Enterobacteriaceae.

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Presentation transcript:

Enterobacteriaceae

Transmitted by the fecal-oral route Primary pathogens Organisms capable of causing disease in anyone Opportunistic pathogens Organisms that can only cause disease under certain conditions or in certain hosts Shigella Salmonella Yersinia Klebsiella pneumoniae E. coli Senatia Morganella Providencia Enterobacter Proteus

Characteristics shared by all members of family Enterobacteriaceae

G-, non-spore forming rods

Peritrichous flagella -shigella: nonmotile Facultative anaerobe Not fastidious

Active metabolism - All ferment glucose; - All reduce nitrates to nitrites; - All oxidase negative - Lactose fermentation: the key measure to isolate and identify the Enterobacteriaceae +: nonpathogenic -: pathogenic

EMB plate 伊红美兰琼脂:弱选择性培养基,用于肠道菌的选择性分离。蛋白胨提供碳源和氮源;乳糖是大肠菌群可发酵的糖类;磷酸氢二钾是缓冲剂;琼脂是培养基凝固剂;伊红和美蓝是抑菌剂和pH指示剂,可抑制革兰氏阳性菌,在酸性条件下产生沉淀,形成紫黑色菌落或具黑色中心的外围无色透明的菌落。

MacConkey agar 麦康凯琼脂培养基:用于肠道致病菌的选择性分离培养。胰酪蛋白胨、蛋白胨提供氮源、维生素和矿物质;乳糖是大肠菌群可发酵的糖类; 牛胆盐抑制非肠杆菌科细菌;氯化钠维持均衡的渗透压;结晶紫抑制革兰氏阳性菌;中性红是指示剂;琼脂是培养基凝固剂。

Triple sugar iron agar (TSI) 用于肠杆菌科细菌的生化反应筛选。蛋白胨、牛肉粉提供氮源、维生素、矿物质;乳糖、葡萄糖、蔗糖为可发酵糖类,其产酸时通过酚红指示剂测出,酸性呈黄色,碱性呈红色;硫代硫酸钠可被某些细菌还原为硫化氢,与硫酸亚铁中的铁盐生成黑色硫化铁;氯化钠维持均衡的渗透压;琼脂是培养基的凝固剂。

Antigenic structures LPS (endotoxin) – O antigen. Most are motile by peritrichous flagella --H antigens. Capsule – K antigen ( Vi for Salmonella). Cell envelope (wall) various outer membrane proteins. Pili - various antigen types, some encoded by plasmids

Enterobacteriaceae: gastrointestinal diseases Escherichia coli Salmonella Shigella Yersinia entercolitica

Serotypes reference laboratory antigens O (lipopolysaccharide) H (flagellar) K (capsular) O6:K15:H6

(I) Escherichia coli

Extraintestinal infection Intestinal tract infection Medical significance Normal flora E. coli is the majority of GI normal flora Opportunistic pathogen Extraintestinal infection Urinary tract infection Septicemia Neonatal meningitis pathogen Intestinal tract infection ETEC EIEC EPEC EHEC EAggEC

Escherichia coli Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin Type III secretion system Adhesions: colonization factors ; both pili or fimbriae; non-fimbrial factors involved in attachment. There are at least 21 different types of adhesions. Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin) Outer membrane proteins

What is the pathogenesis of these five groups of pathogenic E. coli? Gastroenteritis caused by E. coli

Enterotoxigenic E. coli (ETEC) A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Travellers diarrhea and diarrhea in children in developing countries Transmission is via contaminated food or water. diarrhea like cholera milder nursery travellers diarrhea caused by LT, ST, or LT/ST.

Enterotoxigenic E. coli (ETEC) Heat labile toxin like choleragen Adenyl cyclase activated cyclic AMP secretion water/ions Heat stable toxin Guanylate cyclase activated cyclic GMP uptake water/ions

LT vs ST activity

Enteroinvasive E. coli (EIEC) The organism attaches to the intestinal mucosa via pili Outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa. There is lateral movement of the organism from one cell to adjacent cells. Symptoms include fever, severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous, and pus. resembles shigellosis

Enteroinvasive E. coli (EIEC) Dysentery rearrangement of intracellular actin resembles shigellosis elder children and adult diarrhea

Enteropathogenic E. coli (EPEC) Malaise and low grade fever diarrhea, vomiting, nausea, non-bloody stools Bundle forming pili are involved in attachment to the intestinal mucosa. This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin. This is a problem mainly in hospitalized infants and in day care centers.

Enteropathogenic E. coli (EPEC) fever infant diarrhea vomiting nausea non-bloody stools Destruction of surface microvilli - loose attachment mediated by bundle forming pili (Bfp); - Stimulation of intracellular calcium level; - Rearrangement of intracellular actin

Enterohemorrhagic E. coli (EHEC) bloody, copious diarrhea few leukocytes afebrile Hemolytic-uremic syndrome hemolytic anemia thrombocytopenia (low platelets) kidney failure

Enterohemorrhagic E. coli (EHEC) Vero toxin “shiga-like” Hemolysins younger than 5 years old,causing hemorrhagic colitis

Enterohemorrhagic E. coli (EHEC) Usually O157:H7 Transmission electron micrograph

Enteroaggregative E. coli (EaggEC) a cause of persistent, watery diarrhea with vomiting and dehydration in infants. That is autoagglutination in a ‘stacked brick’ arrangement. the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST). The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

Enteroaggregative E. coli (EaggEC) Mucous associated autoagglutinins cause aggregation of the bacteria at the cell surface and result in the formation of a mucous biofilm. The organisms attach via pili and liberate a cytotoxin distinct from, but similar to the ST and LT enterotoxins liberated by ETEC. Symptoms incluse watery diarrhea, vomiting, dehydration and occasional abdominal pain.

Summary of E. coli strains that cause gastroenteritis.

Organism Virulence factors Adherence Toxins invasion serovar ETEC Colonization factors of adherence (CFAs) Type 1 pili Endotoxin Heat-labile enterotoxin (LT) Heat-stable enterotoxin (ST) Noninvasive O6:K15:H6 EIEC Afimbrial adhesins Very invasive Type III secretion system 28 EPEC Bundle-forming pili (BFP) Intimin Poorly invasive 2, 55 EHEC Shiga toxin Probably poorly invasive O157:H7 EaggEC Mucus-associated autoagglutination Cytotoxin (enteroaggregative ST-like toxin) O42

Enterotoxigenic E. coli (ETEC) Small intestine organism Site Disease pathogenesis Enterotoxigenic E. coli (ETEC) Small intestine Traveler’s diarrhea, infant diarrhea in under developed countries, watery diarrhea, cramps, nausea, low-grade fever Heat-stable and/or heat-labile enterotoxins, stimulate guanylate or adenylate cyclase activity with fluid and electrolyte loss Enteroinvasive E. coli (EIEC) Large intestine Fever, cramping, watery diarrhea followed by development of dysentery with scant, bloody stools Plasmid-mediated invasion and destruction of epithelial cells lining colon Enteropathogenic E. coli (EPEC) Infant diarrhea with fever, nausea, vomiting, nonbloody stools plasmid-mediated adherence and destruction of epithelial cells Enterohemorrhagic E. coli (EHEC) Hemorrhagic colitis with severe abdominal cramps, watery diarrhea initially, followed by grossly bloody diarrhea, little or no fever, hemolytic uremic syndrome (HUS) Mediated by cytotoxic “vero-toxin” Enteroaggregative E. coli (EAggEC) Persistent infant diarrhea, sometimes with gross blood, low-grade fever Aggregative adherence mediated by plasmid

Various Types of E. coli

“Nonintimate” association: bacteria attach to host cell by bundle-forming pili Bacterial attachment: signal transduction event stimulated; host cell tyrosine kinase activated; Ca2+ levels increase “Intimate” contact: pedestallike structure (composed of actin fibers) forms in host cell under bacteria (intimin)

Escherichia coli In the diagnostic laboratory generally the groups are not differentiated and treatment would be on symptomatology. Generally fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome). Two major classes of pili are produced by E. coli : mannose sensitive and mannose resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium allowing attachment. This aids in colonization by E. coli.

Sanitary significance Total bacterial number: number of bacteria contained per ml or gm of the sample; the standard of drinking water is less than 100. Coliform bacteria index: the number of coliform bacteria detected out per 1000 ml sample; the standard of drinking water is less than 3

(II) Shigella dysentery bacterium

Genral features Pili. Most strains can not ferment lactose; S. sonnei can slowly ferment lactose. According to O antigen, 4 groups Easily causing drug-resistence.

Classification Serogroup Species Number of serotypes A S. dysenteriae 10 B S. flexneri 13 C S. boydii 18 D S. sonnei 1 The classification is based on O antigens

What disease is caused by Shigella species? Bacillary dysentery shigellosis

Bacillary dysentery Source - humans are the only reservoir Transmission - the fecal-oral route Clinical findings -watery diarrhea -abdominal cramps -tenesmus -bloody stool with mucus and pus -fever Clinical types - acute dysentery acute toxic dysentery -chronic dysentery Tenesmus: Spasmodic contraction of anal or vesical sphincter with pain and persistent desire to empty the bowel with involuntary ineffectual straining efforts

Shigellosis within 2-3 days epithelial cell damage bloody feces intestinal pain pus

Shigellosis Invasiveness Endotoxin Exotoxin: Shiga toxin - S. dysenteriae - neurotoxic, enterotoxic and cytotoxic

Shiga toxin enterotoxic cytotoxic inhibits protein synthesis lysing 28S rRNA

Clinical significance man only "reservoir" mostly young children fecal to oral contact children to adults transmitted by adult food handlers unwashed hands

Clinical significance The infective dose required to cause infection is very low (10-200 organisms). There is an incubation of 1-7 days followed by fever, cramping, abdominal pain, and watery diarrhea (due to the toxin)for 1-3 days. This may be followed by frequent, scant stools with blood, mucous, and pus (due to invasion of intestinal mucosa). Is is rare for the organism to disseminate. The severity of the disease depends upon the species one is infected with. S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.

immunity Primary immunity defense - SIgA Immunity intensity - Limited - reasons surface infection various types

Diagnosis of Shigella infection Specimen: stool. Culture and Identification Quick immunological methods: - Immunofluorescent “ball” test; - Coagglutination.

Prevention & Treatment streptomycin dependent (SD) dysentery vaccine. manage dehydration patients respond to antibiotics , Problem of drug-resistance disease duration diminished

Summary Shigella Shigella (4 species; S. flexneri, S. boydii, S. sonnei, S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer, but does not penetrate. Usually, within 2-3 days, dysentery results from bacteria damaging the epithelium lining layers of the intestine often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus").

Summary Shigella Shiga toxin (chromosomally encoded) is neurotoxic, enterotoxic and cytotoxic plays a role. The toxin inhibits protein synthesis (acting on the 80S ribosome and lysing 28S rRNA). This is primarily a disease of young children occurring by fecal-oral contact. Adults can catch this disease from children. However it can be transmitted by infected adult food handlers, contaminating food. The source in each case is unwashed hands. Man is the only "reservoir". Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.

(III) Salmonella

Salmonella More than 2000 serotypes Transmitted by the fecal-oral routed Salmonellosis may present as one of several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia. Zoonosis

Antigenic composition of Salmonella O antigen H antigen Vi antigen - S. typhi and S. hirschfeldii - Pathogenicity - Screening of carriers (titer≥1:10) - Inhibition of the agglutination of O Ags and the O Abs

The antigenic structures of salmonellae used in serologic typing

Pathogenicity Virulence factors Endotoxin – may play a role in intracellular survival Capsule (for S. typhi and some strains of S. paratyphi) Adhesions – both fimbrial and non-fimbrial Enterotoxin - may be involved in gastroenteritis Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages Flagella – help bacteria to move through intestinal mucous

Pathogenicity Virulence factors Type III secretion systems and effector molecules – 2 different systems may be found: One type is involved in promoting entry into intestinal epithelial cells The other type is involved in the ability of Salmonella to survive inside macrophages

Disease caused by Salmonella pathogens Gastroenteritis S. enteritidis, S. typhimurium, S. choleraesuis Septicemia S. choleraesuis, S. typhimurium, S. enteritidis, S. hirschfeldii Enteric fever S. typhi, S. paratyphi A, S. schottmuelleri, S. hirschfeldii

Gastroenteritis The most common form of salmonellosis and generally requires an 8-48 hour incubation period and may last from 2-5 days. Symptoms include nausea, vomiting and diarrhea (non-bloody stool). Salmonella enteritidis is the most common isolate. poultry, eggs. no human reservoir self-limiting (2 - 5 days)

Gastroenteritis The most common form of salmonellosis (70%) Self-limited (2-5 d) Not enter into blood Culture - blood (-) - stool (+)

Septicemia Salmonella septicemia (bacteremia) may be caused by any species but S. choleraesuis is common. This disease resembles other G- septicemias and is characterized by a high, remittent fever with little gastrointestinal involvement.

Septicemia 5-10% of salmonellosis Intestinal manifestations: often absent Culture - blood (+) - stool (-)

The pathogenesis of typhoid Enteric fever The pathogenesis of typhoid

Enteric or typhoid fever Enteric or typhoid fever occurs when the bacteria leave the intestine and multiply within cells of the reticuloendothelial system. The bacteria then re-enter the intestine, causing gastrointestinal symptoms. Typhoid fever has a 10-14 day incubation period and may last for several weeks. Salmonella typhi is the most common species isolated from this salmonellosis. Human reservoir: carrier state common Contaminated food: water supply Poor sanitary conditions

Typhoid -Therapy Antibiotics essential Vaccines Vi (capsular) antigen :protective

What does the pathogenesis imply in terms of collection of clinical samples?

Diagnosis Specimens Culture and identification Widal test a) Enteric fever: blood, bone marrow, stool, urine. b) Food poisoning: stool, vomitus, suspected food. c) Septicemia: blood. Culture and identification Widal test

Widal test A quantitative agglutination test for typhoid and paratyphoid, in which detects a patient’s antibodies to the specific O antigen of S. typhi and H antigens of S. typhi, S. paratyphi A, S. schottmuelleri and S. hirschfeldii

How to interpret the results of Widal test? Consider the manifestaton, course, history, and local epidemiological conditions

How to interpret the results of Widal test? TO<1:80,TH<1:160, PH<1:80 Normal value TO≥1:80 & TH≥1:160 or TO≥1:80 & PH≥1:80 Typhoid fever Paratyphoid fever TO≥1:80 & TH <1:160 or TO≥1:80 & PH <1:80 Early stage of infection or cross-reaction of O antigen with other salmonellae TO < 1:80 & TH ≥1:160 or TO < 1:80 & PH ≥ 1:80 Vaccination or nonspecific memory reaction

How to interpret the results of Widal test? Dynamic observation -antibody titer give a rise gradually -titer of convalescence serum≥4 times than that of early specimen False negative -pre-antobiotic -immunosuppressed

immunity Primary immunity defense Immunity intensity - CMI - strong and permanent

Salmonella Using appropriate antibodies more than 2000 antigenic “types” have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi). Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs). It does not have a human reservoir and usually presents as gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2-5 days). Like Shigella they invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. choleraesuis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required. .

Salmonella The severest form of salmonella infections "typhoid" (enteric fever), caused by Salmonella typhi. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and during this acute phase, gastrointestinal symptoms are noted. The organism penetrates, usually within the first week, and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person e.g. a food handler can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used

Summary Common members of Enterobacteriaceae to cause human diseases Common properties of Enterobaceriaceae The medical significance of E. coli The opposite traits of Shigella and Salmonella in terms of pathogenesis and immunity Widal test: definition, result determination