GENERAL BACTERIOLOGY

ANTIMICROBIAL SUSCEPTIBILITY TESTING:

Medium used: Mueller Hinton agar/broth

 Prepare Standard inoculum of test bacterium with turbidity 0.5 McF

MC used method: Disc diffusion method---- >

o Kirby Bauer (MC) ---- > Comparison done using standardised tables o Stoke's method ----- > Along with test organism, control organism also put

Less commonly used method but BEST METHOD: As gives exact estimate of MIC is Dilution method: Broth (can be Macrobroth or Microbroth) & agar

E (Epsilometer) test:-Combination of Disc diffusion method and Dilution method

MICROBROTH DILUTION METHOD MACROBROTH DILUTION METHOD

AGAR DILUTION METHOD

STOKE’S METHOD KIRBY BAUER METHOD

EPSILOMETER TEST

BACTERIAL GROWTH CURVE:

LAG PHASE:

o Stage of adaptation o No replication takes place o Metabolically active

LOG PHASE:

o Stage of active replication o Increase in viable count o Metabolically most active

STATIONARY PHASE:

o Gradual depletion of nutrients o Bacteria multiplying = Bacteria dying o Exotoxins formed o Spores formed

DECLINE PHASE:

o Viable count decreasing o Total count constant BACTERIAL CELL:

o Capsule/slime may or may not be formed o Cell wall contains peptidoglycan/ muramic acid o CM doesn’t contain sterols o 70S ribosomes o Mesosomes--- Invaginations of plasma membrane Respiratory chain enzymes present o Pili helps in adhesion o Flagella helps in motility

o Unique amino acid in bacterial cell wall----- > Diaminopimelic acid

o Polymers of NAG and NAM o Linked by peptides o Teichoic acid present of 2 types

GRAM POSITIVE CELL WALL

o Periplasmic space contains two layers of murein monomers o Porins present o LPS/ endotoxin present, released on lysis of cell----recognised by TLR-4 on human cells

GRAM NEGATIVE CELL WALL

Mechanism of β lactams:

“β lactams bind to transpeptidase(PBP) and inhibit crosslinking leading to lysis of cell.”

Mechanism of Vancomycin:

“Vancomycin binds to D-ala- D-ala terminal of cell wall precursors and blocks action of transglycolase---leading to no crosslinking.”

o Yersinia pestis o S.aureus o Bacteroides , Bordetella o Pneumococcus o Haemophilus influenza o Vibrio parahemolyticus o Klebsiella pneumoniae o Bacillus anthracis (polypeptide capsule) o Meningococcus o Clostridium perfringens, Cryptococcus neoformans

Demonstrated by Quellung reaction- >

Swelling of capsule in presence of AntiCapsular Ab

o Bacteriophages can follow either lytic or lysogenic cycle.

o In lysogenic bacterium, integrated phage DNA is called prophage. o Induction of Prophage: Disintegration of phage DNA spontaneously or d/t triggers o A bacterium can never be in both cycles simultaneously o Temperate phages first enters lysogenic cycle and then lytic cycle. o Eg. Gamma phage of E.coli

GENE TRANSFER: TRANSFORMATION VIA PHAGES CONJUGATION Picking up DNA fragments Phage carrying bacterial genes First demonstrated in E.coli K12 through cell wall infects another bacterium- strain Eg. Pneumococcus Transduction Through sex pilus encoded by -MC form of gene transfer in plasmid genes bacteria Horizontal transfer

Phage encoding genes expressed by lysogenic bacterium- Lysogenic conversion Can only be mediated by temperate phage LYSOGENIC CONVERSION: o Prophage encoded genes o Ability of Salmonella to express various antigens o Ability to produce Toxins---Phage mediated toxins DT, CT, Verotoxin of EHEC, Botulinum toxin C & D, Pyrogenic toxins A and C, Panton Valentine Leucocidin toxin

ENTEROBACTERIACEAE:

 GN rods  Facultative anaerobes  Motile by Peritrichous flagella except o Klebsiella o Atypical E. coli o Shigella o S. gallinarum-pullorum o Yersinia pestis  Catalase +ve except S. dysenteriae I  Oxidase -ve  Nitrate reduction +ve  All Ferment glucose (As facultative anaerobe-can utilize glucose both aerobically and anaerobically): aerogenic (acid with gas)/ anaerogenic(only acid)  NON-FASTIDIOUS Selective Media: Mac Conkey Agar-either LF/NLF

Catalase negative- STASH o STreptococcus o All Anaerobes o S. dysenteriae type 1

CATALASE TEST Oxidase negative-CESS o Corynebacterium o Enterobacteriaceae o Staphylococcus o Streptococcus

OXIDASE TEST

Aerogenic bacteria SEEK-(gas bubble)

o Salmonella o E. coli o Enterobacter o Klebsiella

SUGAR FERMENTATION TEST

(Arrow shows Durham’s tube)

 TUBE 1: Aerogenic  TUBE 2: Anaerogenic  TUBE 3: Uninoculated sugar solution

Lactose fermenting pink colonies:

 E. coli  Klebsiella  Enterobacter  Citrobacter

o

MAC CONKEY AGAR: LF

Non Lactose fermenting pale colonies:

 Salmonella  Shigella  Proteus  Yersinia

MAC CONKEY AGAR: NLF

SHIGELLA:

 NM, NC  4 spp. (Serotyping based on O Ag)  Anaerogenic  IMViC -+--  NLF  S. dysenteriae: o only Non-Mannitol Fermenting species o Produces most severe disease o Type l- only serotype to produce Shiga toxin—so called SHIGA’S bacillus  S. flexneri: MC in India  S. sonnei: o LLF o only 1 serotype o MC in world o Mildest shigellosis  S. boydii: Rarely reported species in world

SHIGELLOSIS:  Most communicable bacterial diarrhea  ID50---->10-100  Virulence factor: Plasmid encoded-T3SS & Ipa A-D, VirA — cause Actin polymerisation  Basolateral invasion of colon & rectal mucosa  I.P: 2 days  Invasive Diarrhoea, fever, abd pain; dysentery, tenesmus.  Shiga toxin-inhibits protein syn in endothelial cells  S.dysenteriae Type I---MC cause of HUS In India (EHEC in world)  Complications: Toxic megacolon, Reactive arthritis

 DIAGNOSIS: o Stool microscopy: lots of pus cells seen o Detection of Ag: DFAT, ELISA o Culture on selective media---MOST SPECIFIC o Sereny test---to prove invasive nature, demonstrate keratoconjunctivitis in expt animal o NAATS--- MOST SENSITIVE

 Rx: o Ciprofloxacin x 5 D o If EMPIRICAL Rx: Ceftriaxone X 5D Cefixime x 5 D o Resistance reported to FQ, Macrolides, TMP-SMX

E.coli:

 Motile except Atypical E.coli  Serotyping done on basis of O K & H Ag  Aerogenic except Atypical E.coli  IMVIC++--  LF on MCA  Normal GI flora---but if enter extraintestinal site and have acquired special virulence factors can lead to infections  Single MC cause of UTI----> UPEC  UPEC Virulence Factors: Type 1 fimbriae, P fimbriae  MC cause of neonatal Meningitis: NMEC-K12 capsule DIARRHOEAGENIC E.COLI: Never normal flora

EIEC EPEC Atypical E. coli o MCC of diarrhoea in infants---typical EPEC  NM o Diarrhea regardless of age---atypical EPEC  NLF  Anaerogenic VIRULENCE FACTORS: o EAF plasmid (tEPEC) Also Cause Bacillary dysentery but o LEE plasmid infective dose very high Both lead to Attachment-Effacement effect (ID--- >104-105) o Watery non-bloody diarrhoea

o DIAGNOSIS:  Isolating them from stool  "Localized adherence" on Hep2 adherence assay  NAAT

ETEC MCC Of Diarrhoea ---- Pediatric & adult MCC Of Traveller's Diarrhoea

VIRULENCE FACTORS(plasmid coded): Heterogenous adhesins:- Colonization factor Ag TOXINS: LT (acts by increasing cAMP) & ST (acts by increasing cGMP)

ID50--- >109 (only food and water associated) Acute non bloody diarrhea

DIAGNOSIS: PCR for toxin genes

STEC/EHEC  Shiga toxin/ Verotoxin  Zoonoses (beef animal exposure)  ID50---- > 10-100 bacteria only  VIRULENCE FACTORS: o LEE plasmid----- >A-E effect o Phage mediated Verotoxin (Stx1 and Stx2 variant)---- > inhibit protein syn  MCC of HUS in the world  I.P:-3-8 Days  Bloody diarrhoea, HUS (fever-nt )  Most dangerous strain : E.coli O157:H7  On Sorbitol Mac---- > E.coli O157:H7 produces pale colonies unlike others  NAATS to detect verotoxin in stool directly  Never give antibiotics as they worsen the disease

EAEC  MC cause of persistent diarrhoea  Can cause Traveller's diarrhoea  Plasmid mediated virulence factors: AAF, EAST-1(acts by increasing cGMP)  Acute non bloody diarrhoea  DIAGNOSIS: "stacked brick" adherence on Hep 2 adherence assay

DAEC  Pediatric diarrhea in somewhat older children  Acute non bloody diarrhea  Diffused/Random/ individual adherence on Hep-2 adherence assay

AIEC Possible pathobiont in multifactorial etiology of Crohn's disease A-E EFFECT ON MUCOSAL CELLS Klebsiella pneumonia subsp. pneumoniae:

 Non-motile  capsulated  O & K typing, No H type as no flagella  Aerogenic  IMViC --++  Urease +ve  LF on MCA  Mucoid colonies due to thick capsule  Normal GI, nasopharynx flora  Important Community Acquired Infection : Friedländer disease: Severe type of pneumonia Alcoholism history Other risk factors: Diabetics, COPD, Chronic renal failure Upper lobe involvement Abscess formation  Currant jelly sputum  Bulging fissure sign on X-ray  Can cause UTIs, pneumonia, bacteremia, liver abscess, intraabdominal abscess, splenic abscess  All Resistant to Ampicillin (chromo. encoded β lactamase); MDR plasmids

K. granulomatis

 Causes Granuloma inguinale(Donovanosis)

K pneumoniae rhinoscleromatis

 Causes CGD called rhinoscleroma

K pneumoniae ozaenae

 Causes Atrophic rhinitis (ozaenae) MUCOID COLONIES ON BLOOD AGAR DUE TO THICK CAPSULE

Yersinia pestis:

 Category A Bioterrorism agent  NM  Capsulated  bipolar staining---safety pin appearance  pH range--- 5-9.6  temp range---2-45 degree Celsius, optimum temp-27 degree celsius  Anaerogenic  IMVIC-+--  Urease -ve  NLF  Stalactite growth on Ghee broth  Selective media: CIN  Reservoir: Rodents  Vector: Rat flea

TYPES OF PLAGUE:

BUBONIC PNEUMONIC SEPTICEMIC  Due to bite of rat  Due to inhalation of  Generally secondary to flea bacteria other two  Associated with  80-90% mortality  Symptoms of severe bubos rates endotoxemia  If untreated, 50%  If untreated,go to go to septicemic septicemic plague plague

DIAGNOSIS:

 Wayson stain---Safety pin appearance  Ag detection---DFAT  NAAT  Culture---only done in special labs  Serology---demonstrate 4 fold increase in Ab one week after symptoms start

TREATMENT:

 DOC : Streptomycin BD x 7 D  ALTERNATIVES: Gentamicin OR Doxycycline OR Tetracycline OR Levofloxacin OR Chloramphenicol o Droplet precautions TO BE FOLLOWED for pneumonic plague

OTHER ORGANISMS SHOWING BIPOLAR STAINING:

o Y.pestis o K.granulomatis o B.mallei o B.pseudomallei

Salmonella:

o Motile except S.gallinarum pullorum o Non-capsulated except S.typhi, S.paratyphi C, S.dublin (Vi Ag) o O, H & Vi typing----Kauffman and White scheme---Not species, but serotypes o Aerogenic except S.typhi o IMVIC-+-+ except S.typhi -+-- o urease- o H2S+ except paratyphi A o Enrichment medium: Sel F, TTB, GN broth. o Transport medium: Cary Blair o Selective medium: DCA, XLD, SSA, HEA, WBBS(black colonies due to H2S +ve) o Manifestations milder in paratyphoid

ENTERIC FEVER:

o ID50: 103-106 o I.P-1-2 weeks o Stepladder pyrexia o Pulse-temp dissociation (Pyrexia + Bradycardia)/ Faget’s sign o Rose spots o MC complication in untreated: GI bleed (10-15%) o 2nd MC complication: Perforation

DIAGNOSIS:

o Gold std----culture o BM culture (most sensitive) o Blood culture (more commonly used)----done on Casteneda’s medium o Widal test---tube agglutination test H Ab increase---tells which serotype, O Ab increase---recent infection

TREATMENT: o Preferred t/t- Oflox/Ciproflox x 5-14 D. o Empirical treatment (where decreased FQ susceptibility is common)- Ceftriaxone 14D or Azithromycin x 5-7 D o FQ resistant strains: NARST o Carriers: 3 types- Convalescent, Temporary, Chronic carriers Carrier Rx using FQ or Cotrimoxazole

Non-typhoidal salmonella o Zoonoses o Cause Invasive enteritis I.P---- > 8-24 hrs or sometimes 8-48 hrs o MC serotypes to cause this: S. Typhimurium S. Enteritidis o Symptomatic Rx reqd o In some children, old people, immunodeficient people-can progress to bacteremia---meningitis or osteomyelitis o MCC of osteomyelitis in SCA or Chronic Hemolytic Anemia patients: NTS o MC NTS to be associated with bacterimias are: S. Cholerasuis S. Dublin o Rx- 3rd gen Cephalosporins

PSEUDOMONADACEAE:

Pseudomonas aeruginosa:

o Motile by monotrichous flagella o Slime + (alginate) o Strict aerobe o Only species to grow at 42 °C o But optimum temp: 35-37°C o Catalase + o Oxidase + o Oxidative utilization of sugars o Nitrate + o Non fastidious Pigments----Pyocyanin, Pyoverdin, Pyorubin, Pyomelanin o MCA — NLF o BA-β hemolytic Mucoid colonies o Fruity smell o Selective media: Cetrimide agar o Virulence Factors: LPS, Pili, Flagellum Type IV pili mediate TWITCHING motility independent of flagella Same pili present on Neisseria species Type I, II, III secretion systems Exotoxin A – inhibits protein synthesis Exoenzyme S, T, U, Y , Elastase, phospholipase Quorum Sensing molecules o Envt. Saprophyte (moist envt)

COMMUNITY INFECTIONS NOSOCOMIAL INFECTIONS MCC of Corneal ulcers in contact lens Burn infections users Blood Stream Infections Simple otitis externa (Swimmer's ear) Ecthyma gangrenosum

Malignant otitis externa MC GN causing HAP & VAP (classically seen in uncontrolled Diabetes) Green nail syndrome Shanghai fever Post cathetrization UTI/Post Jacuzzi syndrome/ hot tub folliculitis, lumbar puncture meningitis Hot Hand-Foot 7 Syndrome

RESISTANCE: MDR

o Reduced entry GREEN NAIL SYNDROME JACUZZI SYNDROME o Efflux pumps o Inactivating enzymes

Novel drugs for systemic infections by MDR strains :

o Ceftolozane-tazobactam o Ceftazidime-avibactam

ECTHYMA GANGRENOSUM HOT HAND FOOT SYNDROME Burkholderia cepacia complex:

o Motile o Catalase + o Oxidase + Saprophyte in moist environment o Colonization and infection of LRT in CGD & CF Pulmonary disease with high mortality rates: CEPACIA SYNDROME o DOC: TMP-SMZ o ALTERNATIVES: Ceftazidime, Meropenem, Minocycline o Inherent Resistance to FQ, Polymyxin

Burkholderia pseudomallei:

o Tier 2 agent, BSL-III o Motile, bipolar staining o Catalase + o Oxidase + o Soil & water saprophyte in SEA, China. o Causes: Meliodosis (VIETNAMESE TIME BOMB) Pneumonia/ skin lesion---can lead to septicemia o Reactivation possible o Rx: Intensive Phase: Ceftazidime/ Carbapenem X2 wks. Maintenance phase: Doxycycline or Cotrimoxazole PO for at least (2-3 months)

Burkholderia mallei:

o Tier 2 agent, BSL-III o Non-motile o bipolar staining o Catalase + o Oxidase - o Never found as saprophytes o Found in Equine animals Farcy buds/pipess Equine glanders- Nasal abscesses o Can cause Pneumonia/skin lesion---> septicemia o Rx: Intensive Phase: Ceftazidime/ Carbapenem X2 wks. Maintenance phase: Doxycycline or Cotrimoxazole PO for at least (2-3 months)