10-28-09 Gram Negative Bacilli List the five medically important gram negative bacilli emphasized in this lecture

and the diseases they cause (E. coli, V. cholerae, Salmonella spp., P. aeruginosa, Haemophilus influenza). Explain their modes of transmission, and list their virulence factors. Gram negative bacteria can be classified based on 1. location a. enteric tract: Escherichia, Salmonella, Vibrio, Pseudomonas b. respiratory tract: Haemophilus c. animal sources 2. oxygen requirements a. strictly aerobic: Pseudomonas b. facultatively anaerobic: Escherichia, Salmonella, Vibrio, Haemophilus c. anaerobic The enteric bacteria Enterobacteriaceae: includes Escherichia coli and Salmonella spp. All facultative anaerobes All glucose fermenters All oxidase negative All nitrate reducers (for energy generation) Common pathogenic features: All part of the normal flora of the intestinal tract Pathogenicity usually results from acquisition of virulence factors from a pathogenicity island (PI); however, it can also arise from normal flora strains via trauma Antigenic structures (serogrouping) O-Antigen Polysaccharide core component of LPS endotoxin (recall that lipid A is the domain thats actually responsible for LPS toxicity) Over 1500 serotypes for Salmonella, 173 for E coli K-Antigen Capsular polysaccharide Virulence factor for bacteremia in E. coli Called Vi (virulent) antigen in Salmonella typhi H-Antigen Flagella Both E. coli and Salmonella are flagellated Salmonella can reversibly alternate between to types of HAntigens to evade immune response E. coli Gram negative bacilli ferments lactose (lac+) UNLIKE Salmonella!!

Classified into serogroup by O, K, and H Antigens (i.e. O157:H7 is transmitted in Jack-In-The-Box undercooked meat; O155 and O111 cause neonatal diarrhea) Transmission Found in the GI tract; transmitted endogenously and exogenously Diseases caused: Travelers diarrhea (gastroenteritis), UTIs, neonatal meningitis, bacteremia There are six groups of E. coli that cause gastroenteritis ETEC (enterotoxigenic): stimulates hypersecretion of fluids EIEC (enteroinvasive): destruction of epithelium lining the colon EPEC (enteropathogenic): destruction of epithelium lining the small intestine EAEC (enteroaggregative): aggregation preventing absorption across intestinal epithelium EHEC (enterohemorrhagic): Shiga toxin; may progress to HUS (hemolytic uremic syndrome) and HC (hemorrhagic colitis) DAEC (diffusely adherent): tissue invasion causes watery diarrhea; death associated factor (DAF) is responsible E. coli is the most common cause of bacteremia, making up 45% of cases Virulence factors Adhesins (pili and fimbriae)UTI K-1 antigenneonatal meningitis K-1 antigen is a homopolymer of sialic acid that inibhits phagocytosis and complement Toxins, adhesins, invasive factors that are all plasma encodedgastroenteritis K antigenbacteremia (by decreasing the binding of Abs and complement) Diagnosis ID of species by culture followed by biochemical tests (via fast automoated systems) ID serotypes Use EMB agar to differentiate E. coli from Shigella spp. And other gram (-)s (both appear colorless in normal culture) E. coli takes on a purple color and develops a metallic sheen

Treatment UTIfluoroquinolones, TMP-SMX Neonatal meningitis3rd generation cephalosporin, but antibiotic susceptibility testing is required Gastroenteritissupportive treatment (fluids + electrolytes + glucose)

Bacteremia3rd generation cephalosporin, but antibiotic susceptibility testing required (more so because E. coli bacteremias are usually hospital acquired) Salmonella spp. gram negative bacilli facultative anaerobe 2463 different serotypes Transmission Animal reservoirs including eggs and poultry Transmission is by ingestion of contaminated food and water Exception: Salmonella typhi and Salmonella paratyphi are exclusively human pathogens; transmission of these occurs via food and water contaminated by infected humans Three survival mechanisms: Inhibition of phagosome-lysosome fusion Escape from phagolysosome Replication within the phagolysosome (Salmonella) Diseases caused Gastroenteritis (non-typhoid Salmonella): caused by invasion of epithelial and subepithelial tissues of the small and large intestines; penetration of the lamina propria causes diarrhea Typhoid fever (S. typhi and S. paratyphi): invasion and replication in M cells of Peyers patches, followed by bacteremic spread to liver, gall bladder and spleen Septicemia (rare; greater incidence with sickle cell anemia) Virulence factors Invasions: adherence factors and adhesins Catalase and superoxide dismutase: fighting of ROSes allow for intracellular replication Acid tolerance response (ATR) gene: protection from stomach acid and survival in phagolysosome Carrier state and excretion in feces Diagnosis ID pathogen via culture + biochemical test (BMI or MacConkey, TSI)produces H2S ID serotype (which tells us the species as well as the epidemiologic significance) Treatment S. typhi and S. paratyphi require antibiotic treatment, with fluoroquinones, chloramphenicol, or cephalosporin; antibiotic susceptibility test should be performed first There is a vaccine for S. typhi Enteritis is self-limiting and does not require an antibiotic V. cholerae Gram negative comma shaped bacilli Have single polar flagellum

Facultative anaerobe Transmission Sourced from marine environments, halophilic (requires salt environments); more specifically, found in shellfish, shrimp and oysters Alternative source is water and food with fecal contamination from infected people

Diseases Caused Cholera: severe rice water diarrhea NO abdominal pain Virulence Factors Cholera toxin: hypersecretion of water and electrolytes due to cAMP accumulation Mucinase adhesion factor Siderophores: iron sequestration Virulent agents in CTX phage that are transducible through sex pili Diagnosis ID via culture followed by biochemical tests Colorless on MacConkey Oxidase positive (aerobic) TSI (triple sugar iron): acid slant and butt without H2S or gas production Treatment Empiric choice is antibiotics: tetracycline for adults, furazolidone for pregnant women, and TMP-SMX for children Supportive therapy is also needed for cholera: fluid/electrolyte replacement with glucose Preventive measures include a clean water/food supply and use of tetracycline for prevention in close contact (does not prevent spread) Three vaccines available: killed (short-acting), toxoid, and live attenuated Pseudomonas aeruginosa Gram negative bacilli resembling enterobacteriaceae Secretes a green pigment aerobic Flagella and capsule Non-fermenters Obligate aerobes Oxidase positive Transmission Seen in nosocomial infections,, but can also be community acquired Diseases Caused Seen in nosocomial infections

Bacteremia and endocarditis UTI Pulmonary infections Skin infections at burn + surgical sites Can also be community acquired Recurrent pulmonary infections w/ CF patients Ear infections Skin and soft tissue infections (SSTIs) i.e. diabetic foot and folliculitis Virulence Factors Adhesion factors: pili, neuraminidase Flagellum Toxins (has exotoxin A similar to diphtheria toxin; disrupts mRNA translation): increases tissue damage, invasion, and immune suppression Pigments (pyocyanin: actually destroys tissue and creates bluecolored pus): oxidative effects causing tissue damage and interference with ciliary function Enzymes exoenzyme s: ADP ribosylase causes tissue damage and dissemination; utilizes a type III secretion system with direct injection into host cells elastase: protease destroys elastin, collagen, fibrin, IgG, IgA (thus interfering with complement), while lysozyme degrades tissue and suppresses immunity phospholipase c: hemolysin breaks down lipids and lecithin; tissue invasion Biofilm formation Diagnosis ID pathogen with culture Simple non-fastidious growth requirements Characteristic colonies Blue-green pigment on ordinary agar and fruity odor Biochemical reactions Non-lactose fermenter Metallic sheen of growth on TSI Treatment Avoid nosocomial infections: handwashing and isolation of patients with MDR strains Due to P. aeruginosas alarming trend toward antibiotic resistance, a combo of antibiotics must be used: piperacillin + aminoglycoside or quinolone Susceptibility testing required, of course! Haemophilus influenzae Gram negative pleomorphic bacilli Facultative anaerobes

Transmission Human reservoir only: present on mucus membranes and less so in GI and GU tracts Consists largely of non-encapsulated H. influenzae and H. parainfluenzae Exogenous transmission of H. influenzae Sexual transmission of H. ducreyi (fyi) Diseases caused Encapsulated form causes meningitis Nonencapsulated form causes pneumonia Otitis media Sinusitis Epiglottitis H. ducreyi causes chanchroids/STDs Virulence factors Polysaccharide capsule containing PRP (polyribitol phosphate) Serotype b (HIB) is most pathogenic and is responsible for 95% of H. influenzae infections Adhesins: allow for adherence to oropharynx LPS: impairs ciliary function and causes epithelial damage IgA protease Diagnosis Fastidious growth on chocolate agar (enriched with X and V factors) X factor: protoporphyrin IX (heme precursor) V factor: NAD, NADP Treatment 3rd generation cephalosporin Vaccine (highly effective!): PRP conjugated to a protein carrying tetanus or diphtheria toxoid

General points about gram negative bacteria: Gram negative bacilli are major pathogens Pathogenesis is multifactorial Pathogens often employ several virulence properties Host responses All require specific biochemical and serological tests to establish identity; important for treatment and epidemiology; i.e. Gram staining is not enough