Bacterial Pathogens

1. Bacterial Pathogens ENVR 421 Mark D. Sobsey Spring Semester, 2008

2. Some Important Enteric Bacterial Pathogens and Their Sources Bacterium/Group Animal FecesNon-fecal sources Salmonella spp. yes no (except S. typhi) no no Campylobacter spp. yes yes Escherichia coli yes no Helicobacter pyloriunknown unknown Aeromonas hydrophila yes yes Yersinia enterocolitica yes yes Listeria monocytogenes Vibriocholerae yes yes some other Vibrio sp. yes yes Shigella spp. no no

3. Enterobacteriaceae Family • Small gram-negative rods (2-5 by 0.5 microns) • Most motile with peritrichous flagella • Shigella and Klebsiella arenonmotile • Oxidase-negativefacultative anaerobes • Reduce nitrate • Ferment glucose and other carbohydrates • Many genera • Escherichia, Salmonella, Shigella,Klebsiella,Proteus,Enterobacter,Yersinia, etc. • Some strains opportunistic pathogens • Some strains true pathogens • Salmonella, Shigella, Yersinia, some strains of E. coli

4. Distinguishing Properties of All Enterobacteriaceae • Ferment glucose • Reduce nitrates • NO3to NO2 or all the way to N2 • Oxidase negative

5. Distribution of Pathogenicity within Enteric Bacteria E. coli Shigella Salmonella Citrobacter Klebsiella Erwinia Serratia Yersinia Proteus based on this distribution, virulence is the derived state Pathogens have virulence genes not present in non-pathogenic relatives, and this distribution suggests that bacteria evolve to become pathogens by acquiringvirulence determinants

6. Bacterial Toxins • ENDOTOXIN • Integral part of cell wall • Endotoxin is LPS; Lipid A is toxic component • Heat stable • Antigenic • Toxoids cannot be produced • Many effects on host • Fever • activate complement • kinin and clotting factors • Produced by gram-negative bacteria only • EXOTOXIN • Released from the cell before • or after lysis • Protein • Heat labile • Antigenic and immunogenic • Toxoids can be produced • Specific in effect on host • Produced by gram-positive and gram-negative organisms

7. (from Redfield, Nat. Rev. Genet. 2001) Genes for pathogenic traits can get transferred laterally Transduction: via bacteriophage Conjugation: direct contact Transformation: integrating free DNA or plasmids

8. Typical Procaryotic Cell

9. Gram Negative Outer Cell Membrane (Wall) and Lipopolysaccharide Structure

10. Shigellosis - Illness • Persistent diarrhea with frequent and painful passage of stools consisting mostly of blood, mucus and pus • accompanied by fever and stomach cramps. • Blood and mucus in stools are likely signs of shigellosis • Shigella infect, invade cells lining the large intestine (colon) • Cause breaks (ulcers) in mucous membrane lining of intestine • Inflammation and tissue damage • Causes painful straining to pass stools; • can lead to rectal prolapse • Ulcers commonly in the rectum • results in increased production of mucus • loss of blood and serum proteins into intestinal cavity • Result: symptoms of dysentery: blood and mucus in the stool (bloody diarrhea); fever is also common

11. Shigella and Shigellosis • Gram-negative rods; do not ferment lactose • Biochemistry and genetics are similar to E. coli • Fecal-oral transmission • person-to-person, fomites, food, water, ect. • Waterborne and water-washed • Reservoirs: humans and primates • Infectious dose: low; as few as 10 cells to infect • Incubation period: 1 to 7 days; typically, 1-3 days • Duration of illness: • untreated: severe symptoms for about two weeks • Antibiotic treatment shortens illness and prevent spread to others

12. Shigellosis - Epidemiology • Four species of Shigella: flexneri, sonnei, dysenteriae, boydii • Major public health problem in many developing countries • causes about 5 to I0% of childhood diarrhea • up to 25% of all diarrhea-related deaths attributed to Shigella Developing countries: • Sh. flexneri is endemic (always present) in most communities • Sh. dysenteriae type 1 often occurs in an epidemic pattern • organism can be absent for a number of years, then reappear and infect a large proportion of the population. • These two species generally produce the most severe illness. Developed countries: • Sh. sonnei is the most common and is the least virulent • Sh. boydii causes disease of intermediate severity • Least common, except in the Indian sub-continent

13. Shigellosis - Epidemiology • Worldwide distribution; infections occur throughout year • Mostly in children aged under five • Rates of infection are highest where sanitation is poor • As few as 10 cell can initiate infection • Transmission influenced by: • nutritional status • environmental factors affecting transmission: • rainfall and temperature • Water-washed as well as Waterborne • Incidence highest in dryer climates in hot and dry weather • Scarcity of water limits handwashing and other hygiene measures to reduce transfer of the few bacteria that cause infection • Incidence in wet climates is often highest in rainy season

14. Shigellosis - Complications • Severe anorexia (loss of appetite) • Hypoproteinaemia (a low concentration of blood protein) • Hyponatraemia (a low concentration of blood sodium) • Dilation of the large intestine • Seizures • Anaemia • Kidney damage • Persistent diarrhea • Weight loss and malnutrition • Rectal prolapse

15. Shigellosis - Treatment, Control and Prevention Treatment: • Continue to eat (feed) • to prevent weight loss and hypoglycemia • include foods rich in potassium (bananas) • Replace fluids • Oral or IV rehydration with fluids and electrolytes • Treat with antibiotics: • trimethoprim/ sulfamethoxazole • ampicillin Prevention and Control: • Handwashing, especially after defacation • Improved sanitation and hygiene • improve water, waste treatment/disposal and food sanitation • reduce overcrowding, etc. • No effective vaccine

16. Escherichia - Taxonomy and Biochemistry Five accepted species: • E. coli, E. blattae, E. fergusonii, E. hermanii and E. vuneris Can be differentiated on the basis of the following reactions: • Indole, citrate, lysine decarboxylase, growth in KCN and malonate utilization Biochemistry: • frequently produce indole (except E. blattae and E. vulneris) • ferment glucose by mixed acid fermentation • do not produce H2S, phenylalaninedeaminase or urease, do not utilize citrate as sole carbon source (except some strains of E. blattae , and E. fergusonii. ) • Most are motile, ferment a variety of carbohydrates and decarboxylae arginine, lysine and/or ornithine.

17. E. coli Genetics and Serology Genetics: • Single, circular DNA molecule, ~4 x 106 base pairs • Molecular weight of 4 x 109 • Total length of about 1.4mm. • Some strains completely sequenced and genomic organization is now being characterized • many of the genes have been mapped. Serology: • E. coli can be subdivided by somatic (cell-wall) or O antigens and flagellar or H antigens. • >160 recognized O types and 55 recognized H types • over 8000 possible OH serotypes. • also capsular (K) and fimbrial antigens.

18. Some Virulence Properties of E. coli Enterotoxins: • at least two types: Heat Stable (ST) and Heat Labile (LT) • Verotoxins or Shiga-like toxins (interchangeable terms): • Verotoxin term is based on the reactions of toxins on Vero cells • at least two families of these toxins: • VT1 (SLT I): similar to Siga-toxin (produced by some strains of Shigella dysenteriae) • VT2 (SLT II) which is only about 50% realted Shiga toxin. • Other Toxins: • Cytolethal distending toxin (CLDT), VirCytotoxin, Cytotoxic necrotising factors (CNF), a possible Enteropathogenic E. coli (EPEC) enterotoxin and a possible E. coli Sudden Infant Death Syndrome (SIDS)-toxin. • Haemolysins: • extracellular haemolysin known as alpha-haemolysin (many strains) • cell-associated haemolysin, beta-haemolysin, (some strains) • enterohaemolysin: extracellular; Enterohaemorrhagic E. coli (EHEC)

19. Virulence Properties of E. coli • Fimbriae: CFAI/CFAII, Type 1 fimbriae, P fimbriae, S fimbriae • Most important: K88, K99 and CFA fimbriae associated with enterotoxigenic E. coli (ETEC). • p-fimbriae: associated with urinary tract pathogens. • E. coli also produce common fimbriae not associated with virulence. • Adhesins: • Intimin: non-fimbrial adhesin; causes the intimate association with target cells in enteropathogenic and enterohaemorrhagic E. coli . • Associated with the 'attachment and effacement' phenomenon • Causes destruction of the intestinal surface cells. • Other outer membrane proteins can act as adhesins.

20. Pathogenic E. coli Enteric Infections: • Enteroadherent E. coli (EAEC) • Enteroaggregative E. coli (EAggEC) • Enterohaemorrhagic E. coli (EHEC) • Enteroinvasive E.coli (EIEC) • Enteropathogenic E. coli (EPEC) • Enterotoxigenic E. coli (ETEC) Extraintestinal Infections: • Uropathogenic E. coli (UPEC): urinary tract infections • Neonatal Menigitis E. coli (NMEC).

21. Enterohemorrhagic E. coli Harbor genes for one or more of the virulence attributes known to associated with the EHEC. • Shiga toxin(s) • Adherence factor(s) • Enterohemolysin • Somatic antigens characteristic of many EHEC serogroups, such as O111 or O157. • An E. coli must cell carry a sufficient number of such genes to cause disease. • Magnitude of exposure or size of infectious dose is also important. • Dose is very small in comparison with those for most other enteric pathogens; a few bacteria per dose has high probability of causing infection and illness

22. Enteropathogenic E. coli • Cause infantile gastroenteritis. • Certain serotypes and serogroups are associated with infantile diarrhea. • Infantile gastroenteritis with dehydration is an important problem. • EPEC have declined in the developed world as major causes of infantile diarrhea • EPEC remain very important in the developing world. • EPEC adhere to the intestinal mucosa to produce a characteristic "attaching and effacing" lesion in the brush border microvillous membrane.

23. Enterotoxigenic E. coli • Major cause of travellers' diarrhea (Montezuma’s revenge, Delhi belly, Aztec two-step, etc.) and of diarrhea in children in the developing world • Produce an enterotoxin similar to cholera toxin. • They are involved with a condition known as "Non-Vibrio cholerae cholera-like diarrhoea". • Produce one or both of two enterotoxins: • heat stable enterotoxin (ST) : survives boiling for 30 minures • heat labile enterotoxin (LT): does not survive such boiling • LT response is sensitive to acid pH; ST was not. • LT is closely related to choleragen (CT) the enterotoxin of V. cholerae. • Most ETEC isolated from humans produce colonization factor antigens which are human specific fimbrial antigens • also a common cause of diarrhea in young animals.

24. Enteroinvasive E. coli (EIEC) • Cause positive reaction in the Sereny test (ability to cause keratoconjunctivitis in guinea pig eyes). • a characteristic shared with strains of Shigella • By DNA probes the invasiveness plasmids of both E. coli and Shigella are identical. • 120-140 mD invasiveness plasmids encode all the genes necessary for the virulence of the EIEC. • Many EHEC are non-motile and anaerogenic. • Account for only a small proportion of diarrhea in non-tropical countries • but, cause high proportions of illness and death, mostly in warmer seasons • Also important causes of dysentery-like diarrhea in tropical countries.

25. Enteroadherent E. coli (EAEC) • Different patterns of adherence to cultured epithelial cells. • Localized adherent E. coli (LAEC) by some serotypes • form adherent microcolonies on HEp-2 cells • associated with acute, non-bloody diarrhoea in children. • Diffusely adhering E. coli (DAEC) • a cause of diarrhea in some studies

26. Enteroaggregative E. coli (EAggEC) • 3rd type of adherence: enteroaggregative adherence • Bacteria align in parallel rows to cells or glass ('stacked brick-like’). • persistent childhood diarrhea in South America and India • Illness lasts >14 days. • Produce a heat-labile toxin antigenically related hemolysin but not hemolytic • Produce plasmid-encoded heat stable toxin (EAST1) unrelated to the heat stable enterotoxin of ETEC.

27. Uropathogenic E. coli (UPEC) • Common cause of urinary tract infections (UTI). • Severity: from asymtomatic to bacteriuria, cystitis and pyelonephritis. • Women more frequently affected than men. • Same serotypes are found in feces and urine of patients, but, UPEC have virulence factors which enhance their ability to cause infection. • Only some O groups cause UTI • Only some K antigens cause UPEC • Certain pili (fimbrae), the p-pili, are an important virulence factor • bind to the P-antigen, a blood grouping antigen • bind more to uroepithelial cells of persons with the P or P2 phenotype • other adhesins may also be involved. • Production of alpha-haemolysin • Production of aerobactin • Several virulence factors enhance an E. coli's ability to cause UTI • an infecting strain may only express some of them to cause infection.

28. Neonatal Meningitis E. coli (NMEC) • Neonatal menigitis in about 1/2500 live births. • Up to 80% of cases of neonatal menigitis are due to E. coli. • ~80% of the isolates possess the K1 capsular antigen. • masks the underlying structures of the bacterial cell surface, preventing specific antibody responses and the activation of the alternate complement system from being activated. • poor immunogen, maybe due to it resembling extracellular matrix proteins. • serotypes isolated from meningitis cases often found in maternal feces; probable source; infection occurs at birth. • Reasons for susceptibility of neonates to NMEC is not known.

29. Salmonella and Salmonellosis • In Enterobacteriaceae family • Gram-negative bacilli; facultative and flagellated (motile). • 3 major antigens: • "H" or flagellar antigen (phase 1 & 2) • "O" or somatic antigen (part of the LPS moiety) • "Vi" or capsular antigen (called "K" in other Enterobacteriaceae). • Posess LPS endotoxin characteristic of Gram-negative bacteria • Evoke fever and can activate complement, kinin and clotting factors.

30. Salmonella Illness Gastroenteritis or salmonellosis: less severe disease • Contaminated food or water • Often from contaminated poultry (turkeys and chickens). • S. enteritidis or S. choleraesuis: most commonly isolated species Enteric fever: more severe disease • Transmitted from person to person • S. typhi (no animal reservoirs) • Reservoirs: • Contaminated food or water with human feces • asymptomatic human carrier • “Typhoid Mary”, a food service worker who infected many patrons

31. Salmonella enteritidis Salmonella typhi

32. Salmonella typhimurium DT 104 • A commonly reported phage type of S. typhimurium • Resistant to many antibiotics: ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. • also some additionally resistant to trimethoprim and to quinolone antibiotics such as ciprofloxacin • Antibiotic resistance emerged as a result of widespread use both prophylactically and therapeutically. • Effective antibiotics to control its spread is severely limited. • Associated with cattle, pigs and chickens and other animals. • May infect animals without showing any signs of illness. • Spreads from farm to farm by raw water and other sources • Once a farm becomes contaminated it is hard to eradicate • Survives in dry as well as in wet environments • Illness is more difficult to treat than from other salmonellae • Has a higher mortality rate

33. Campylobacters • Gram-negative • Curved (spiral) rod • about 1.5-3 microns • motile via polar flagella • Microaerophilic • Prefer high CO2 • C. jejuni: • Highly prevalent • Relatively fragile • But, persistent in feces and water • Adapted to birds

34. Campylobacteriosis • Due to Campylobacter jejuni or sometimes C. coli. • Normal intestinal flora of many warm-blooded animals • chickens and turkeys; also in raw water and raw milk. • Illness in sheep (abortion), dogs and cats (gastroenteritis) • Causes illness in humans • Cause 5-11% of all diarrhea cases in the United States. • Symptoms: from mild to severe: • bloody diarrhoea is the most characteristic symptom) • also fever, nausea, abdominal cramps and (seldom) vomiting • duration of illness usually 2-10 days • abdominal cramps, may recur for up to 3 months after infection • Complications such as septicaemia, may arise. • The infectious dose may be very low, i.e. 100s of cells • Infants, young children and debilitated people at highest risk

35. Campylobacteriosis • Campylobacter enteritis or gastroenteritis. • Illness is watery or sticky diarrhea • Contain blood and fecal leukocytes. • Other symptoms: • Fever • Abdominal pain • Nausea • Headache • Muscle pain.

36. Illness typically occurs 2-5 days after ingestion of contaminated food or water Generally lasts 7-10 days. Most infections are self-limiting; not treated with antibiotics C. jejuni Infectious at low dose 400-500 cells have high probability of causing illness Major cause of bacterial diarrhea in the U.S An estimated 2.1 - 2.4 million cases per year Most retail chicken is contaminated with C. jejuni Most chickens in commercial operations are colonized by 4 weeks of age Campylobacteriosis Epidemiology

37. Campylobacteriosis Epidemiology, Continued • C. jejuni infection is most frequent in children under 5 years and young adults (15-29) • Transmission is usually not person-to-person • but it can occur if the infected person is a child or is producing a large volume of diarrhea • Illness can occur in single, sporadic cases • Also occurs as outbreaks • Hyperendemic in some countries (New Zealand) • Increased human infections caused by antimicrobial-resistant strains of C. jejuni • Makes clinical management of cases more difficult. • Antimicrobial-resistant enteric infection rates are highest in the developing world • Antimicrobial use in humans and animals is relatively unrestricted

38. C. jejuni pathogenicity • Many likely virulence factors • Suspected determinants of pathogenicity: • chemotaxis and motility (flagella) for attachment, invasion and colonization • After colonization, other possible virulence determinants are: iron acquisition, host cell invasion, toxin production, inflammation and and epithelial cell lysis

39. Complications and Sequelae of Campylobacteriosis: Guillain-Barré Syndrome (GBS) • Develop a rare disease of the nervous system beginning several weeks after the diarrheal illness. • A demyelinating disorder resulting in acute neuromuscular paralysis. • Also called Acute Post Infective Polyneuritis • Person's immune system is "triggered" to attack the body's own nerves • Peripheral nerves show the presence of mononuclear cells, demyelination, and macrophages. • Pathology is most severe in spinal roots. • Can lead to paralysis lasting several weeks; usually requires intensive care • About 1 per 1000 reported campylobacteriosis cases leads to GBS. • Perhaps 40% of Guillain-Barrsyndrome cases in this country may be triggered by campylobacteriosis.

40. Ang CW, Jacobs BC, Laman JD. (2004) The Guillain-Barre Syndrome: a true case of molecular mimicry. Trends in Immunology. 25(2):61-66 “Carbohydrate mimicry between human ganglioside GM1 and Campylobacter jejuni lipooligosaccharide causes Guillain–Barre ́ syndrome” Yuki, N. et al (2004) PNAS Vol. 101 No. 31 p. 11404-11409.

41. Immune system destroys the protective covering of the peripheral nerves (myelin sheath) disables the nerves from transmitting signals to the muscles. the primary cause of the symptoms elicited by GBS. Begins with paresthesias in the toes and fingertips, followed by rapidly advancing weakness and areflexia Weakness reaches a plateau within 4 weeks, then recovery begins. Some cases are fulminant, evolving in one or two days muscles become unable to respond to nerve commands Weakness, numbness or paralysis may occur. The brain also receives fewer sensory signals, resulting in the inability to feel heat, cold, pain and other sensations. Many patients are completely paralyzed and unable to breathe. Approximately 5% of patients die from respiratory paralysis, cardiac arrest, sepsis, and other complications. 10% of those who recover have residual weakness GBS

42. Campylobacteriosis Prevention and Control • Proper food handling and kitchen hygiene practices • Thorough cooking of all poultry and other foods of animal origin • Reduction of endemicity in food animals like poultry and sheep • Probiotics: colonize the intestinal tract of the animals with other bacteria to prevent or overcome colonization by campylobacters

43. Some Readings and References • http://www.cdc.gov/ncidod/eid/vol5no1/altekruse.htm • http://www.cdc.gov/ncidod/dbmd/diseaseinfo/campylobacter_g.htm • http://www.cfsan.fda.gov/~mow/chap4.html • http://www.mayoclinic.com/health/guillain-barre-syndrome/DS00413/DSECTION=1