Laboratory Identification of Salmonella & Shigella & E. coli [PDF]

Laboratory Identification of Salmonella & Shigella & E. coli & Campylobacter & Vibrio & Aeromonas& Plesiomonas

Babak Valizadeh , DCLS [email protected]

1389/07/01 2010.09.23 1

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Foodborne diseases / NIH News 2010 CDC estimates 76 million people suffer from foodborne illnesses each year in the US

Accounting for 325,000 hospitalizations and more than 5,000 deaths There are more than 250 known foodborne diseases

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Bacterial diarrhea  More than 5.2 million cases of bacterial diarrhea that occur each year in the US  80% are a result of foodborne transmission  Person to-person spread occurs if only a small amount of a pathogen is required for infection;  Shigella  Shiga toxin–producing Escherichia coli  Viral agents 4

Bacterial diarrhea  Bacterial enteropathogens lead to an estimated 46,000 hospitalizations and 1500 deaths each year in the US  Campylobacter  Nontyphoid salmonella  Shiga-toxin–producing E. coli  Shigella  Lower number of organisms required to cause clinical infection in infants than in older children and adults 5

Bacterial diarrhea When bacterial enteropathogens are suspected, a stool culture or toxin assay will help to establish the diagnosis

Indications for stool culture include the presence of severe diarrhea (passage of six or more unformed stools per day) 6

Bacterial diarrhea Diarrhea of any severity that persists for longer than a week, fever, dysentery, and multiple cases of illness that suggest an outbreak. When multiple stool samples are obtained from patients with diarrhea, the increased yield of bacterial pathogens is approximately 20% (one in five additional samples is positive) 7

Acute Watery Diarrhea  Diarrhea caused by salmonella and campylobacter < 3%  Many of the potentially important agents that cause watery diarrhea are not detectable by means of routine diagnostic laboratory tests:

   

Enterotoxigenic E. coli Enteroaggregative E. coli Enteroinvasive E. coli Noncholeraic vibrios

 Noroviruses & Other Virus

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Dysentery  Four major causes of bloody diarrhea in the United States  Shigella  Campylobacter  Nontyphoid salmonella  Shiga toxin–producing E. coli  Aeromonas species  Noncholeraic vibrios  Yersinia enterocolitica

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Traveler’s Diarrhea Bacterial enteropathogens cause up to 80% of cases The diarrhea producing E. coli (enterotoxigenic E. coli, enteroaggregative E. coli, and possibly diffusely adherent E. coli) account for more than half of cases Shigella, Salmonella, Campylobacter, Aeromonas species, noncholeraic Vibrios, and Plesiomonas also cause this condition 10

Staphylococcus aureus 185,000 (Estimated No. of U.S. Cases/Yr) Outbreak of vomiting lasting ≤12 hr, with an incubation period of 2–7 hr Characteristic clinical manifestations; food may be cultured for staphylococcus or enzyme immunoassay may be performed for enterotoxin in food

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Clostridium perfringens  250,000 (Estimated No. of U.S. Cases/Yr)  Potentially very large foodborne outbreaks of watery diarrhea without fever or vomiting;

 Incubation period of 8–14 hr  Confirmed in foodborne outbreaks by detecting ≥106 C. perfringens spores/g of feces in affected persons or ≥105 organisms/g in food

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Bacillus cereus 27,000 (Estimated No. of U.S. Cases/Yr) Foodborne outbreaks of gastroenteritis Two syndromes resembling S. aureus with vomiting after 2–7 hr or C. perfringens disease with watery diarrhea after 8–14 hr Confirmed in foodborne outbreaks by detecting >105 organisms in food 13

Clostridium difficile Associated Disease (CDAD)  Historically, specific agents such as Clindamycin, Ampicillin, Amoxicillin or Cephalosporins were the most often associated with an increased risk of CDAD  Stool test for C. difficile toxin  Enzyme immunoassay for toxins A and B  Cytotoxicity tissue-culture assay for toxin B  Microbiologic culture 14

Clostridium difficile Associated Disease (CDAD)

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Clostridium difficile Associated Disease (CDAD)

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Treatment - Bacterial diarrhea / 2009

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Treatment - Bacterial diarrhea / 2009

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SPECIMEN COLLECTION,TRANSPORT AND HANDLING .

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Stool Culture  Majority of laboratories (99.3%) included Salmonella and Shigella in the routine stool workup / US  96% routinely included Campylobacter / US  30-60% of laboratories surveyed also included other organisms such as Aeromonas, Plesiomonas, Yersinia, Vibrio & Escherichia coli O157

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Specimen collection  Transfer at least 5 ml of diarrheal stool  1 g of stool  Buffered glycerol saline (preferred for Shigella spp. but inhibits Campylobacter)  Modified Cary-Blair medium pH : 8.4

 AGAR ; 1.5 G  Shigella and Campylobacter

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Specimen collection & Timing and transport  Rectal swabs  Pass the tip of a sterile swab approximately 1 in. (2.5 cm) beyond the anal sphincter

 Submit specimen during the acute stage of infection (usually 5 to 7 days)  Submit and culture fresh stool within 30 min of collection to allow for isolation of Shigella spp., which are extremely fragile

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Timing and transport Store and transport stool in transport medium at 4'C and submit within 24h for best recovery of pathogens Generally submit two stools per patient from different days to diagnose bacterial causes of gastroenteritis 24

Rejection criteria

Reject stools not in transport medium received >2 h after collection as changes occur that are detrimental to most Shigella spp

If specimen in transport medium is delayed for more than 3 days at 4 C or is delayed for more than 24 h at 25 C 25

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REPORTING RESUTS Final reports : No Salmonella, Shigella, or Campylobacter spp. Isolated

Preliminary reports : to date ,……..

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REPORTING RESUTS No normal enteric gram-negative rods isolated

Identify numerous P.aeruginosa & S. aureus organisms; do not perform AST Report yeast, if found in pure or predominating culture, without genus or species identification Do not report enterococci in stool 29

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Positive Urease

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Lysine Iron Agar (LIA)

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SS

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XLD

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HEA

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E. coli .

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?!?!

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Escherichia coli E.coli is the predominant aerobe of the human colonic flora The organism typically colonizes the infant gastrointestinal tract within hours of life Anaerobe / Aerobe = 1000 / 1 in Stool E.coli number 108 per gram stool Klebsiella & Proteus & Enteroccci 105-107 42

Diarrheagenic Escherichia coli  Culturing stools for most categories of diarrheagenic E. coli should be performed in cases of persistent diarrhea, especially in  Travelers  Children  Immunocompromised  Outbreak  E. coli can be isolated from the stool and sent to a qualified reference laboratory for definitive identification

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Diarrheagenic E.coli / Laboratory Diagnosis

Fecal specimens should be plated on a differential medium of low selectivity (e.g.; MaC) .

Between 5 and 20 colonies , mostly lactose fermenting but with a representative sample of nonfermenting colonies ,should be selected and inoculated onto a nonselective agar slant 44

Diarrheagenic E.coli / Laboratory Diagnosis

This identification process may include HEp-2 cell adherence, DNA hybridization, and PCR assays to detect the presence of specific virulence traits or the genes encoding these traits. 45

Serogrouping  Serogrouping  Determination of O serogroups associated with the cell wall lipopolysaccharides  e.g. ;O111 in EPEC & STEC  Rough strains that lacked side chains, thus being nontypeable with regard to O antigen

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Serotyping Determination of O serogroups associated with the cell wall lipopolysaccharides and H of the flagella

E. coli are serotyped on the basis of their O (somatic), H (flagellar), and K (capsular) surface antigen profiles  e.g. O111:H2 in EPEC & O111:H8 in STEC

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Serotyping / E.coli

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Serotyping Serotypic markers correlate, sometimes very closely, with specific categories of diarrheagenic E.coli

These markers are rarely sufficient to reliably identify a strain as diarrheagenic

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Diarrheagenic Escherichia coli  Six categories of Escherichia coli have been well associated with diarrhea  Enterotoxigenic E. coli (ETEC)  Typical and Atypical Enteropathogenic Escherichia coli (EPEC)  Enteroaggregative E. coli (EAEC)  Diffuse adherent E. coli (DAEC)  Enteroinvasive E. coli (EIEC)  Enterohemorrhagic E. coli (EHEC) Or Shiga toxin-producing E. coli (STEC) isolates

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Enterohemorrhagic E. coli (EHEC) Shiga toxin-producing E. coli (STEC) / Verotoxin & E. coli O157:H7

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Enterohemorrhagic Escherichia coli (EHEC)

The infection is potentially fatal, especially in young children and elderly persons in nursing homes Meats (beef) , such as undercooked hamburgers served at fast-food restaurants, unpasteurized dairy products and apple cider 54

EHEC / STEC - E. coli O157:H7

Infectious dose : 102

Inoculum to cause infection with E. coli O157:H7 is low so that person-to-person spread can occur 55

Shiga toxin–producing E. coli Shiga toxin–producing E. coli strains cause watery diarrhea that becomes bloody in 1 to 5 days in 80% of patients

Characteristic features of this condition include severe abdominal pain and cramps and passage of five or more unformed stools per 24 hours in the absence of fever 56

Shiga toxin–producing E. coli  Watery diarrhea progressing to passage of bloody diarrhea  Infection acquired from food (beef or contaminated produce) (52% of patients)  Person to- person spread ( 14%)  water and wading pools ( 9%)  Contact with animals ( 3%)  Laboratories ( 90% occur in developing countries  In developing countries 69% of episodes occur in children under five years of age  1.1 million deaths attributed to Shigella infections in developing countries  60% of deaths occur in the under-five age group 98

Shigella First isolated the organism in 1896

 Japanese microbiologist Kiyoshi Shiga

Fewer than 200 bacilli are needed to initiate the disease in some healthy individuals

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Shigella Four species of Shigella i.e. S. dysenteriae, S. flexneri, S. boydii and S. sonnei

Shigella dysenteriae type 1 produces severe disease

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Shigella Humans are the only known reservoir of Shigella organisms

No animal reservoir has been identified Infections caused by Shigella spp. are associated with human carriers responsible for spreading the disease

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Shigella / Clinical Infections Children younger than 10 years of age seem to be most affected, and those 1 year of age and younger are the most susceptible The initial symptoms, marked by high fever, chills, abdominal cramps, and pain accompanied by tenesmus, appear approximately 24 to 48 hours after ingestion of the organisms 103

Shigella / Clinical Infections The organisms, originally multiply in the small intestine, move toward the colon, Shigella bacteria multiply within colonic epithelial cells, cause cell death They may be isolated 1 to 3 days after the infection develops 104

Shigella / Clinical Infections  S. sonnei is the predominant isolate (77%), followed by S. flexneri

 In the US and other industrialized countries  S. sonnei is more resistant and survive better in environment (e.g. 5 days in feces dried on cloth in cool, damp & dark condition)  S. sonnei infection is usually a short, self-limiting disease characterized by fever and watery diarrhea 105

Shigella / Clinical Infections In developing countries, S. dysenteriae type 1 and S. boydii are the most common isolates

S.dysenteriae type 1 remains the most virulent species, with significant morbidity and high mortality (rates of 5% to 10% )

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Antibiotic resistance among Shigella serogroups isolated in Tehran, Iran (2002-2004)

 Ref; Reza Ranjbar et al;JIDC 2009; 3(8):647-648

 Of a total of 200 Shigella spp. isolates studied, 110, 84, 5, and 1 were identified as S. sonnei, S. flexneri, S. boydii, and S. dysenteriae, respectively

 Overall, 93.5% of tested isolates were resistant to sulfamethoxazole-trimethoprim  More than 72.6% of S. flexneri isolates, but 2.7% only of S. sonnei isolates, were resistant to ampicillin  Nine (8.2%) isolates of S. sonnei were resistant to nalidixic acid 107

Stool Culture Even the best technique with fresh specimens may miss fragile organisms such as shigella.

Fecal cultures failed to yield shigella in 40% of volunteers with inflammatory diarrhea from experimental shigella infection. 108

Stool Culture  Shigella spp. are highly fragile organism

 Fecal specimens should be collected in the early stages of the disease when pathogens usually are present in the stool in high numbers

 Positive cultures are most often obtained from blood-tinged plugs of mucus in freshly passed stool specimens obtained during the acute phase of disease 109

Method of streaking plating medium for isolation of Shigella

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Isolation and Identification of Shigella / WHO

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Appearance of Shigella colonies on selective plating media

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S. dysenteriae 1 colonies on XLD

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Shigella  Gram-negative bacilli  Nonmotile  Gas from glucose : Negative  Urease : Negative  H2S : Negative  Lysine decarboxylase / LDC : Negative  Oxidase negative  IMViC = - + - - or (+ + - - )

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Reactions of Shigella in screening biochemicals

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Antigenic Structures  The genus consists of four species  Shigella spp. are also divided into four major O antigen groups , Serogroup (A,B,C and D)  These species are subdivided into Serotypes on the basis of O-specific polysaccharide of the LPS  Several serotypes exist within each species with the exception of S. sonnei, which has only one serotype

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Antigenic Structures  Certain strains may possess K antigens.  Shigella K antigens, when present, interfere with the detection of the O antigen during serologic grouping  The K antigen is heat labile and may be removed by boiling the organism in a cell suspension  The shigellae are nonmotile and therefore lack H antigens 118

Antigenic Structures

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Shigella antiserum

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Shigella Shigella dysenteriae type 1 : Catalase – Negative  1-Touch the center of well-isolated young colony (18-24 hrs.) on SBA or MacConkey with a wooden stick to transfer to a clean ,dry glass slide.  Dose not test from Muller-Hinton Agar (MHA)  Place 1 drop of 3% H2O2 and observe immediately bubbles 121

Shigella sonnei  IMViC = - + -  LDC= -

 Ornithine decarboxylase / ODC : Positive  ONPG : Positive  Slowly ferments lactose, forming pink colonies on MacConkey agar after 48 hours of incubation 122

Shigella sonnei

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Shigella serogroups A,B & C + S.sonnei

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Shigella serogroups A,B & C + S.sonnei

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Shigella serogroups A,B & C + S.sonnei

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Shigella serogroups A,B & C + S.sonnei

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Salmonella .

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Salmonella / New Classification  Salmonella enterica DNA group1,2,3,4,6  Salmonella bongori DNA group 5  Salmonella enterica subspecies enterica (DNA group1)****** > 90 % of human infections  e.g;Salmonella enterica subspecies enterica serotype Typhi  Salmonella serotype Typhi  Salmonella Typhi  More than 2400 of Salmonella serotype

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Typhoid and paratyphoid salmonella  800 (Estimated No. of U.S. Cases/Yr)  Systemic toxic effects and fever, abdominal symptoms (pain, diarrhea, constipation)  Most infections acquired during international travel  Organism reservoir is infected humans

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Nontyphoid salmonella  1.4 million (Estimated No. of U.S. Cases/Yr)  Acute watery diarrhea, often with fever, occasionally with dysenteric characteristics  95% of cases are a result of foodborne transmission (from poultry or hens’ eggs);  Commonly seen in infants

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Salmonella Salmonella produce significant infections in humans and in certain animals Many Salmonella serotypes are usually found in coldblooded animals ( e.g. turtles, snakes) as well as in rodents and birds Typhi and Paratyphi have no known animal reservoirs, and infections seem to occur only in humans 135

Clinical Infections  An acute gastroenteritis or food poisoning characerized by vomiting and diarrhea  Enteric fever, caused by Salmonella serotype Typhi and other Salmonella serotypes (mild)  Nontyphoidal Bacteremia  Carrier state following Salmonella Infection

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Culture and serologic diagnosis of typhoid fever

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Nontyphoidal Bacteremia  Most commonly associated serotypes of Salmonella are Typhimurium, Paratyphi, and Choleraesuis  Young children, who experience fever and gastroenteritis with brief episodes of bacteremia  Adults, Transient bacteremia during episodes of gastroenteritis  Adults, Septicemia without gastroenteritis e.g. underlying illnesses, such as malignancies and liver disease .e.g. Choleraesuis 138

Carrier state following Salmonella Infection

The carrier state may be terminated by antimicrobial therapy if gallbladder infection is not evident.

Otherwise, cholecystectomy has been the only solution to the chronic state of enteric carriers 139

Antimicrobial Therapy

Antimicrobial therapy is believed to prolong the carrier state

Antidiarrheal agents are also restricted in cases of salmonellosis because these agents may encourage adherence and further invasion 140

Antimicrobial Susceptibility Testing For patient with

Invasive Salmonella Typhoidal infection Child is under the age 6 years Extra intestinal isolates of Salmonella Antimicrobial Susceptibility Testing should be reported 141

Antigenic structures of salmonella

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Antigenic Structures Somatic O antigens Heat-stable Lipopolysaccharide (LPS) More than one O antigen may also be found in a particular strain 143

Antigenic Structures Flagellar H antigens Heat labile Proteins phase 1, the specific phase phase 2, the nonspecific phase 144

Antigenic Structures  Capsular K surface antigens , Vi  Polysaccharide  Heat-labile  Vi antigen often blocks the O antigen  Vi antigen is important in identifying Salmonella serotype Typhi &  Few strains of Salmonella serotype Choleraesuis & Citrobacter 145

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Serotyping / Serogrouping Typing Should be performed from a non-sugar containing medium ,such as 5% sheep blood agar (BAP) or LIA

Use of sugar containing media , such as MacConkey or TSI agars , can cause the organism to autoagglutinate 147

Salmonella  Gram-negative bacilli  Motile except Gallinarum & Pullorum  Gas from glucose : Positive except Typhi  Urease : Negative  Indole : Negative  H2S : Positive  Lysine decarboxylase / LDC : Positive except Paratyphi A  Oxidase negative

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Salmonella  Salmonella serotype Typhi

 IMViC = - + - - ,

LDC = +

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 Nontyphoidal Salmonella group I  IMViC = - + - + , LDC=+  Salmonella serotype Paratyphi A  IMViC = - + - - , LDC= -

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ODC= -

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ODC = +

ODC = +

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External Quality Assurance System results from 2000 to 2004 WHO

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Distribution of Salmonella serotypes reported to the Country Databank, 2000-2004 / WHO

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Distribution of human Salmonella serotypes by region, 2000-2004 / WHO

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Campylobacter .

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Campylobacter

Fecal samples from chicken 83% of the samples yielded more than 106 colonyforming units Campylobacter, per gram of feces Usually transmitted via contaminated food ( chicken ), milk, or water. 162

Campylobacter

In contrast to other agents of foodborne gastroenteritis, including Salmonella and Staphylococci, Campylobacter does not multiply in food Within the genus Campylobacter, C. jejuni (90)% and C. coli are most often associated with infections in humans 163

Campylobacter Gastroenteritis caused by Campylobacter spp. is usually a self-limiting illness and does not require antibiotic therapy Erythromycin / Azithromycin is the drug of choice then Ciprofloxacin (10-40% = R) Use of quinolones in the poultry industry has increased resistance of campylobacters to quinolone 164

Campylobacter Currently, 17 species and various subspecies are recognized in the genus Campylobacter

Motile / Darting Motility across the field in a zigzag fashion in fresh stool (