Bacteriology Review

Beginning definitions

• Obligate Aerobe – require oxygen (20%) to grow• Obligate Anaerobe –>30 min of oxygen exposure can be toxic• Facultative anaerobes – grow in aerobic and anaerobic

conditions, most “aerobic” bacteria are actually facultative• Microaerophilic – Grow better with reduced oxygen and

elevated Carbon dioxide %

• Aerotolerant anaerobes– anaerobe not killed by prolonged exposure to oxygen – example: Clostridium tertium

• Lag Phase - >24 hrs old, growth slowing, not appropriate for biochemical or susceptibility testing

• Log Phase – Exponential growth – appropriate for all testing• Stationary phase – appropriate for transporting specimens

Specimen collectionfor Aerobic Bacteriology

Throat / Wound collected using swab1. Swab placed in Stuart’s or Amies transport media buffer

solution with peptones – 2. Preserve viability but not promote growth of bacteria3. Swabs made of polyester or sponge like material, cotton

traps organisms/potentially toxic

Urine – two methods suggested1. Boric acid to place organism in stationary phase2. Refrigerate within one hour, Specimen stable for 24 hours

• Purpose to maintain original colony count• Maintain viability of organisms

Gram stain Procedure

10 seconds-1 minute

Primary stain

Mordant 10 seconds-1 minuteRinse

5-10 secondsRinse

Rinse

Decolorization

Counter stain 10 seconds-1 minute

Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color

Gram positive cocciin pairs and chains Streptococcus

Gram negative bacillusResembling anEnteric

Gram positive rodmost likely a Bacillus species

Gram positive cocci in clusters, Staphylococcus

Gram negative rod – FusiformShaped – Fusobacterium species

Some Gram stains toremember! Visit the Gram stain Tutorial for more organisms!

Gram stain is used to assess quality of Sputum specimen for culture

• Expectorated sputum specimen is rejected for bacterial culture if judged to be “spit”:– Perform Gram stain of sputum specimen – If >= 25 epithelial cells /field – Sputum is judged to be spit / not a sputum specimen– Bacterial culture is not performed / it is rejected– Request made for a new “deep cough” specimen

Bad Sputum Good Sputum

10X objective 10X objective

Most used Agar Media in BacteriologyBlood agar- 5% sheep’s blood agar

Gauge hemolytic reaction of bacteria (alpha, beta, gamma)Grow variety of Gram positive & Gram negative bacteria and Yeast

Chocolate agar – “carmelized” blood agar – more enriched than blood agar medium. Will support the growth of the sameorganisms as blood agar Plus the more fastidious bacterialike Haemophilus influenzae and Neisseria gonorrhoeae

Most used agar media in Bacteriology

• MacConkey agar – Supports growth of Gram negative rods (GNR) only (crystal violet inhibits Gram positive organisms)Selective (only GNR grow ) and Differential (Lactose fermentation can be determined)Lactose fermentation = pink (neutral red indicator) (+)non-lactose fermentation = no color (-)

• After media is plated incubate in aerobic and/or CO2 incubators at 35˚ C for 24 – 72 hours

Newest Method for Organism Identification

• MALDI-TOF mass spectrometry– Matrix-assisted laser desorption/ionization– Identification of organisms by analyzing proteins

Gram Positive Cocci

Staphylococcus

Streptococcus

Enterococcus

StaphylococcusGram positive cocci inclusters

due to bound coagulase or “clumping factor”

All Staphylococcus are Positive for Catalase Enzyme

Staphylococcus aureus Coagulase Negative StaphCoagulase Enzyme Positive Coagulase Enzyme Negative

+/- Yellow colonyBeta hemolysis

White colonyMost not hemolytic

Catalase Enzyme ReactionNegative Positive

Hydrogen Peroxide plus bacteria

Avoid blood agar when collecting Staph – blood has innate catalase activity and will give false positive catalase reaction

Rabbit plasma inoculated with organismIncubate at 35˚C

Read at 4 hours and if negative read again at 24 hours

Negative tube coagulase =no clotCoagulase negative Staph

Positive tube coagulase [at 4 or 24 hrs] = Clot = Staph aureus

Tube Coagulase Reaction

Slide Agglutination Test to identify Staphylococcus aureus

• Clumping factor (coagulase) and protein A is found on the S. aureus cell wall.• Fibrinogen and IgG to protein A is on the surface of the Staphaurex latex beads• S. aureus will clump in the Staphaurex latex bead solution and positively identifies Staphylococcus aureus.• Coagulase negative Staphylococcus species remain milky and do not clump in the solution.•Advantage over tube coagulase – reaction time is 30 seconds vs. 24 hours

clumping

No clumping

Staphylococcus aureus • Primary virulence factor:

– Protein A – surface protein, ability to bind immunoglobulin and combat the immune response

• Diseases and associated toxin: – Toxic shock syndrome (TSST-1 toxin)– Scalded skin syndrome (Exfoliatin (SSS) toxin)– Soft tissue infection (Panton valentine leucocidin toxin – PVL)– Septic arthritis – primary cause– Food poisoning / Enterotoxins – stable to heating at 100*C for 30 minutes– Bacteremia and endocarditis

• Toxins act as superantigens, recruit hostdefense cells that liberate cytokines with systemic effects

Susceptibility issues –(1) MRSA• Methicillin Resistance (MRSA =Methicillin resistant S. aureus)

– Altered Penicillin binding protein (PBP2) produced by the mecA gene codes for resistance to oxacillin/methicillin/nafcillin

– All MRSA are considered resistant to semisythetic penicillins and cephalosporins

– Vancomycin is drug of choice – although reduced susceptibility reported

– Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance and provides better detection of MRSA than oxacillin testing. Shown is a Kirby Bauer (KB) disk test that is resistant to cefoxitin.

(2) The “D” Test • To accurately determine if Staph aureus/MRSA is susceptible To accurately determine if Staph aureus/MRSA is susceptible

to Clindamycinto Clindamycin• During therapy, S aureus isolates resistant to Erythromycin During therapy, S aureus isolates resistant to Erythromycin

possess enzymes capable of inducing Clindamycin to become possess enzymes capable of inducing Clindamycin to become resistant and therefore not useful for therapy.resistant and therefore not useful for therapy.

• Kirby Bauer zone around Clindamycin will be blunted to form Kirby Bauer zone around Clindamycin will be blunted to form a “D” if Clindamycin can be induced by Erythromycin to be a “D” if Clindamycin can be induced by Erythromycin to be resistant – so called INDUCIBLE RESISTANCE. resistant – so called INDUCIBLE RESISTANCE.

• Clindamycin should be reported as resistant by clindamycin Clindamycin should be reported as resistant by clindamycin induction and not used for therapy.induction and not used for therapy.

D test positiveInducible resistance

D test negativeClindamycin canbe used for therapy

Nares is a colonization site for MRSA and the most common surveillance site cultured.

ChromAgar is commonly used to detect MRSA. It is a selective and differential media with chromogenic substrates that turn specific colors to ID Staph aureus. Cefoxitin is added to the medium to select for methicillin resistant Staph aureus (MRSA).

Molecular assays (MA) can also be used to screen nares for MRSA. MAs increase the sensitivity of detection (@5-10%) but increases laboratory costs.

Mupiricin can be used to eliminate carriage

Staphylococcus aureus (MRSA)Hospital Epidemiology Issue:Nasal surveil lance for MRSA

Coagulase negative Staph (CNS)@ 15 species infect humans

• Staph epidermidis – is the most common– Major aerobic component of normal skin flora – Common cause of subacute bacterial endocarditis – Pathogenicity from cell adhesion factors forming biofilm on biologics and plastic (Catheters and replacement joints) – can be

difficult to treat• Staph saprophyticus –

– Urinary tract infections in the child bearing age female, – This species of CNS adheres in greater #’s to epithelial cells– Novobiocin resistant by KB disk test can be used as test to identify

Staph saprophyticus

White non-hemolyticcolony

resistant

CNS & Related Cocci• Staph hemolyticus –

– Can cause line related sepsis– This CNS is hemolytic on blood agar– Do not confuse with Staph aureus

• Staph lugdunensis– Positive PYR test– Perhaps greater pathogenicity than other species

• Micrococcus species– Mustard yellow colored colony– Catalase positive / Gram positive cocci in tetrads– Tube coagulase negative – Does not ferment glucose

• Staphylococcus ferment glucose– Environmental contaminate

Pos Neg

Streptococcus

Gram positive cocci in chains and pairs

Catalase enzyme negative

Grouped by hemolytic pattern on 5% sheep’s blood agar– Alpha – greening of agar, partial hemolysis of RBCs• Viridans Streptococcus, Strep pneumoniae,

Granulicatella/Abiotrophia– Beta – clearing of agar, complete hemolysis of RBCs• Beta hemolytic Streptococcus groups A - G

– Gamma – no clearing of agar, intact RBCs• Streptococcus bovis (gallolyticus)

Beta hemolytic Streptococcus

• Beta Streptococcus are grouped using the “C” (CHO) carbohydrate in the cell wall of the bacteria

• Identifies the Beta Strep groups – A, B, C, F, and G that are most common cause of human infections

• Lancefield grouping system uses the “C” CHO in the cell wall of Streptococcus (antigen) in a slide agglutination reaction with purchased monoclonal antibody attached to latex beads

See below slide for a positive and negative reaction.• Immunologic typing is more accurate than biochemical

testing to group Streptococcus

+-

Streptococcus pyogenes• Genus/species for Group A beta Streptococcus [GAS]

Biochemical identification:

– Bacitracin KB sensitivity test – inhibited, no growth @ disk– This test is not specific for Group A , it x-reacts with group C– “A” disk is the Bacitracin disk

– PYR (pyrrolidonyl arylmidase) reaction• Organism spotted on moist disk• 2 min – RT incubation• Add Cinnamaldehyde reagent• Look for color reaction• Pink = positive = Strep pyogenes• This test is not exclusive for Strep pyogenes – Enterococcus and

Staph lugdunensis are also positive for PYR

– Therapy : Penicillin or Cephalosporin antibiotics

– Streptolysin O and Streptolysin S toxins • Comprise the ASO titer dtermination• Cell toxins lead to evasion from the immune system• O toxin is oxygen labile S toxin is oxygen stable• When both are active - most hemolysis occurs in the 5% Sheep’s

blood agar

Primary virulence factors:

M Protein – preventsphagocytosis

Capsule – hyaluronic capsule protects from phagocytosis

Streptococcus pyogenes Diseases

• Diseases – – Pharyngitis– Impetigo (1)– Erysipelas (2)– Cellulitis (3) – Necrotizing fascitis (4)– Puerperal sepsis– Toxic Shock– Scarlet fever (5)

1

2

3

4 5

Sequelae of Strep pyogenes infections

• Rheumatic fever– Cause: inadequately treated Strep throat– Rise in the 1990s due to EIA tests being used as the only diagnostic test for

pharyngitis – EIA tests are @ 60-70% sensitive – and led to children not receiving antimicrobial therapy. All negative EIA specimens on children must be confirmed with culture

– Similarity between the proteins of the Strep A and muscle tissue cause immune system confusion which leads to immune system to attack heart, joint, and bones

– Anti-streptolysin O measures the amount of antibodies against Streptococcus group A in the serum

• Glomerulonephritis– 10-14 days following skin infection or pharyngitis– Renal disease with inflammation of the glomeruli– ASO titers will be positive– Usually resolves with therapy

Streptococcus agalactiae (Group B)Lancefield agglutination test demonstrates “B” cell wall CHO

Biochemical tests for identification:Camp test – performed using a Staph aureus strain with Camp

factor streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin and produces arrow shaped hemolysis

Rapid hippurate hydrolysis -4 hr identification of GBS - Hippurate disk inSaline inoculated with Group B StrepNinhydrin reagent added for color formation

Positive = Purple

pos

Arrow-like

Staph aureus

Strep group B

Strep agalactiae [GBS]• Pathogen of the elderly – mostly bacteremia and urinary tract infection• Pathogen of neonate – in utero or perinatal organism acquisition during

birthing process – Early onset – within 7 days – Late 7 – 28 days from birth process.

• Treatment: Penicillin or Cephalosporin (3rd generation)

• Pregnant women carry organism in the cervix and/or rectal area. All pregnant should be tested at 35 – 37 weeks of pregnancy.– Enrichment methods for GBS is standard of practice and must be used

• Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto Blood agar. This broth can also be used as an enrichment method to enrich for molecular testing.

• Carrot enrichment broth turns orange with growth of group B Strep

Carrot Broth

It used to be Streptococcus bovis Taxonomy update – S. gallolyticus

1) Streptococcus gallolyticus ssp. gallolyticus (formerly S. bovis biotype 1) associated with colonic cancer and endocarditis

2) Strep gallolyticus ssp. pasteurianus (formerly S. bovis biotype II) associated with neonatal meningitis

S. gallolyticus (both ssp) have same biochemical reactions

Bile esculin slant – positive (turns black)

6.5% NaCl – no growth

PYR reaction - negative

• Susceptible to Penicillin

• Two most common species– E. faecium– E. faecalis

• No well defined virulence factors• Possesses the Group “D” CHO in the cell wall

• Biochemical tests:– Bile esculin positive – growth and black precipitin– Growth in 6.5% salt– PYR positive (Group A beta Strep and Staph

lugdunensis also positive)*– E. faecium = arabinose fermentation positive – E. faecalis = arabinose fermentation negative

Enterococcus

Bile esculin

PYR

+ -

Neg Pos

Neg Pos

6.5% Salt

PYR

Enterococcus

• Variety of infections / Pathogen of opportunity• Intestinal normal flora• Cause UTI, bacteremia, and abdominal infections• Antimicrobial therapy:

– Natural resistance to cephalosporin antibiotics– Ampicillin plus Aminoglycoside can be synergistic for therapy in

cases of endocarditis

• Unique susceptibility issues – Acquired resistance to vancomycin known as vancomycin resistant

enterococcus or VRE• Van A (E. faecium) resistance genes• Van B (E. faecalis) resistance genes

Alpha hemolytic Streptococcus

Streptococcus pneumoniaeGram stain = Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor, antiphagocytic

Identification:– Bile soluble – colonies dissolve in sodium deoxycholate (bile)– Optochin sensitive – 14mm or greater zone of inhibition around the

6mm optochin disk – “P” = pneumococcus

Inhibition >=14mm

NOInhibition

Colonies dissolved

Streptococcus pneumoniae• Upper and Lower respiratory tract infection, sepsis and

meningitis • Multi-serotype vaccine aids preventing invasive infections• Susceptibility issues:– Acquired Resistance to Penicillin due to Penicillin binding

proteins (PBP) – Minimum inhibitory concentration (MIC) testing necessary

to detect resistance to penicillin – in broth or Etest method– Testing of CSF isolate a result of – Penicillin = >2 mcg/ml is resistant

• Therapy – – If susceptible 1st line therapy is either– Penicillin or 3rd generation Cephalosporin

MIC

MIC

Viridans Streptococcus

Several species of alpha hemolytic Streptococcus are NF in mouth and upper respiratory tract

S. mutans S. salivarius S. sanguis S. mitis• Bile esculin slant = negative• Not bile soluble• Optochin resistant with zone size <=13 mm • Cause 30 – 40% cases of sub acute endocarditis / native valve• Can cause abscess and various infections throughout the body

especially in the immune suppressed host• Variable susceptibility patterns, some isolates with elevated

MICs to Penicillin

Viridans Streptococcus

Viridans Streptococcusunique species

• Streptococcus anginosis group: includes– S. anginosus S. constellatus S. intermedius

• Normal flora in human mouth• More virulent than “normal” viridans

Streptococcus, perhaps due to capsule• Cause deep tissue abscess, bacteremia,

endocarditis, intra abdominal infections…• Variable susceptibilities – so best to do

susceptibility testing

Nutritionally Variant Streptococcus• Vitamin B6 (pyridoxal) deficient – so will not grow on media without B6

supplementation

• Will grow in a patient blood culture bottle due to vit B6 in patient’s blood• Will not subculture onto 5% Sheep’s blood agar plate ( no vit B6)• Requires Staph aureus streak (supplies vitamin B6) • Nutritionally variant Strep will satellite @ S. aureus streak (see pix)

• There are 2 species:– Abiotrophia spp– Granulicatella spp

• Endocarditis – – More destructive to valve than

than “regular” viridans Strep– Higher MIC’s to Penicillin

Satellite streptococcus

Gram Negative Cocci

Neisseria

Moraxella

Gram Negative Cocci

• Neisseria species and Moraxella catarrhalis– Small kidney bean shaped cocci in pairs– Oxidase enzyme positive

• CTA (Cysteine Trypticase Agar) carbohydrate fermentations for identification – glucose, maltose, lactose, sucrose– N. gonorrhoeae Gluc + Mal - Lac - Suc -– N. meningitidis Gluc + Mal + Lac - Suc -– N. lactamica Gluc + Mal + Lac+ Suc-– M. catarrhalis all negative Dna’ase +

• N. gonorrhoae will NOT grow on 5% Sheep’s blood agar• N. meningitids will grow on 5% Sheep’s blood agar

Miniature CHO fermentation reactions for Neisseria and Moraxella catarrhalis

Compare (+) yellow reactions to negative (red) control well

+

+ +

Oxidase enzyme spot test:Detects presence of enzyme cytochromeoxidase Add reagentN,N dimethyl-p-phylenediamine oxalate to filter paper with organism positive = purple/ blue

control

Full size tube CHO fermentation test

Neisseria meningitidis• Meningitis in children and young adults• Hallmark is petechia (organisms crowd into capillaries) tissue

necrosis and DIC (endotoxin) /infection can be rapidly fatal• Carriage in Nasopharynx/ Susceptible to Penicillin• Capsular polysaccharide is the primary virulence factor• Complement deficiencies in 7,8,and 9 can predispose to

chronic type disease• Adrenal necrosis is referred to Waterhouse Friderichsen syndrome• Type C is the current endemic strain in the USA – 1*problem

in college Freshmen living in dorm• Immunization available for all serotypes except serotype B

Neisseria gonorrhoeae– Acute urethritis, endocervix, ocular, rectal, oropharynx,

septic arthritis– 10 – 20 % female ascend to PID but only 0.5% disseminate– Gram stain of urethral discharge useful for male diagnosis,

however, cervix Gram stain not specific for females– Transport for culture – charcoal swabs, No refrigeration– Media: Selective Thayer Martin or Martin Lewis agar– Amplification methods [PCR] increase sensitivity of

detection and superior to culture – Produces a beta lactamase enzyme and also Chromosomal

resistance : Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent resistance

Neisseria speciesgram negative diplococcus – intra and

extra cellular

Acinetobacter species –Cocci are larger and more round than Neisseria, not usually in pairs – normal f lora in female genital tract

Primary reason why female Gram stainsCannot be trusted.

Moraxella catarrhalis

• Pneumonia, ocular, sinusitis, otitis media• Gram stain of sputum can be helpful in

diagnosis of Moraxella pneumonia– Polys with gram negative diplo-cocci

• Hockey puck colony – able to push around on agar surface– Oxidase enzyme positive– DNA’ase enzyme positive– Resistant to ampicillin by beta lactamase enzyme

production – Cefinase test can be used– Therapy: Augmentin or 2nd or 3rd generation Cephalosporin

Gram Positive RodsCorynebacteria

BacillusListeria

Erysipelothrix

Corynebacterium

•Over 20 species, most not pathogenic•Gamma hemolytic gray colonies •Catalase + •Diphtheroid morphology – Gram positive Chinese letter forms•No spores produced

Corynebacterium diphtheriae• Agent of Diphtheria• Diphtheritic adherent pseudo membrane produced in throat• Phage mediated exotoxin is distributed from the membrane

causing respiratory paralysis (virulence factor)• Toxin detected by Elek immuno-diffusion test • Grows well on 5% Sheep’s BAP• Selective medium Cysteine Tellurite agar

– Produces black colonies with brown halos• Grow on egg containing Loeffler medium then stain

with methylene blue to observe metachromatic granules - colorful storage granules characteristic of C. diphtheria

Elek plate

Tellurite agarMetachromatic granules

Other Corynebacterium• Corynebacterium jeikeium – – normal skin flora bacteria / thrives on lipid – Infects patients with plastic catheters and indwelling

devices by tunneling into the device from the skin– Biofilms are formed on the plastic, protecting the

organism from antibiotic therapy– Very resistant to most antibiotics– Susceptible to vancomycin and tetracycline only

• Corynebacterium urealyticum – – Rapidly urease positive diphtheroid– Urinary tract infection in post renal transplants – Resistant to many antibiotics – vancomycin susceptible

Red is (+) forUrease reaction

Bacillus species• Large gram positive rods boxcar shaped with square

ends• Can over-decolorize easily and appear reddish• Spores produced – causes clearing in rod• Catalase enzyme positive• Most are motile

Bacillus anthracis

• Agent of Anthrax• Natural infection of herbivores – infected animals can have

fatal infection and contaminate the environment for years• Virulence factors: anthrax toxin and capsular polypeptide• Skin (Woolsorters’ disease) is the most common

presentation • Unique black eschar skin lesions • Pneumonia, sepsis, and meningitis

uncommon

Bacillus• Bacillus anthracis culture:• Medusa head colonies on BAP – non-hemolytic on 5% Sheep’s blood agar

• Non-motile • Penicillin susceptible• With these reactions – Alert public health department for assistance

• Bacillus cereus – food poisoning with rapid onset (1 – 6 hr) of vomiting

• Preformed emetic toxin in food • Fried rice is one of the most common food sources• Traumatic wound infection from contaminated soil• Beta hemolytic colony on Sheep’s blood agar• Motile

Listeria monocytogenes• Small gram positive rod• Catalase positive / No spores produced• Subtle beta hemolysis on Blood agar • Tumbling motility on wet mount• Umbrella motility in tube media • More motile at 25˚C than 35˚C• Cold loving – Grows well at 4˚C and reason it is

abundant in refrigerated foods• Found in dairy products and deli case meats• Infections: Bacteremia in pregnant women and can induce still births• Infections: Bacteremia and CNS in immune suppressed• Ampicillin is drug of choice/ resistant to Cephalosporins

Erysipelothrix rhusiopathiae • Small gram positive rod• Catalase enzyme negative• Alpha hemolysis on blood

– Only G+R that produces hydrogen sulfide (H2S) in a TSI agar slant– Human skin infection acquired from swine with erysipelas or

poultry• Infection most common in butchers

– Bacteremia uncommon • most common in drug addict• High % of endocarditis with extensive damage to valve

– Intrinsically resistant to Vancomycin

H2S production on Triple sugar iron agar (TSI)

Gram negative bacilli

Enterics

Non fermenters

Fastidious

Gram negative cell Wall

Rod shaped

Simple flow chartIdentification

Strategy

Enteric Gram Negative Rod

Ferment GlucoseFerment LactoseEx. Escherichia

coli

Enteric GramNegative Rod

Ferment GlucoseDo NOT ferment

LactoseEx. Proteus species

Gram negative BacilliDo NOT ferment

GlucoseDo not ferment

LactosePositive OxidasePseudomonasBurkholderia

Gram negative BacilliDo NOT ferment

GlucoseDo NOT ferment

LactoseNegative Oxidase

AcinetobacterStenotrophomonas

EnterobacteriaceaeEnteric Gram Negative BacilliFerment glucoseAlmost all are oxidase negativeNitrate reduced to nitrates

Non fermentersGlucose is not fermentedOxidase positive and negativespecies

Enterics that Ferment Glucose• Escherichia coli

– Major aerobic NF in intestine– #1 cause of UTI [@80%]– Bacteremia, neonatal meningitis– Abdominal cavity infections– Spot indole reaction = positive– Green sheen produced on EMB agar

• Pathogen of diarrhea– Traveler’s diarrhea - travel to developing countries– Enterohemorrhagic E. coli [0157:H7 as well as other serotypes]

• Bloody diarrhea usually acquired from eating undercooked cow meat from an infected cow

• HUS – hemolytic uremic syndrome [hemolytic anemia, thrombocytopenia, and renal failure] particularly in children

• Does not ferment sorbitol – most other E. coli types ferment Sorbitol

Green sheen onEMB agar

Mucoid colony

Indole positive =Robin’s egg blue

Enterics that ferment Glucose

• Enterobacter species• Enterobacter cloacae and E. aerogenes most common• Environmental organism with low pathogenicity• Enterobacter (Cronobacter) sakazakii associated with

neonatal meningitis

• Klebsiella species – • K. pneumoniae most common• Mucoid colony• Currant jelly sputum in alcoholics due to blood mixed with

capsular polysaccharide in sputum

Enterics that do NOT ferment lactose!No fermentation = colorless colony on Mac

• Proteus species Swarming colonies in layers on agar surface

• Indole positive – Proteus vulgaris• Indole negative – Proteus mirabilis

• Serratia marcescens – Produces a red pigment that intensifies at room temp– Causes infections in

• immune suppressed• Ventilator associated pneumonia• Bacteremia

Gram negative bacillus MacConkey agar

Lactose fermenter

Lactose non fermenter

Gram stain enteric baci l l i – plump

No lactosefermentation

Lactose fermentation

Glu/lac/suc fermentedwith gas

Glucosefermentedonly

Glucosefermentedwith H2S

No CHO fermentationNon fermenter

Triple Sugar Iron Agar – Detect fermentation of glucose, lactose and/or Sucrose and the production of hydrogen sulf ide [H2S]

Fermentation= yellowGas = Disruption of the agar

H2S

No fermentation =red

Extended Spectrum Beta Lactamase Extended Spectrum Beta Lactamase Enzymes – “ESBL”Enzymes – “ESBL”

• ESBL enzymes are produced by numerous species of gram ESBL enzymes are produced by numerous species of gram negative rodsnegative rods– These enzymes confer resistance to Cephalosporins and These enzymes confer resistance to Cephalosporins and

synthetic Penicillinssynthetic Penicillins• Plasmid mediated Tem 1 beta lactamase is the most common Plasmid mediated Tem 1 beta lactamase is the most common

ESBL enzyme producedESBL enzyme produced– Possible plasmid dissemination makes this an infection Possible plasmid dissemination makes this an infection

control issuecontrol issue• ESBL producing GNR are usually treated with:ESBL producing GNR are usually treated with:– Carbapenemases – Meropenem, & Imipenem most Carbapenemases – Meropenem, & Imipenem most

commoncommon

Carbapenamase Resistance (CRE)

• Resistance enzymes conferring resistance to carbapenamase antibiotics (meropenem and imipenem)

• Most common in Klebsiella pneumoniae – where this enzyme is known as “KPC”

• Cross resistance to virtually all classes of antibiotics – so very difficult to treat

• Treatment: Colistin or antibiotic combinations

Salmonella species• Salmonella spp. – Diarrhea with +/- fever – polys in the stool– Infection from food - Eggs, meats and contaminated

uncooked vegetables, must ingest large #’s of organisms to make you ill (1,00,000 bacteria), stomach acid protective

– Does not ferment lactose/ produces Hydrogen sulfide– Selective agars for growth: SS and Hektoen– Identification based on biochemical reactions & serologic

typing – Kaufman White serologic typing scheme

• O Somatic (cell wall) antigen – Salmonella group “B”• H flagellar antigens – 2 phases [h1 & h2] aids in speciation

Salmonella• Vi capsular antigen – Found in S. typhi only

Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless due to lactose not being fermented

Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black coloniesShigella – green coloniesNormal flora – orange colored due to fermentation of lactose

Salmonella typhi• Typhoid fever – fever, sepsis, high fatality rate

• Vi capsular antigen surrounds the D cell wall antigen• Boil solution of organism for 15 minutes to destroy the

Vi capsular antigen and expose the cell wall D antigen • Serotyping can then be performed for the D antigen• Moustache of H2S in the TSI tube

– Carrier state in gallbladder– Ingest organism, it clears the bowel

then in @ 1 week enters

Blood stream & Bone Marrow

Shigella• Diarrhea, +/-vomiting, fluid loss, polys and blood in stool ,

tenesmus, usually treated, Quinolone therapy• Human to human transmission /control with good hygeine• Low #’s of organisms to make you ill [10 – 100 bacteria]• Non motile and No H2S produced (differ from

Salmonella) – Green colonies on Hektoen• Does not ferment lactose • 4 species based on somatic antigen – S. boydii Group C– S. dysenteriae Group A– S. flexneri Group B– S. sonnei Group D

Yersinia enterocolitica

• Diarrhea• Major reservoir – swine• Human usually infected by non pasteurized milk• Causes septicemia in iron overload syndromes• Unique infection Mesenteric adenitis – RLQ pain

which mimics appendicitis• Grows well at 4 °C (like Listeria)• CIN agar (Cefsulodin-irgasan-novobiocin) is

selective for Y. enterocolitica• Has been associated banked blood infections related

to transfusion of infected products

Yersinia pestis - Plague

• Category A agent of bioterrorism• Obligate flea/ rodent/ flea cycle • Infected Flea bite - leads to Bubonic form/ painful buboes (lymph node swelling)• Pneumonic form +/- bacteremia spread • Fatality >=50%• Endemic Southwestern USA• Grows on blood agar – catalase +, oxidase -– Bipolar staining “safety pin”

Glucose FermentersOxidase Positive

• Vibrio cholerae– Rice water stool/ mucus flecks in diarrhea – Natural environment is saltwater– Virulence due to enterotoxin – receptor on epithelial cell –

activates adenyl cyclase – increases cAMP with hyper secretion of NaCl and H20 – death from dehydration and metabolic acidosis– Halophilic bacteria which means it is a salt loving / 1% salt

actually enhances growth– Curved appearance to gram negative rod– Selective media – TCBS = thio citrate bile sucrose agar,

colony turns yellow due to sucrose fermentation

TCBS Agar

Other Vibrio species

• Vibrio parahaemolyticus• Colony on TCBS medium is green = sucrose negative• Diarrhea from ingestion of raw fish and shellfish

• Vibrio vulnificus• Highly virulent Vibrio species – infection leads to formation of

painful skin lesions on lower extremities with muscle necrosis• Most prevalent in patients with pre existing liver disease • 50% fatality rate• Ingestion of raw oysters and shellfish

from coastal waters• Usuallly Green colonies TCBS

Classic Gram Negative Rod Gram Stains

Campylobacter

Vibrio species

Enteric gram negative rod

Sea gull wings C shaped

Plumb and rectangular

Do not ferment Glucose Oxidase negative

• Acinetobacter species – isolated from a hospital environment and human skin, large gram negative cocco-bacilli– Ac. baumannii is a major nosocomial pathogen

• Glucose oxidizer• Acquires antibiotic resistance with pressure

– Ac. lwoffi – • glucose non oxidizer

• Stenotrophomonas maltophilia – Rapid maltose oxidizer– Long Gram negative bacillus– Naturally resistant to many antibiotics– Gun metal gray pigment– Nosocomial pathogen – super-colonizer when on long term Imipenem

therapy due to natural resistance to Imipenem

Glucose non-fermenterOxidase Positive

Pseudomonas aeruginosa • Fluorescent & blue-green

pigment(pyocyanin)• Grape-like odor • Growth at 42˚C

– Ps fluorescens/putida – no growth at 42°C

• Major pathogen in cystic fibrosis – Mucoid strains [polysaccharide capsule]– in combination with Burkholderia cepacia – can cause major lung damage

• Nosocomial pathogen – associated with water & moisture• Intrinsically resistant to many antibiotics

Glucose Non Fermenters/oxidase +

• Burkholderia cepacia– Dry , yellow colony – Oxidase negative– Problem organism in cystic fibrosis

colonization and infection with extensive lung damage• Flavobacterium (Chryseobacterium) meningosepticum– Associated with fatal septicemia in the neonate– Low virulence– Environmental source – Yellow pigment

Haemophilus species Haemophilus influenza– Variety of infections– Transmission – close contact secretions– Polysaccharide “B” capsule = virluence factor– Requires 2 nutritional factors for growth

• X = hemin• V= NAD (nicotinamide adenine dinucleotide)• Demonstrate by satellite phenomenon or X/V strips

– Small pleomorphic GNR / Grows on chocolate agar – Will not grow on 5% sheep’s blood agar– Requires high level C0₂ [5 – 8%] for growth– Vaccine targets the H. influenza type B– Resistance to Ampicillin by beta lactamase production [15-

20 %], Cefotaxime becomes the antibiotic of choice+

Disk test for Beta lactamase Disk test for Beta lactamase DetectionDetection

• Add bacteria to filter paper impregnated with Nitrocefin or Add bacteria to filter paper impregnated with Nitrocefin or Cefinase test (yellow colored/chromogenic cephalosporin)Cefinase test (yellow colored/chromogenic cephalosporin)

• Incubate at room temp for @ 1 minuteIncubate at room temp for @ 1 minute

• Positive result is color change from Positive result is color change from yellow to redyellow to red - beta - beta lactamase enzyme breaks down beta lactam ring to form lactamase enzyme breaks down beta lactam ring to form hydrolyzed red end producthydrolyzed red end product

• Detects resistance to Amp/Pen/Cephalosporin in Detects resistance to Amp/Pen/Cephalosporin in Haemophilus species, N. gonorrhoea , Moraxella catarrhalis, Haemophilus species, N. gonorrhoea , Moraxella catarrhalis, and anaerobic gram negative rodsand anaerobic gram negative rods

• Does NOT detect the ESBL enzymeDoes NOT detect the ESBL enzyme

More Haemophilus species

• H. parainfluenza – – requires V (NAD) factor only– Usually normal flora in the upper respiratory tract– One of the HACEK organisms of endocarditis

• H. (Aggregatibacter) aphrophilus – (new taxonomy)– No factor requirements for growth– Infections: abscesses (liver, lung, brain) & endocarditis

• H. ducreyi – – Requires X factor– Cause of Chancroid - venereal disease– “school of fish” appearance on stains

Satellite phenomenaDemonstrates need for X and V factor

Small pleomorphic GNR

The HACEK group

• Oral flora can be pathogens of endocarditis caused by poor detention or dental procedures

• Requires 2-4 days to grow in patient blood cultures• Haemophilus species = oxidase neg, catalase neg • Actinobacillus actinomycetom-comitans = oxidase

neg, catalase pos• Cardiobacterium hominis = oxidase pos• Eikinella corrodens = oxidase pos, colony pits BAP• Kingella kingii = oxidase pos, hemolytic on BAP, also

cause infections in small children (septic joints)

Bordetella pertussis

Whooping cough – three stages of disease– (1) Prodromal – flu like disease – most contagious stage– (2) Catarrhal - cough - with classic whoop in small children Toxin adheres to bronchial epithelial cells and cough

continues until toxin wears off – can be months– (3) Paroxysmal - recovery phase

• Gram stain = Tiny gram negative coccobacillus• Inhabits Nasopharynx – specimen source• PCR most sensitive and specific for diagnosis• Charcoal containing media for transport and culture –

Regan Lowe Charcoal agar • Reservoir for infection – young adults due to waning

immunity. Reason behind booster shots for young adults

Pasteurella multocida

• Primary cause of Cat and Dog bite wound infections• Human pneumonia from close animal contact

– Infections can disseminate to blood stream

• Found as normal flora in animal’s mouth• Small gram negative coccobacilli• Growth on 5% Sheep’s blood agar

– Non hemolytic grey colony

• Will not grow MacConkey agar (big clue)• Oxidase Positive• One of very few Gram negative rods

sensitive to penicillin

Capnocytophaga• Fusiform shaped gram negative rods – very pleomorphic• Gliding motility, fingerlike projections from colonies• Oxidase negative, Catalase negative• C. canimorsus – dog bites with bacteremia – high% of these

infections lead to endocarditis

• Other Capnocytophaga species are normal flora in the human mouth and

• Can infect mouth ulcers induced by chemotherapy – • Blood stream can be invaded from infected mouth ulcer

Brucella species

• Disease – Fever of unknown origin, significant joint pain• Small gram negative coccobacilli – slow and difficult to grow from blood

cultures – intracellular pathogen of the RES system– Castaneda biphasic blood culture held for 21 days [old school]– Current: Automated Blood culture systems with growth within 5 days

• Specimens: blood and bone marrow most profitable– Granuloma formation in bone marrow

• Serology can be used for genera/species diagnosis• Brucella species in clinical specimen related to animal

species the patient was exposed: – B. abortus – ingestion of raw cow milk– B. melitensis – ingestion raw goat milk, feta cheese– B. suis – contact with pigs B. canis - contact with dogs

Granuloma in bone marrow

Campylobacter• Small curved bacilli – shaped like sea gull wings• C. jejuni – agent of diarrhea

– Related to undercooked poultry ingestion– Requires selective media containing antibiotics -Skirrow’s blood agar– Thrives at 42˚C - can use to selectively culture Campylobacter– Requires microaerophilic atmosphere (high CO₂, low O₂)– Significant % Guillain-Barre syndrome as sequelae of infection

• C fetus – Bacteremia in the immune suppressed host / does not cause diarrheal disease

• C. jejuni – grows at 37˚C and 42˚, hippurate positive• C. fetus - grows at 37˚C and 25˚C hippurate negative

• Reservoir – rabbits, rodents, ticks and flies. Humans infected by bug bites or directly from exposure to animal blood

• Strongly associated with skinning rabbits with bare hands• Bacteria can penetrate small breaks in skin:– cause painful skin lesions – – enlarged lymph nodes –– leading to bacteremia (ulceroglandular tularemia)– Pneumonia

• Great hazard to lab workers• Fastidious small gram negative cocco-bacillus• Culture media requires cysteine*

Francisella tularensis

Helicobacter pylori

• Small curved Gram negative bacilli• Cause of acute gastritis…..gastric adenocarcinoma• Human to human transmission/ fecal oral route• H. Pylori, Rapidly!! And strongly urease positive – used for

detection directly from gastric biopsy tissue• Difficult to grow in culture• Stool antigen/diagnosis and test of cure ]

Serum antibody for exposure• Treatment – Antibiotics and acid

suppression

Histologic exam of gastricbiopsy

Hematoxylin-eosin stain orWarthin-Starry Silver stain

Legionella

Legionella pneumophila most common species [6 serotypes]• Requires cysteine in culture medium for growth • Buffered Charcoal Yeast Extract agar -colonies form in 3-5

days• Will not show on Gram stain of specimens • Use silver impregnation stains in tissue • Pulmonary disease associated with water,

cooling towers, shower heads, stagnant water• Urinary antigen test will detect L. pneumophila type I

infection only• Treatment: Erythromycin (macrolide)

BCYE

Bacteria without cell walls• Mycoplasma and Ureaplasma – have cell membranes only! Media and

transport contain sterols to protect the membrane• Do not form discreet colonies on agar plates – must read plates under a

microscope to visualize organisms [up to 14 days of incubation]

• M. pneumoniae –– community acquired pneumonia– Serology and PCR for diagnosis– Cold agglutinins produced

• Genital mycoplasmas– M. hominis – fried egg colony, vaginitis, cervicitis, postpartum sepsis,

neonatal infections , pre rupture of membranes– Ureaplasma urealyticum – rapid urea hydrolysis in broth, NGU &

upper genital tract infection, spontaneous abortion, neonatal infections

Ureaplasma

Mycoplasma hominis

Difficult to grow• Bartonella henselae – Diagnose by PCR and/or serology

– Cat scratch disease – exposure to cat and cat excrement– Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV

• Bartonella quintana – cause of trench fever/ vector is the body louse/Diagnose by Serology

• Chlamydia trachomatis Serovars L1,L2,& L3 agent of Lymphogranuloma venereum

– Serology / clinical findings – lymphatics and lymph nodes involved

• C. pneumoniae (TWAR agent)- Pneumonia, Diagnose by PCR• C. psittaci- psittacosis, pneumonia, exotic parrot exposure/ Serology, PCR and epidemiology

Molecular Testing for Neisseria gonorrhoeae Molecular Testing for Neisseria gonorrhoeae and Chlamydia trachomatisand Chlamydia trachomatis

• Amplification of DNA by Polymerase Chain Reaction (PCR)Amplification of DNA by Polymerase Chain Reaction (PCR)– Urine, Cervix, and urethral most often testedUrine, Cervix, and urethral most often tested– More sensitive than any culture based systemMore sensitive than any culture based system• Sensitivity/Specificity @ 96%/99%Sensitivity/Specificity @ 96%/99%• Females most sensitive specimen = cervixFemales most sensitive specimen = cervix• Males good sensitivity with urine and urethralMales good sensitivity with urine and urethral

• PCR replaced culture methods as “Gold” standard of PCR replaced culture methods as “Gold” standard of detectiondetection

Iodine staining of inclusions in McCoyCell culture

Elementary bodies in Fluorescent antibody stain

More difficult to grow

• Ehrlichiosis – Rickettsia bacteria– Zoonotic intracellular pathogens– Anaplasma spp, inclusion in the PMN– Ehrlichia spp inclusion in the Monocytes– Vector = Ixodes tick – Fever, leukopenia, thrombocytopenia, – Elevated serum aminotransferases,– No rash (differs Rocky Mountain Spotted Fever)

• Found in the south central, southeast , midwest USA• PCR and serology for diagnosis

Spirochetes

• Borrelia burgdorferi - Lyme’s disease– Primarily found in NE part of USA– Vector = Ixodes tick– Serology and PCR

• Borrelia recurrentis - Relapsing fever Vector = human body louse Blood smear observe spirochete

Spirochetes• Treponema pallidum

– Syphilis– RPR and VDRL for antibody detection– Molecular methods

• Brachyspira – intestinal spirochete found

on the brush border of the intestine,

?? Role in disease• Leptospira interrogans – Leptospirosis

– Fever with rash and renal involvement– Urine from rats and other animals contaminate water supplies

Darkfield from chancre lesion

Spirochete – Shepherd’s crook

Unusual Bacterial diseases

• Granuloma inguinale– Klebsiella (Calymmatobacterium) granulomatis– Rare STD – causes ulcerative genital lesions

• Streptobacillus moniliformis – Rat bite fever or Haverhill fever– L form - cell wall deficient bacteria– Inhibited by SPS in blood culture media– Needs serum supplementation to grow, will not grow on

routine bacteriology media– Infection obtained from rat bite

Anaerobes• Anaerobic infections can occur in virtually

every organ and region of the body• Polymicrobial – both aerobic and anaerobic species in the

infections• Endogenous organisms (commensal flora) cause most

anaerobic infections– Due to trauma, vascular or tissue necrosis cutting off the oxygen

supply to the involved tissue• Surgery plus antibiotics often necessary• Anaerobic culture collection – must be oxygen free

– Gel containing swabs – ESwab, – Evacuated vials (port o cult)/ oxygen free collection– Do not refrigerate specimens – absorb oxygen and kill anaerobes

Anaerobes

• PRAS media – pre reduced anaerobically sterile– Media packaged in oxygen free environment

• Most common media used– CDC anaerobic blood agar– Kanamycin-vancomycin blood agar– Bile Esculin agar– Thioglycollate broth– Chopped meat glucose broth

• Anaerobic gas pack jars – incubate cultures in anaerobic conditions• Wet pack – add 10 ml water to hydrogen and CO2 generating

envelope/ requires palladium coated catalysts – generate heat• Dry pack – (Anaeropack) Absorbs O2 and generates CO2

• Anaerobic bacteria can be poorly staining with bizarre & irregularly shape

Anaerobic Gram Negative Rods

Bacteroides fragilis groupPleomorphic irregular staining gram negative rod–Grows in the presence of bile–Esculin positive – turning media black–Resistant to Penicillin and Kanamycin–Common normal flora in GI tract –Infections foul smelling/gas produced–B. fragilis* - most common–B. ovatus–B. thetaiotamicron – indole positive–B. uniformis B. vulgatus

• Resistant to Penicillin by beta lactamase enzyme• Metronidazole is one antibiotic of choice

Growth on bileBlack pigment

Gram Negative Anaerobic Bacillus

• Prevotella and Porphyromonas species– Will not grow in the presence of bile– Will not turn black on esculin media– Brick red fluorescence and black pigment– Normal commensal flora of the upper respiratory tract– Respiratory tract infections

Fusobacterium spp.Long thin gram negative bacilli –

spindle shaped with pointed ends • Normal flora in upper respiratory tract• Associated with mouth and respiratory tract abscess

formation and liver abscess • Vincent’s angina – necrotizing oral infection caused

by Fusobacterium species and spirochetes• Lemierre’s syndrome - oropharyngeal infection that

leads to thrombosis in jugular vein, septicemia, high fatality rate, caused by Fusobacterium necrophorum

Anaerobic Gram Positive Rods

Clostridium species

• Gram positive bacilli (boxcar shaped ) with spores– Bacilli may over-decolorize and appear red

• Clostridium perfringens – Predominate anaerobic G+R in intestine – Double zone of beta hemolysis on BAP– Lecithinase produced on egg yolk agar– Reverse camp test positive

LecithinaseReverse Camp Test

Clostridium• Clostridium botulinum –

– Adult disease – Preformed heat labile toxin ingested in mass produced or home-canned foods

– Infant disease - spore ingested from product produced in nature, neurotoxin produced in gut • Begins with constipation and difficult sucking bottle • Associated with Honey, soil, household dust

– Life threatening neuroparalytic disease• Clostridium tetani

– Tetanus– Gram stained cells appear like Tennis racket – Toxin enters human due to penetrating skin injury– Disease occurs in Non-immunized– Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw

(trismus)

Clostridium • C. septicum – – Bacteremia or Gas Gangrene in patient with underlying

malignancy• C. difficile – – Normal Colonization in 5% adults and 70% healthy infants– Disease = Antibiotic associated colitis,

pseudomembranous colitis due to toxin production [Virulence Factor]• Toxin A – enterotoxin causing fluid accumulation• Toxin B – potent cell cytotoxin • Binary toxin (Nap1) strain is unique and produces larger amount of

toxins A and B and is more virulence– Diagnosis of infection:

• EIA methods [toxin A/B] are not sensitive and are discouraged• PCR methods [toxin B] are more sensitive and for infection• Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA]

Actinomyces

• Branching gram positive bacilli - do not form spores • Normal flora in the mouth• Capable of forming sulfur granules

in tissue – often found on normal tonsil• Actinomyces israelii – associated with oral, thoracic,

and abdominal infections, IUD infectionsB

read crumb colonies in broth

Molar tooth colony on agar plates

Penicillin susceptible.

Branching Gram positive rods of Actinomyces – antler like

Molar tooth colony

Sulfur granule

Propionibacterium acnes

• Pleomorphic gram positive rod• Catalase positive• Indole positive• Normal flora - skin, oral, GU and GI• Potential contaminate in blood cultures• Pathogen in cerebral shunt infections • Firmly established as significant cause of prosthetic joint

infection – particularly shoulder joints• Cultures should be held up to 7- 14 days • Therapy - Ampicillin

Bacterial vaginosis

• Clue cells are diagnostic• Mixed anaerobic/aerobic bacterial infection - the two most common

organisms in vaginosis are:– Gardnerella vaginalis (aerobic gram variable rod) and Mobiluncus

(anaerobic curved gram negative rod)– Human blood agar used to culture Gardnerella, it

is beta hemolytic and distinctive morphology• Nugent score – gram stain scoring system to diagnose

bacterial vaginosis [BV]– Healthy = Lactobacillus-like Gram positive rods– Intermediate = mixed bacterial types– BV = Gardnerella and Mobiluncus on smear

Clue Cell