DNA extraction and electrophoresis of bacterial and fungal genome using modified protocol of kirby method

ARINOLA O.T1 , OLOWOOKERE B E1, ELUWOLE O.O2

TITILAYO O.E2

FUNBELL MEDICAL DIANOSTICS EGBEDA ,AKOWOJO. LAGOS STATE NIGERIA

DEPARTMENT OF MEDICALMEDICAL LABORATORY SCIENCES, ACHEIVER'S UNIVERSITY OWO, ONDO STATE2

CORRESPONDING AUTHOR: arinolatus@yahoo.com

ABSRACT

This study describes a modified, rapid and cost-effective method (combination of boiling and phenol

chloroform) for the extraction of genomic DNA from pure culture of Staphylococcus aureus and Candida

albicans on agarose gel electrophoresis and to quantify the amount of DNA obtained by this protocol. For

Candida albicans and drug-sensitive Staphylococcus aureus, DNA extraction using this modified Kirby method

was found to be efficient while the method was found to be inefficient in DNA extraction from drug-resistant

Staphylococcus aureus until further treatment and modification were made. The concentrations of the DNA

extracted from Drug-sensitive Staphylococcus aureus (gram positive bacteria), Drug-resistant Staphylococcus

aureus and Candida albicans (gram positive fungi) were 320.05 ng/µl, 613.06 ng/µl and 143.13 ng/µl

respectively. The extracted DNA was further qualitatively analyzed using two different concentrations (1% and

0.8%) of agarose gel via gel electrophoresis. There was clear separation in the 0.8% agarose gel

electrophoresis showing high molecular weight DNA bands while the bands were not clear in the 1% agarose

gel. This modified Kirby DNA extracted method seemed to be efficient and effective. Based on the results

observed using this modified method, it can also be used for extracting the DNA of other gram positive and

gram negative microbes of medical importance.

Abbreviation: DNA:, TSST-1:Toxic shock syndrome toxin, TSS: Toxic shock syndrome, VISA: vancomycin

insensitive S. aureus, PBP: penicillin-binding proteins, MRSA: methicillin resistant S. aureus, 5-FOA:

antimetabolite 5-fluoroorotic acid

key word: Bacteria, fungi, DNA,

INTRODUCTION

Staphylococcus aureus is a prominent human pathogen that was reported in 1881 by Alexander Ogston (Ogston,

1881). After microscopic analysis of purulent infections, Ogston discovered grape-like clusters of round, golden

cells. Following this observation, he re-plicated the original infections by injecting experimental subjects with

the putative infectious organism. It is often found as a commensal associated with skin, skin glands, and mucous

membranes, particularly in the nose of healthy individuals (Crossley & Archer, 1997). It has been estimated that

approximately 20–30% of the general population are S. aureus carriers (Hayman, 2004). The first genome

sequences of S. aureus strains Mu50 and N315 were published in 2001 (Kuroda et al., 2001). At present,

complete genomic sequences of ten S. aureus strains are available, and the genomes of several others have been

partially determined (Baba et al., 2002; Gill et al., 2005; Diep et al., 2006). The genome of S. aureus is a

circular chromosome that is 2.8–2.9 Mbp in size, with a G+C content of about 33% (Crossley & Archer, 1997).

Staphylococcus aureus is typically carried asymptomatically by human and animals. It is responsible however

for a wide range of medical conditions that require treatment. The nature and extent of disease depends on the

pathogenic characteristics of the infecting strain, host susceptibilities and the rate of entry. The most common

ailments are skin and soft tissue infections that include abscesses, cellulitis, impetigo and postoperative surgical

wounds (Cohen, 2007); with osteomyelitis also known to occur (Cunningham et al., 1996). Strains carrying

particular toxins can be associated with specific disease states, including staphylococcal scalded skin syndrome

(SSSS) (Ladham et al., 1999), Toxic shock syndrome (TSS) (Novick and Subeda, 2007), food poisoning (Le

Loiu et al., 2005) and necrotizing pneumonia (Labandeva-Rey et al., 2007). Septicaemia is a serious progression

of S. aureus disease that occurs when the blood stream becomes infected. The genus Staphylococcus plays an

important role in public health causing food poisoning by the production of a wide variety of enterotoxins (SE).

Toxic shock syndrome toxin (TSST-1) also plays a role in pathogenicity being involved in the production of

skin lesions in neonates (DeBuyser et al., 2001). Up to 52% of the strains isolated from bovine mastitis

produced enterotoxins (Kenney et al., 1993; Aarestrup et al., 1995; Ichikawa et al., 1996). The excessive use of

antibiotics has led to the emergence of multiple drug resistant S. aureus strains (Lowy, 1998). Penicillin was

introduced for treating S. aureus infections in the 1940s, and effectively decreased morbidity and mortality.

However, by the late 1940s, resistance due to the presence of Penicillinase emerged (Eickhoff, 1972). The

staphylococci are very capable of evolving resistance to the commonly used antimicrobial agents, such as,

erythromycin (Wallmark and Finland, 1961), ampicillin (Klein and Finland, 1963), and tetracycline (Eickhoff,

1972). In most cases, resistance to antibiotics is coded for by genes carried on plasmids, accounting for the rapid

spread of resistant bacteria (Morris et al., 1998). Soon after the introduction of methicillin, Jevons (1961)

described the emergence of methicillin resistant S. aureus (MRSA), which have since spread worldwide as

nosocomial pathogens. The Central Public Health Laboratory, UK (2000) found that 61% of nosocomial S.

aureus infections in the 96 hospitals studied were methicillin resistant. Penicillin, a ß-lactam antibiotic works by

inhibiting bacterium cell wall synthesis by inactivating the penicillin-binding proteins (PBP). MRSA strains

produce a distinct PBP, designated PBP2/, which has a low affinity to ß-lactam antibiotics, hence PBP2/ can still

synthesize the cell wall in the presence of the antibiotic (Hiramatsu, 1995). This is the basis for ß-lactam

resistance in MRSA strains. PBP2/ are products of the gene mecA, which is located in mec, foreign

chromosomal DNA found in methicillin resistant strains but not in methicillin susceptible strains (Hiramatsu et

al., 1997). Vancomycin, a glycopeptides has been the most reliable antibiotic against MRSA infections;

however, in 1996 the first MRSA to acquire vancomycin intermediate resistance was isolated in Japan

(Hiramatsu et al., 1997). Unfortunately, several vancomycin insensitive S. aureus (VISA) strains have been

reported in the USA, France, Scotland, Korea, South Africa and Brazil (Hiramatsu, 2001). Upon exposure to

vancomycin, certain MRSA strains frequently generate VISA strains, called hetero-VISA (Hiramatsu, 2001).

VISA resistance appears to be associated with thickening of the cell wall peptidoglycan, and due to an increase

in the target for the glycopeptide in the cell wall, therefore requiring more glycopeptide to inhibit the bacteria

from growing (Hanaki et al., 1998). All VISA strains isolated appear to have a common mechanism of

resistance, which differs from that found in Vancomycin resistant enterococci, in that enterococcal van genes are

not present (Walsh, 1993). However in 2002, the first Vancomycin resistant S. aureus (VRSA) infection was

documented in a patient in the United States (Walsh, 1993). This strain was shown to carry a van gene,

suggesting that the resistance determinant might have been acquired through the genetic exchange of material

between vancomycin resistant enterococci and S. aureus. The spread of vancomycin resistance worldwide is

now inevitable, and could potentially result in a return to pre-antibiotic era. Hence, the identification of novel

targets on the bacteria seems to be a pre-requisite in the search for new antibiotics and prophylaxis, e.g.

vaccines.

Fungi are eukaryotic organisms with approximately 300 000 different species. Of these, about 200 are potential

parasites, with only a few of these affecting humans (Ashman et al., 1995). Fungal diseases of mammals,

mycoses, range from the common mild cutaneous or subcutaneous skin infections, such as athletes foot, to the

potentially lethal acute or chronic infection of deep tissues that are typically caused by Candida species. Of the

Candida species afflicting humans, Candida albicans is by far the most common. Yeast can live as harmless

commensal in many different body locations, and is carried in almost half of the population. However, in

response to a change in the host environment, C. albicans can convert from a benign commensal into a

disease-causing pathogen, causing infections in the oral, gastrointestinal and genital tracts (Cole et al., 1995).

The infection caused by C. albicans can be defined in two broad categories, superficial mucocutaneous (Bailey

et al., 1996) and systematic invasive, which involves the spread of C. albicans to the blood stream (candidemia)

and to the major organs (Hobson et al., 2004). Systemic candidemia is often fatal. The incidence of vulvo-

vaginal candidiasis (thrush) has increased approximately two folds in the last decade. Approximately 75 % of all

women experience a clinically significant episode of vulvovaginal candidiasis (VVC) at least once during the

reproductive period (Pitarch et al., 2002). The role of prostaglandins during the infection is not very clear,

however it has been demonstrated that mononuclear cells from the patients suffering from recurrent vaginal

candidiasis produce higher levels of PGE2 as compared with cells from control women, indicating the

important role of PGE2 during infection. Recurrent vaginal candidiasis is also common in female patients with

acquired immune deficiency syndrome (AIDS), suggesting a role for depressed cell-mediated immunity in

candidiasis (Ishibash et al., 2005). Numerous virulence factors have been attributed to the pathogenicity of C.

albicans (Grimme et al., 2004). These include dimorphism, phenotypic switching and immune interference.

Several studies suggest that the ability of C. albicans to switch between the yeast and mycelial forms is an

important virulence factor (Grimme et al., 2004). Moreover, C. albicans can not only change its cellular

morphology in response to growth conditions, but can also irreversibly switch its cellular phenotype both in

vitro and in vivo (Alloush et al., 1997). C. albicans classical genetics had a great impact in the 1980s. Now, in

spite of its being out of fashion, it may still offer clues to the understanding of this yeast. The diploid nature of

C. albicans was deduced from the determination of its DNA content and the kinetics of reassociation of

denatured total DNA. In addition, it was found that many clinical isolates of C. albicans displayed a strongly

biased auxotroph spectrum after ultraviolet (UV) irradiation (Scherer and Magee, 1990). A major advance in C.

albicans genetic manipulation was achieved when Fonzi and Irwin (Fonzi and Irwin, 1993) adapted a disruption

strategy used in S. cerevisiae. The method involves the use of C. albicans URA3 flanked by Salmonella

typhimurium hisG genes to provide flanking recombination regions. Following homologous recombination in

the first chromosomal allele, intrachromosomal excision of the URA3 marker is selected with the antimetabolite

5-fluoroorotic acid (5-FOA), thus allowing the recovery of ura3 auxotrophy and enabling a second round of

homologous recombination to disrupt the second chromosomal allele.

The aim of this research is to evaluate for increase in quantity and quality of bacterial and fungal genome DNA

extraction by modified protocol of Kirby method

MATERALS AND METHODS

This research work was carried out at University of Lagos , Nigeria. The procedures and methods of the study

were as follow.

The bacterial strains used were collected from the Microbiology department, Nigerian Institute of Medical

Research.The srains used are gram positive bacteria viz. drug-sensitive S. aureus, drug-resistant S. aureus and

fungi viz. Candida albicans. Chocolate agar was the primary culture medium for S. aureus strains while

Saboraud agar was used for Candida albicans. Nutrient agar was used for short-term preservation as well as for

sub culturing and producing seed cultures. Equipments used were Micropipette, Incubator, Beakers, Autoclave

for sterilization, Bunsen burner, Petri dishes, Graduated measuring cylinders, Weighing balance, and Broad

spectrum antibiotics discs.The antibiotics panel used for testing the sensitivity and resistance of S. aureus were

Cefuroxime, Cefixime, Ceftazidime, Nitrofurantoin, Augmentin, Ciprofloxacin and Ofloxacin. DNA from drug

sensitive and resistant S. aureus and Candida albicans was extracted from pure culture using a modified

protocol of Kirby Phenol/Chloroform method (Kirby, 1956; Kirby, 1957).

The reagents used for the extraction are extraction buffer (pH 8.0, which constitutes 50 mM NaCl, 50 mM Tris-

HCl (pH 7.6), 50 mM EDTA and 5% SDS), Phenol (pH 8.0), Chloroform:Isoamyl alcohol (24:1),

Phenol:Chloroform:Isoamyl alcohol (25:24:1), Chloroform, 100% isopropanol (filter sterilized), 70% ethanol

(filter sterilized) and sterile microcentrifuge tubes.

50mg each of pure culture from Candida albicans, drug-sensitive and drug-resistant S. aureus was taken in 3

different test tubes labelled DC (Candida albicans pure culture), DS (drug-sensitive S. aureus pure culture) and

DR (drug-resistant S. aureus pure culture).

EXTRACTION OF DNA

DNA was extracted from drug-sensitive s. aureus, drug-resistance s. aureus and candida albicans using modified

protocol Kirby method developed for the research.

1 ml of extraction buffer was added into the labeled tube DR and mixed promptly by inversion. The DS

suspension was incubated at 80oC for 45 minutes. Further incubation was done for another 45 minutes. It was

then centrifuged at 14000 x g for 3 minutes. 300 µl of phenol: chloroform: isoamyl alcohol (25:24:1) was added

to the mixture, vortexed for 8 seconds until the solution appeared milky forming an emulsion. The emulsified

DR solution was centrifuged for another 3 min at 14,000 x g until all phases were well separated.

Very carefully top phase was collected with a 200 μl pipette and transferred the solution into fresh eppendhorf

tube which was later treated again with 300µl of the phenol/chloroform/isoamyl alcohol working reagent and

centrifuged for another 10 minutes at 14000 x g. 300 µl of the supernatant was again transferred into a new

eppendhorf tube in which 500 µl of chloroform was added, centrifuged for 10 minutes at 13000 x g. The clear

phase was further transferred into a graduated 1.5ml eppendhorf tube. The volume of the extract was measured

as 165 µl, 0.1 volume of 3M Sodium Acetate solution and 0.7 volume of isopropanol was added to the

supernatant, mixture mixed well by inverting the tube several times but not vortexed, after which it was

centrifuged at 14000 x g for 30 minutes in other to precipitate the DNA.

The aqueous phase was discarded. A pellet was formed. The pellet was washed by adding 0.5ml ice cold 70%

ethanol, inverting the tube gently and making sure that the ethanol covers the pellet inside the tube. The DNA

was re-pelleted again with centrifugation for 5 minutes. The ethanol was removed again by inverting the tube

gently and making sure that the ethanol covers the pellet inside the tube being careful not to discard the pellet.

The pellet was then finally allowed to dry for 4 minutes. The pellet was re-suspended by adding exactly 100µl

of the ultra-distilled water and stored at -20oC.

DNA PURITY AND CONCENTRATION ASSESSMENT

The extracted DNA was assessed and analyzed by checking for the purity and quantity. This was done using a

nano-particle principled spectrophotometer from the Nigerian Institute of Medical Research, Lagos, Nigeria.

Absorbance measurements made on the NanoDrop™ 8000 Spectrophotometer were the absorbance of DNA

molecules in the sample that absorb at the wavelength of 260nm. Absorbance was also taken at 280nm so that

the 260/280 ratio can be estimated. 2 µl of the purified DNA was collected using a micropipette and applied to

the sample port of the NanoDrop 8000 spectrophotometer having initially blanked with TE buffer. The ratio

between the readings at 260 nm and 280 nm (OD260 /0D280) provides an estimate of the purity of the DNA.

Pure DNA preparations have OD260/OD280 values of 1.5 and above. For the qualitative analyses of extracted

DNA, gel electrophoresis was carried out.

DNA ELECTOPHORESIS

Electrophoresis of the isolated DNAs was carried out via the agarose gel electrophoresis since it is a separation

technique that can be used to separate DNA based on molecular weight. The concentration of the agarose

used was dependent on the size of DNA to be separated, here it was size of 100kbp. The materials used for the

electrophoresis were agarose powder, 1Kb DNA ladder, 0.5- 10µl micropipette and tips, Ethidium Bromide

(1mg/ml) and loading dye.

RESULTS

Quantitative Analysis of Extracted Genomic DNA

The concentration of DNA and the ratio between the absorbance at 260 nm and 280 nm of extracted genomic

DNA from drug-sensitive Staphylococcus aureus, Candida albicans and drug-resistant Staphylococcus aureus

using the modified protocol of Kirby method is given in Table 1 while the concentration of the DNA (ng/µl) is

shown in Figure 1.

The DNA concentrations of Staphylococcus aureus, Candida albicans and drug-resistant Staphylococcus aureus

are 320.03, 143.13 and 1324.06 ng/µl respectively.

CLASS OF ORGANISM

NAME OF ORGANISM

DNA CONCENTRATION IN ng/µl

ABSORBANCE AT 260mm

ABSORBANCE at 280nm

ABSORBANCE RATIO (260:280)

BACTERIA (Gram- positive)

Drug-sensitive Staphylococcus aureus (DS)

320.03 0.864 0.484 1.79

FUNGICandida albicans (DC)

143.13

2.863

1.103

2.60

BACTERIA (Gram-positive)

Drug-Resistant Staphylococcus aureus (DR)

613.06 26.481 12.317

2.15

Table 1: Concentration and purity ratio of extracted genomic DNA from drug-sensitive

Staphylococcus aureus, Candida albicans and drug-resistant Staphylococcus aureus

Drug-Sensitive Staphy-lococcus aureusCandida albicansDrug-Resistant Staphy-lococcus aureus

Figure 1: DNA concentrations (ng/µl) of drug-sensitive Staphylococcus aureus, Candida albicans

and drug-resistant Staphylococcus aureus.

3.2 Qualitative Analysis of Extracted Genomic DNA

The agarose gel electrophoresis pattern of genomic DNA extracted from drug-sensitive

Staphylococcus aureus, Candida albicans and drug resistance Staphylococcus aureus in Figure 2 and

3.

1 2 3 4 5

Figure 2: Agarose gel (1%) electrophoresis of the Extracted Genome using a smaller

electrophoretic tank. No clear separation was seen at this initial stage.

Lane 1: Drug-sensitive Staphylococcus aureus,

Lane 2: Drug-resistant Staphylococcus aureus,

Lane 3: Candida albicans

Lane 4: Candida albicans

Lane 5: 50bp Marker

1 2 3 4 5 6 7 8 9 10 11

Figure 3: Agarose gel (0.8%) electrophoresis of the Extracted Genomic DNA using a larger

tank and higher number of combs.

Lane 1: 1 kb marker

Lane 2: Drug-sensitive Staphylococcus aureus

Lane 3: Drug-sensitive Staphylococcus aureus

Lane 4: Drug-sensitive Staphylococcus aureus

Lane 5: Drug-sensitive Staphylococcus aureus

Lane 6: Candida albicans

Lane 7: Candida albicans

Lane 8: Drug-resistant Staphylococcus aureus

Lane 9: Drug-resistant Staphylococcus aureus

Lane 10: Drug-resistant Staphylococcus aureus

Lane 11: Negative control

DISCUSSION/CONCLUSION

This study, so far, has described a modified protocol of Kirby’s DNA Phenol-Chloroform extraction method by

analyzing the purity, quantity and yield of bacteria and fungi of medical importance viz. Drug-sensitive Staphylococcus

aureus, Candida albicans and Drug-resistant Staphylococcus aureus.

This modified method produced high yield DNA as observed in the result. Extraction of other types of bacteria genomic

DNA is also possible based on the outcome of this particular research. It is efficient, cost-effective and simple and can be

performed in small to medium laboratories unlike other Extraction protocol kits produced by some companies which are

expensive and can even deteriorate during transport.

Extraction of drug-sensitive S. aureus and Candida albicans DNA was easier compared with drug-resistant S. aureus.

Since proteins are subjected to chemical denaturation and/or enzymatic degradation while most common technique of

protein removal involves denaturation and extraction into an organic phase consisting of phenol and chloroform, Drug-

resistant S. aureus phenol-chloroform treatment had to be carried out several times (up to four times) before a clear

aqueous phase (supernatant) could be observed. This, to an extent, could be due to a number of unique mechanisms that

have evolved in Gram-positive bacteria that allow them to immobilize proteins on their surface, either by a covalent

binding of protein to the peptidoglycan or the non-covalent binding of protein to the peptidoglycan or secondary wall

polymers, such as teichoic acids (Navarre and Schneewind, 1999). These molecules have been implicated in the

resistance of Gram-positive cell to lysis by chemical treatment alone; a reason for the lack of good quality DNA for

polymerase chain reaction (PCR).The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A

ratio of ~1.5 and above is generally accepted as “pure” for DNA. If the ratio is appreciably lower, it may indicate the

presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm.Figure 1 shows the DNA purity

assessments, concentration and absorbance ratios of the DNAs isolates at wavelengths 230nm, 260nm and 280nm.

Furthermore, drug-resistant S. aureus was incubated for 90 minutes at 80oC because cell lysis was not observed after 45

minutes even up to 60 minutes while cell lysis was observed with Candida albicans after 25 minutes, hence, the variation

in incubation time.

It may be summarized that the modified Kirby method extracted DNA with a good value of DNA concentration and

extracted DNA were of high molecular weight for both gram positive. Although no single method will be appropriate for

all types of samples, however, from this result it could be suggested that the modified Kirby method might be a suitable

choice for extracting DNA from the three strains of micro-organisms as it was attractive because of its fastness and

needing lesser time to handle a large number of samples. In addition, it did not require lysis enzyme (Proteinase-K or

Lysozyme)

REFERENCE

Aarestrup, F. M., Wegeber, H. C. and Rosdhal, V. T. (1995). Lack of staphylococcal enterotoxin production among

strains of Staphylococcus aureus from bovine mastitis in Denmark. Acta Veterinaria Scandinavica. 36: 273-275.

Alloush, H. M., Lopez-Ribot, J. L., Masten, B. J. and Chaffin, W. L. (1997). 3-phosphoglycerate kinase: a glycolytic

enzyme protein present in the cell wall of Candida albicans. Microbiology. 143: 321–330.

Ashman, R. B., Fulurija, A., Robertson, T. A., and Papadimitriou, J. M. (1995). Rapid destruction of skeletal muscle

fibers by micelial growth forms of Candida albicans. Experimental and Molecular Pathology. 62:109–117.

Baba, T., Takeuchi, F., Kuroda, M., Yuzawa, H., Aoki, K., and Oguchi, A.(2002). Genome and virulence

determinants of high virulence community-acquired MRSA. Lancet. 359: 1819–1827.

Bailey, D. A., Feldmann, P. J. F., Bovey, M., Gow, N. A. R., and Brown, A. J. P. (1996). The Candida albicans

HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall

proteins. Journal of Bacteriology. 178:5353–5360

Cohen, P. R., (2007). Community-acquired methicillin-resistant Staphylococcus aureus skin infections: a review of

epidemiology, clinical features, management and prevention, International Journal Dermatology. 46:263-9.

Cole, M. F., Bowen, W. H., Zhao, X. J. and Cihlar, R. L., (1995). Avirulence of Candida albicans auxotrophic

mutants in a rat model of oropharyngeal candidiasis. Microbiology Letter. 126:177–180.

Crossley, K. B. and Archer, G. L., (1997). The Staphylococci in human disease. Churchill Livingstone.

Cunningham, R., Cockayne, A. and Humphreys, H., (1996). Clinical and molecular aspects of the pathogenesis of

Staphylococcus aureus bone and joint infection, Journal of Medical Microbiology. 44: 157-64.

Debuyser, M. L., Dufour, B., and Maire, M. (2001). Implication of milk and milk products infood-borne diseases in

France and in different industrialized countries. International Journal of Food Microbiology. 67: 1-17.

Diep, B. A., Gil,l S. R., Chang, R. F., Phan, T. H., Chen, J. H., and Davidson, M. G. (2006). Complete genome

sequence of USA300 an epidemic clone of community-acquired methicillin-resistant Staphylococcus aureus. Lancet.

367: 731–739.

Eickhoff, T. C. (1972). Therapy of staphylococcal infection. Staphylococci. Wiley, New York. 517-541.

Fonzi, W. A., (1999). PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-

linking of b-1,3- and b-1,6-glucans. Journal of Bacteriology. 181: 7070–7079.

Fonzi, W. A., and Irwin, M. Y. (1993). Isogenic strain construction and gene mapping in Candida albicans. Genetics.

134:717–728.

Gill, S. R., Fouts, D. E., Archer, G. L., Mongodin, E. F., Deboy, R. T., and Ravel, J. (2005). Insights on evolution of

virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus

strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain. Journal of Bacteriology. 187:

2426–243.

Grimme, S. J., Colussi, P. A., Taron, C. H., and Orlean, P. (2004). Deficiencies in the essential Smp3

mannosyltransferase block glycosylphosphatidylinositol assembly and lead to defects in growth and cell wall

biogenesis in Candida albicans. Microbiology. 150: 3115–3128.

Hanaki, H., Labischinski, H., Inaba, Y., Kondo, N., Murakami, H., and Hiramatsu, K., (1998). Increase in

glutaminenon-amidated muropeptides in the peptidoglycan of vancomycin-resistant Staphylococcus aureus strain

Mu50. Journal of Antimicrobial Chemotherapy. 42: 315-320.

Heyman, D. (2004). Control of Communicable Diseases Manual. 18th edition. American Public Health Association,

Washington DC.

Hiramatsu, K. (2001). Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet

Infectious Diseases. 1: 147-155.

Hiramatsu, K., Hanaki, H., Ino, T., Yabuta, K., Oguri, T., Tenover, F. C. (1997) Methicillin-resistant Staphylococcus

aureus clinical strain with reduced vancomycin susceptibility. Journal of Antimicrobial Chemotherapy. 40: 135-136.

Ichikawa, M., Ichikawa, T., and Mizomoto, T. (1996). Productivity of enterotoxins and toxic shock syndrome toxin-

1, and coagulase type of Staphylococcus aureus strains isolated from bovines and humans in the same district. Animal

Feed Science and Technology. 67: 780-786.

Ishibashi, K., Yoshida, M., Nakabayashi, I., Shinohara, H., Miura, N. N., Adachi, Y., and Ohno, N. (2005). Role of

anti-b-glucan antibody in host defense against fungi. Immunology of Medical Microbiology. 44: 99–109.Kenney, K.,

Reiser, R. F., and Bastidacorcuera,F. D. (1993). Production of enterotoxins and toxic shock syndrome toxin by

bovine mammary isolates of Staphylococcus aureus. Journal of Clinical Microbiology. 31: 706-707.

Klein, J. O., and Finland, M. (1963). The new penicillin's. New England Journal of Medicine. 269:1019-1025.

Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I., Cui, L., Oguchi, A., Aoki, K., Nagai, Y.,

Lian, J., Ito, T., Kanamori, M., Matsumaru, H., Maruyama, A., Murakami, H., Hosoyama, A., Mizutani-Ui, Y.,

Takahashi, N. K., Sawano, T., Inoue, R., Kaito, C., Sekimizu, K., Hirakawa, H., Kuhara, S., Goto, S., Yabuzaki, J.,

Kanehisa, M., Yamashita, A., Oshima, K., Furuya, K., Yoshino, C., Shiba, T., Hattori, M., Ogasawara, N., Hayashi,

H., and Hiramatsu, K. (2001). Whole genome sequencing of methicillin-resistant Staphylococcus aureus. Lancet.

357: 1225-1240.

Labandeira-Rey, M., Couzon, F., Boisset, E. L., Bes, M., Benito, Y., Barbu, E. M., Vazquez, V., Hook, M., Etienne,

J., Vandenesch, F., and Bowden, M. G. (2007). Staphylococcus aureus Panton- Valentine leukocidin causes

necrotizing pneumonia. Science. 315: 1130-113l

Ladhani, S., Joanna, C. L., Lochrie, D. P., Evans, R. W., and Posten, S. M. (1999). Clinical, microbial and

biochemical aspects of the exfoliative toxins causing Staphylococcal scalded-skin syndrome, Clinical Microbiology

Reviews. 12: 224-42.

Lowy, F. D. (1998). Is Staphylococcus aureus an intracellular pathogen. Trends in Microbiology. 8: 341-344.

Navarre, W. W., and Schneewind, O. (1999). Surface proteins of Gram-positive bacteria and mechanisms of their

targeting to the cell wall envelop. Microbiology and Molecular Biology Reviews. 63: 174- 229.

Novick, R. P., and Subedi, A. (2007). The SAPIs: mobile pathogenicity islands of staphylococcus. Chemical

Immunology and Allergy. 93: 42-57.

Ogston, A., (1881). Report upon micro-organisms in surgical diseases. British Medical Journal. 1: 369-75.

Patti, J. M., Bremell, T., Krajewska-Pietrasik, D., Abdelnour, A., Tarkowski, A., Ryden, C., and Hook, M. (1994).

The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infection and

Immunity. 62:152–161.

Scherer, S., and Magee, P. T. (1990). Genetics of Candida albicans. Microbiology Reviews. 54:226–241.

Walsh, C. T. (1993). Vancomycin resistance: decoding the molecular logic. Science. 261: 308-309.