with other agents in the treatment of high-risk patients with invasive group C and G

infections who cannot be treated with penicillin, which remains the drug of choice

(Zaoutis et al., 2001). They remain uniformly sensitive to vancomycin, teicoplanin,

trimethoprim, chloramphenicol, and refampicin (G mez-Garcés et al., 1994; Doern

et al., 1996).

Results in Table (3-7) showed a resistance rate 45% to erythromycin this result

is much higher than that reported by Horodniceanu et al. (1981) who mentioned that

resistance to erythromycin-lincomycin-clindamycin group, although currently rare,

is transferable among streptococci, including the SMG, and can be as high as 12-

14%.

For ciprofloxacin, seven anginosus group isolates (Table 3-7) were able to grow

in concentrations one fold of the break point ( 4µg/ml). This result is in accordance

with the data of Asmah et al. (2009) who showed that even in ciprofloxacin-

susceptible isolate, the ciprofloxacin MICs are very close to the resistance

breakpoints and suggest that ciprofloxacin is inadequate for tratment of SAG

infections. Limia et al.(1999) observed a steady increase in the susceptibility to

ciprofloxacin over the study period, while some authors reported that ciprofloxacin

was noticebly the less active agent than the other agents tested, it was the least

active quinolones, especially against all species of streptococci (Citron et al., 2007).

Conclusions and Recommendations

69

Conclusions and Recommendations

Conclusions: The results of the present study yield the following conclusions:

1. The genus streptococcus was the predominant type among bacterial isolates

recovered from patients with of pharyngitis and the rate of infection with

pharyngitis caused by group C Streptococcus anginosus was 6.2% among other

groups of streptococci.

2. The anginosus group streptococci are highly variable in their biochemical and

physiological characteristics, which make the diagnosis of these organisms difficult.

This group cannot easily be identified to the species level by conventional or

traditional ways.

3. Most bacterial isolates in this study have the ability to produce more than one

virulence factor such as capsule, hemolysin, colonization factor antigen III, and

lipase enzyme.

4. There was a high rate of resistance among isolates in the present study for -lactam

antibiotics, but they were highly susceptible to vancomycin, ofloxacin, and

clindamycin.

Recommendations: According to the results obtained in the present study, thefollowing recommendations are put forward:

1. Most isolates were highly sensitive to vancomycin, ofloxacin and clindamycin so

these antibiotics can be used in the treatment of pharyngitis caused by this group of

streptococci.

2. Further studies are required for detection of antibiotic resistance genes (especially

for vancomycin) using genetic methods.

3. Further studies are required on the molecular analysis like PCR for the identification

of isolate of Strep.anginosus group.

4. Further studies are needed to investigate the clinical significance of the unusual trait

of this group (gliding motility).

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a

Appendix (1): Tests of API 20 STREP system usefor identification of Streptococcus anginosus isolates

ResultBiochemical tests

+Production of acetoine

(VogesProskauer)-Acid hippuique hydrolyse+Esculine hydrolysis-PYRA--Galctosidase--Glucuronidase+-Galactosidase-Phosphatase Alcaline+Leucine Aminopeptidase-Arginine dihydrolase-Acidification of Ribose-Acidification of arabinose-Acidification of mannitol-Acidification of sorbitol+Acidification of lactose+Acidification of trehalose-Acidification of inuline+Acidification of Raffinose+Acidification of amidon-Acidification of glycogene+Beta-hemolysis

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Group C and G Streptococci, their role in acute pharyngitis

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