Medically Important Gram negative bacilli (Part 1) Dr Ekta Chourasia Lecturer, Microbiology

Representative Gram-negative Bacilli 1. Gram-negative rods a. Family Enterobacteriaceae: Medically important species Escherichia coli, Salmonella typhi, Shigella sp, Klebsiella spp, Proteus spp Yersinia spp. b. Pseudomonas sp. & related organisms c. Bordetella pertussis d. Haemophilus spp 2. Curved rods Vibrio spp Campylobacter spp Helicobacter spp 3.Miscellaneous Legionella spp Chlamydia spp Rickettsia Dr Ekta  Page 2

General Features of Enterobacteriaceae Present in large intestine Gram negative bacteria Aerobic or facultative anaerobic Motile by peritrichate flagella Grow on ordinary media (non fastidious) Ferments glucose with acid & gas or only acid Catalase + ve & oxidase -ve Dr Ekta  Page 3

Classification of Enterobacteriaceae Based on lactose fermentation – oldest method : Lactose fermenters e.g. Escherichia coli, Klebsiella sps Late lactose fermenters e.g. Shigella sonnei Non lactose fermenters e.g Salmonella, Shigella sps other than Shigella sonnei Commensal intestinal bacteria: LF Intestinal pathogens: NLF LF NLF Dr Ekta  Page 4

Escherichia coli Named after Escherich, first to describe colon bacillus Normal flora of the human & animal intestine. Remains viable in the feces for few days. Detection of E. coli in the drinking water – indicates recent pollution with human or animal feces. Dr Ekta  Page 5

Antigenic Structure of Gram –ve Bacteria Three antigens – serotyping of E.coli H – flagellar antigen O – somatic antigen K – capsular antigen Majority do not possess K Ag. Dr Ekta  Page 6

Virulence Factors - Two types of virulence factors: Surface Ags & Toxins Surface Antigens LPS surface O Ag – endotoxic activity Envelope or K Ag – protects against phagocytosis Fimbriae – colonisation, found in strains causing diarrhoea and urinary tract infections Toxins (Exotoxins) – two types Enterotoxins – pathogenesis of diarrhoea - 3 types : LT (heat labile toxin), ST (heat stable toxin) & VT (verocytotoxin or shiga- like toxin) Hemolysins Dr Ekta  Page 7

Heat Labile Toxin (LT) Resembles cholera toxin in its structure, function and mode of action Complex of polypeptide subunits. LT: one subunit of A (action- enzymic), five subunits of B (binding) Dr Ekta  Page 8

Escherichia coli / Vibrio cholerae Heat Labile Toxin (LT) Escherichia coli / Vibrio cholerae Gut lumen Intestinal epithelial cell Dr Ekta  Page 9

Pathogenicity / Clinical Infections Urinary tract infection Diarrhoea Infantile diarrhoea Traveller’s diarrhoea Bloody/ Hemorrhagic diarrhoea Pyogenic infections Wound infection, especially after surgery of lower intestinal tract. Peritonitis. Biliary tract infection. Neonatal meningitis. Septicemia – can lead to fatal conditions like Septic shock Dr Ekta  Page 10

Lab Diagnosis of UTI Specimens Urine Mid stream urine (MSU) Catheter specimen urine (CSU) Supra pubic aspiration (SPA) Microscopy Wet mount Pus cells / hpf Bacteria Gram stain Gram negative bacteria (1bacteria / oil field is significant) Urine Culture To know significant bacteriuria Dr Ekta  Page 11

Lab Diagnosis of E. coli UTI Significant bacteriuria > 105 organism / ml of MSU Culture BA / MAC : LF (flat) CLED medium Identification tests I M Vi C test: + + - - TSI agar Acid, no gas Dr Ekta  Page 12

Diarrheagenic E.coli Enteropathogenic E. coli (EPEC) – infantile diarrhea, nontoxigenic Enterotoxigenic E. coli (ETEC) – traveller’s diarrhea, resembles cholera Enteroinvasive E. coli (EIEC) – bloody diarrhea (blood, mucus & leucocytes with stool) Enterohemorrhagic E. coli (EHEC) or Verocytotoxigenic E. coli (VTEC):- O157:H7 serotype (food poisoning) - Hemorrhagic colitis, Hemolytic uraemic syndrome Enteroaggregative E. coli (EAEC) : “stacked brick” appearance- persistent diarrhea in children Diffusely adherent E. coli (DAEC) Dr Ekta  Page 13

Klebsiella pneumoniae General features Normal gut flora in the intestine Gram negative bacilli (short & plump) Capsulated, non-motile, produces mucoid LF colonies on MAC Pathogenicity Pneumonia: hospital & community acquired Meningitis & enteritis in infants Urinary Tract Infection Septicemia Dr Ekta  Page 14

Proteus Normal gut flora in the intestine Gram negative bacilli, pleomorphic Motile, Non lactose fermenter (NLF) on MAC Swarms on BA, Urease +, H2S + Species P. mirabilis P. vulgaris UTI Pneumonia Wound infections Urease converts urea to NH4 & CO2 causing alkalinization of urine leading to renal calculi (stones) Proteus antigens are used in the Weil - Felix test to diagnose Rickettsial diseases Dr Ekta  Page 15

Shigella Classification – 4 species : biochemical & serological characteristics. - Sh.dysenteriae - Sh.flexneri Non Lactose Fermenter - Sh.boydii - Sh.sonnei - Late Lactose Fermenter Dr Ekta  Page 16

Shigella species- Mannitol fermentation Non Fermentation Fermentation S. dysenteriae - 12 S. flexneri- 6 S. boydii - 18 S. sonnei (Late lactose fermenter) Dr Ekta  Page 17

Epidemiology & Clinical Syndromes Causes Bacillary Dysentery – frequent passage of blood stained, mucopurulent stools. Incubation period: 1-7 days, usually 48 hrs Low Infectious dose: 10-100 bacilli Feco-oral transmission Common in pediatric age group (1-10 years) – leading cause of infantile diarrhea. Sh.dysenteriae type I : most serious form of dysentery. Shigellosis : whole spectrum of disease caused by Shigella. Complication: Hemolytic Uremic Syndrome Dr Ekta  Page 18

Pathogenesis Early stage Second stage Two-stage disease Watery diarrhea attributed to the enterotoxin activity of Shiga toxin in the small intestine Second stage Dysentery due to adherence and tissue invasion of large intestine (cytotoxic activity of Shiga toxin) Fever attributed to neurotoxic activity of toxin Shiga toxin Enterotoxic, neurotoxic and cytotoxic Similar to Shiga-like toxin of Enterohemorrhagic E. coli (EHEC) Dr Ekta  Page 19

Laboratory Diagnosis Treatment Specimen: fresh feces – mucus flakes (buffered glycerol saline – transport medium) Microscopy: Gram–ve, nonmotile bacilli Culture MacConkey agar: NLF colonies Enrichment broth – Selenite F, Gram-ve broth Selective media – Deoxycholate agar (DCA), Salmonella-Shigella (SS) agar, XLD (Xylose Lysine deoxycholate) Slide agglutination with polyvalent & monovalent sera. Treatment Oral rehydration Antibiotics for severe & toxic cases – Nalidixic acid or Norfloxacin. Dr Ekta  Page 20

Salmonella Gut of domestic animals & poultry. Divided into 2 groups : Enteric fever group – typhoid & paratyphoid bacilli. Food poisoning group – usually animal parasites, producing gastroenteritis, septicemia or localized infections. Dr Ekta  Page 21

Pathogenesis Source of infection - Carriers , Cases, Poultry, dairy Transmission - Ingestion of contaminated water or food High infectious dose - 108 CFU Incubation period - 7-14 days Dr Ekta  Page 22

Infection pattern of Salmonella Salmonella are ingested in contaminated food or water Organisms reach the terminal ileum Enteritis Organisms invade the gut wall & cause ulcertion, perforation & hemorrhage Organisms spread to intestinal lymphatics & are phagocytosed by macrophages Organisms disseminate to bones, kidneys, lungs,liver, brain & blood Enteric fever or typhoid fever Dr Ekta  Page 23

Pathogenicity Enteric fever – Typhoid & paratyphoid fever. Clinical features: nausea, vomiting, fever, bradycardia, toxemia, splenomegaly, hepatomegaly, diarhoea alternating with constipation. Septicemia with or without local suppurative lesions. Gastroenteritis Dr Ekta  Page 24

Lab diagnosis of Enteric fever Specimens Blood, Bone marrow, urine, stool, pus, CSF Blood culture BHI broth 1st week Antibody detection (serum) 2nd week Widal test 3rd week Urine culture Use selective & enrichment medium 4th week Stool culture Dr Ekta  Page 25

Morphological & Cultural characteristics Motile, gram negative bacilli Non lactose fermenting (NLF – pale colored) colonies on MacConkey & Deoxycholate citrate agar (DCA). Enrichment broth - Selenite F, Tetrathionate broth Selective media – Wilson & Blair (jet black colonies due to H2S), XLD, SS agar. XLD Dr Ekta  Page 26

Serology - Widal Test Tube agglutination test To detect antibodies in patient serum Test is performed after 2 wks To diagnose Enteric fever Antigens used TO O antigen of S typhi TH H antigen of S typhi AH H antigen of paratyphi A BH H antigen of paratyphi B CH H antigen of paratyphi C O is group specific Enteric fever H is species specific Typhoid or paratyphoid Dr Ekta  Page 27

Prevention & Treatment Detection of Carriers Food handlers & Cooks Repeated stool cultures Vi agglutinins indicates carrier status Vaccines TAB Typhoral Typhim Treatment Ciprofloxacin Dr Ekta  Page 28

Pseudomonas aeruginosa – General characteristics Obligate aerobe small gram-negative rods with a single polar flagellum, produce oxidase & catalase common inhabitant of soil & water (ubiquitous-wide spread) grapelike odor greenish-blue pigment (pyocyanin) resistant to soaps, dyes, quaternary ammonium disinfectants, drugs, drying frequent contaminant of ventilators, IV solutions, anesthesia equipment opportunistic pathogen multidrug resistant Dr Ekta  Page 29

Pathogenicity common cause of nosocomial infections in hosts with burns, neoplastic disease, cystic fibrosis Can cause: pneumonia, UTI, abscesses Septicemia can lead to: endocarditis, meningitis, bronchopneumonia Corneal ulcers from contaminated lens solutions Ear infections (Otitis) “swimmer’s ear” Skin rash (contaminated hot tubs, saunas, swimming pools) Dr Ekta  Page 30