Laboratory Identification of Salmonella &
Shigella & E. coli & Campylobacter & Vibrio &
Aeromonas& Plesiomonas
Babak Valizadeh , DCLS
Babak_Valizadeh@hotmail.com
1389/07/01
2010.09.23
1
2
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
3
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
8
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
9
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
11
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
12
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)
15
Clostridium difficile Associated Disease (CDAD)
16
Treatment - Bacterial diarrhea / 2009
17
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
21
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
22
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
23
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
26
27
REPORTING RESUTS
Final reports : No Salmonella, Shigella, or
Campylobacter spp. Isolated
Preliminary reports : to date ,……..
28
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
30
Positive Urease
31
Lysine Iron Agar (LIA)
32
33
34
SS
35
XLD
36
HEA
37
E. coli
.
38
39
?!?!
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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
43
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
46
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
47
Serotyping / E.coli
48
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
49
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
50
51
52
Enterohemorrhagic E. coli (EHEC)
Shiga toxin-producing E. coli (STEC)
/ Verotoxin &
E. coli O157:H7
53
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 ( <1%)
 Unknown sources (21%)
 Important reservoir in cattle
57
Shiga toxin–producing E. coli
 Infection by Shiga toxin–producing E. coli is the main cause of
renal failure in childhood
 In the hemolytic– uremic syndrome, Shiga toxin released in
the gut enters the bloodstream and reaches the renal
endothelium
 Two thirds of children with the hemolytic–uremic syndrome
require dialysis
 Associated mortality rate is 3 to 5%
58
Shiga toxin–producing E. coli
Shiga toxin–producing Escherichia coli,
including E. coli O157:H7 and non-O157 strains
100,000 (Estimated No. of U.S. Cases/Yr)
59
Shiga toxin–producing E. coli
 It appears that Shiga toxin 2 is more important in the pathogenesis of the
hemolytic–uremic syndrome than Shiga toxin 1
 Laboratory evaluation of bloody stools should include assays
for sorbitol-negative E. coli
 Serotyping for O157:H7 strains
 As well as examination of the stools for Shiga toxins 1 and 2 by means of
commercial enzyme immunoassay
60
Shiga toxin–producing E. coli
 Approximately 40% of Shiga toxin–producing E. coli infections
in the US are non-O157 strains
 There are at least 100 serotypes of STEC
 E.coli O111 is the second most common Non-O157 STEC
 Non-O157 Shiga toxin–producing E. coli can cause the same
spectrum of disease as O157 strains
 Unlike most O157:H7 strains, the non-O157 strains are usually
sorbitol-fermenting
61
Shiga toxin–producing E. coli
In cases in which fecal testing for Shiga toxin is
positive but testing for E. coli O157 is negative
Isolates can be examined in a reference
laboratory for a non-O157 Shiga toxin–
producing E. coli serotype
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Non-0157: H7 EHEC
63
E. coli O157:H7
Stool cultures have a low positivity rate
Isolation of E.coli O157:H7 is possible only during the
acute phase of illness, and the organisms may not be
detectable 5-7 days after onset
Varied geographic distribution - evaluate
prevalence for the need of routine workup
64
Laboratory Diagnosis / E. coli O157:H7
Only one serotype, namely E. coli
O157:H7 can be detected in clinical
laboratories
Selective media: sorbitolMacConkey agar (SMAC)
65
Laboratory Diagnosis / E. coli O157:H7
E. coli O157:H7 does not ferment sorbitol in 24 (48)
hours, a characteristic that differentiates it from most
other E. coli
E. coli O157:H7 appears colorless on Sorbitol
MacConky agar (SMAC)
66
67
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Laboratory Diagnosis / E. coli O157:H7
Confirm by latex agglutination / Antiserum
 Agglutination test for rapid presumptive detection of E. coli
0157 from SMAC / Serogrouping
 Sorbitol-negative colonies are subsequently subculture for
Serotyping using E. coli 0157:H7 antiserum
69
Laboratory Diagnosis / E. coli O157 / WHO
70
Laboratory Diagnosis / E. coli O157:H7
 When testing colonies taken directly from the SMAC plate, the test for the
H7 antigen may be initially negative
 It is helpful to grow these isolates in motility media first to enhance
flagella production
 Following the serotyping, isolates are tested for the presence of Stx
EIA for detection of STEC (MaC Broth or GN Broth)
71
E. coli O157:H7 / Antimicrobial therapy
The use of antimicrobial therapy in patients with E.
coli O157:H7 infection showing that increase the
production of Shiga-like toxin & bacterial cell lysis &
release toxin
Hemolytic-uremic syndrome is more likely to develop in
patients treated with an antimicrobial agent, & or
Antimotility agents
72
E. coli O157:H7 / Antimicrobial therapy
Determination of the antimicrobial susceptibility
pattern is usually not meaningful.
Except for cases of cystitis and pyelonephritis or
for Epidemiological Study
73
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CHROM agar O157
75
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Shiga Toxin–Producing E.coli Infections / 2009
In U.S. studies, STEC were detected in 0%–4.1%
of stools submitted for testing at clinical
laboratories, rates similar to those of
Salmonella species (1.9%–4.8%)
 Shigella species (0.2%–3.1%)
Campylobacter species (0.9%–9.3%)
77
Shiga Toxin–Producing E.coli Infections / 2009
All stools submitted for testing from patients with
acute community-acquired diarrhea (i.e., for
detection of the enteric pathogens Salmonella,
Shigella, and Campylobacter) should be cultured
for O157 STEC on selective and differential agar
These stools should be simultaneously assayed
for non-O157 STEC with a test that detects the
Shiga toxins
78
EHEC / 2010
ImmunoCard Stat, EHEC, for the
detection of Shiga toxins
An immunochromatographic rapid
test for the qualitative detection of
Shiga toxins produced by E. coli
93.8% sensitivity and 99.7%
specificity
Meridian Diagnostics
79
Enteroinvasive E. coli (EIEC)
Most enteroinvasive E. coli (EIEC) isolates will
be missed in the clinical laboratory
80
Enteroinvasive E. coli (EIEC)
 EIEC is less common
 Infectious Dose : 108 vs. Shigella : 102
 Congo red Agar
 Lactose : Negative
 Motility : mostly Negative but some positive
 Gas : Negative
 LDC : mostly Negative but some positive
 Xylose : POSITIVE vs. Shigella : mostly Negative
 ipaH & ipaC for EIEC isolates & Shigella
81
Enterotoxigenic E. coli (ETEC)
79,000 (Estimated No. of U.S. Cases/Yr)
Acute watery diarrhea; cause of nearly half of cases of
traveler’s diarrhea, important cause of diarrhea in
children in developing regions
Stool culture for E. coli, followed by assay for heatlabile cholera-like enterotoxin and heat-stable
enterotoxins by ELISA, DNA hybridization, or PCR
methods
82
Enterotoxigenic E. coli (ETEC)
ETEC have been shown to be the most common
bacterial enteric pathogen in developing countries
 ETEC is a major cause of traveler’s diarrhea
Among the six recognized diarrheagenic categories of
Escherichia coli, ETEC is the most common,
particularly in the developing world
ETEC is more difficult to recognize
83
Laboratory Diagnosis
Phenotypic methods
 ETEC must be recognized by the enterotoxins it produces,
diagnosis must depend upon identifying either LT and/or ST
 Serotyping was found to be of limited use
 ETEC belongs to a heterogeneous family of lactose-fermenting
E. coli, belonging to a wide variety of O antigenic types
84
Enteropathogenic E. coli (EPEC)
1940
85
Enteropathogenic Escherichia coli (EPEC)
86
Enteropathogenic Escherichia coli (EPEC)
Enteropathogenic Escherichia coli
(EPEC) is an important category
cause of infantile diarrhea in
developing countries ,usually
associated with infants from the
poorer social groups
The central mechanism of EPEC
pathogenesis is a lesion called
attaching and effacing (A/E)
87
Enteropathogenic Escherichia coli (EPEC)
 In symptomatic patients, EPEC can be isolated from stools up to 2 weeks
after cessation of symptoms
 EPEC infection is primarily a disease of infants younger than 2 years
 in Less than 6 months
 In some studies, as many as 17 to 20% of healthy infants
younger than 2 years shed E. coli of EPEC serotypes in their
stools
 The most prevalent serogroups are O111, O119, and O55
88
Enteropathogenic Escherichia coli (EPEC)
Use of commercial antisera to the classical somatic (O)
and capsular (K) antigen yields many false-positive
results.
Further testing with H (flagellar) antisera would
reduce the number of false-positive reports.
This type of testing is not practical for the average
clinical microbiology laboratory
89
Enteropathogenic Escherichia coli (EPEC)
eae and bfpA for EPEC / Multiplex PCRs
Detection of eae confirms the presence of typical and/or
atypical EPEC strains
Testing for bfpA confirms the presence of the bundleforming pilus major subunit that is found only in
typical EPEC strains
90
Enteroaggregative E. coli (EAEC)
Enteroaggregative Escherichia coli (EAEC) is
increasingly recognized as a cause of diarrhea
worldwide
EAEC is defined by its characteristic “stacked brick”
aggregative adherence (AA) pattern of adherence to
HEp-2 cell
91
Enteroaggregative E. coli (EAEC)
92
Diffuse adherent E. coli (DAEC)
93
94
Multiplex PCR assays
95
Shigella
.
96
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Shigella
 Shigellosis is a global human health problem
 The estimated 165 million cases of Shigella diarrhea that occur annually
 ~ 500,000 in US
 >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
99
100
Shigella
Four species of Shigella i.e. S. dysenteriae, S. flexneri,
S. boydii and S. sonnei
Shigella dysenteriae type 1 produces severe disease
101
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
102
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% )
106
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
110
Isolation and Identification of Shigella / WHO
111
Appearance of Shigella colonies on selective plating media
112
S. dysenteriae 1 colonies on XLD
113
Shigella
 Gram-negative bacilli
 Nonmotile
 Gas from glucose : Negative
 Urease : Negative
 H2S : Negative
 Lysine decarboxylase / LDC : Negative
 Oxidase negative
 IMViC = - + - - or (+ + - - )
114
Reactions of Shigella in screening biochemicals
115
116
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
117
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
119
Shigella antiserum
120
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
123
Shigella serogroups A,B & C + S.sonnei
124
Shigella serogroups A,B & C + S.sonnei
125
Shigella serogroups A,B & C + S.sonnei
126
Shigella serogroups A,B & C + S.sonnei
127
Salmonella
.
128
129
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
130
131
132
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
133
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
134
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
136
Culture and serologic diagnosis of typhoid fever
137
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
142
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
146
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
148
149
150
Salmonella
 Salmonella serotype Typhi
 IMViC = - + - - ,
LDC = +
,
 Nontyphoidal Salmonella group I
 IMViC = - + - + ,
LDC=+
 Salmonella serotype Paratyphi A
 IMViC = - + - - ,
LDC= -
151
ODC= -
,
,
ODC = +
ODC = +
152
153
154
155
156
External Quality Assurance System results from 2000 to 2004
WHO
157
Distribution of Salmonella serotypes reported to the Country Databank,
2000-2004 / WHO
158
Distribution of human Salmonella serotypes by region, 2000-2004
/ WHO
159
Campylobacter
.
160
161
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 ( <30 minute) & from colony in
broth (e.g.. TSB)
Emulsify a loopful of 24 to 48-h bacterial growth in
broth ,not saline or distilled water
165
Campylobacter
 Gram-negative bacilli, faintly staining (Safranin)
 Gram stain with carbol fuchsin or 0.1% basic fuchsin
 Curved, seagull-winged
 Acute phase of diarrhea ; Sensitivity :66 to 94%
 Report: Campylobacter-like organism
 Coccoidal forms may be seen in the Gram stain, especially in
older cultures
166
167
Specimen transport
Process within 1 to 2 h or place in transport
medium
Modified Cary-Blair medium
168
Cultivation / Filtration method
 Nonselective medium to recover Campylobacter and Arcobader spp.
 A filter (0.65-mm pore-size cellulose acetate) is placed on the agar
surface, and a drop of stool is placed on the filter.
 The plate is incubated upright.
 After 60 minutes at 37° C, the filter is removed and the plates are
reincubated in a microaerobic atmosphere
169
Cultivation
 For optimum recovery, the inoculation of two selective agars is
recommended
 The use of more than one type of selective medium increases
the yield from stools by as much as l5%
 Two sets of selective plates should be incubated, one at 42° C /
(40 ° C ) for 72 h and one at 37° C for 4-5 days
170
171
Microaerobic environment
172
173
174
Presumptive Campylobacter
 Gram Stain
 Growth at 42 C
 Darting Motility
 Oxidase & Catalase positive colonies
Campylobacter jejuni
Hippurate hydrolysis : positive
175
176
177
178
Non-culture methods for direct detection of Campylobacter in stool / 2010
179
Vibrio
.
180
181
VIBRIONACEAE
 Oxidase –positive
 Glucose-fermenting
 Gram-negative bacilli
 Grow on MacConkey agar
 HALOTOLERANT
 MARINE/AQUATIC ENVIRONMENT
182
VIBRIONACEAE
VIBRIO
AEROMONAS
PLESIOMONAS
183
Vibrio
The genus Vibrio resides in the family
Vibrionaceae and encompasses more than
30 species
Although to date only 12 of these species
have been found in human clinical
specimens
184
Vibrio
The four Vibrio spp. likely to be encountered in the
clinical laboratory are
 V.cholerae (01 and non-O1)
 V. parahaemolyticus
 V. vulnificus
 V. alginolytieus
185
CHOLERA
In acute cases as a severe gastroenteritis accompanied
by vomiting followed by diarrhea
The stools produced by cholera patients are described
as "rice-water"
Number of stools, may be as many as 10 to 30 per day
186
V.Cholerae non-O1 / NAG
 Strains are phenotypically similar to toxigenic
V. cholerae 01, but most lack the cholera toxin
gene and appear to cause a milder form of gastroenteritis or cholera-like
disease.
 These non-O1 strains also have been implicated in a variety of
extraintestinal infections,




187
Cholecystitis
Ear infections
Cellulitis
Septicemia
Vibrio
 Oxidase-positive
 Required 3% to 6% NaCI for growth
 All species, except for V.cholerae and V.mimicus, are
halophilic, or salt-loving and require the addition of Na' for
their growth
 Gram-negative rods , curved
 Morphology is often seen in the initial Gram stain of the
clinical, specimen
 Highly pleomorphic, especially under suboptimal growth
conditions
188
Vibrio cholerae
189
190
Laboratory Diagnosis
Specimen Collection and Transport
 Vibrios are not fastidious
 Stool / Rectal swab should be collected as early as possible in the course of
the illness
 Rectal Swab : Pass tip of sterile swab approximately 2-3 cm
 Cary-Blair
 Buffered glycerol saline is not recommended
 Strips of blotting paper soaked in liquid stool and
placed in airtight plastic bags are considered viable specimens
191
Culture Media
 SBA plate should be examined for the presence of hemolysis
 Determine the oxidase
• Subculture any oxidase positive colonies to SBA &
either TSI or KIA
 On MacConkey agar, the pathogenic vibrios grow as
nonlactose fermenters ( except ;V. vulnificus )
192
TCBS
 TCBS (Thiosulfate Citrate Bile Salts Sucrose Agar) is the most
widely used selective medium
 Screen TCBS at 24 and 48 h
 TCBS differentiates sucrose-fermenting (yellow) from the
nonsucrose-fermenting (green) vibrios
 Proteus is yellow & Enterococci may grow
• Quality-control :there is great lot to lot variation in
performance and not all Vibrio spp.grow on TCBS
193
194
195
Alkaline peptone water
Enrichment procedure enhance isolation of vibrios
Alkaline peptone water/ APW : 1% NaCl , pH 8.5;
Subculture to TCBS at 24 h at 35 C
 Subculture at is not necessary 5-8h (optional)
 Vibrio & Aeromonas
196
Oxidase test
Oxidase test must be performed from 5% sheep blood
agar or another medium without a fermentable sugar
Acidification of medium if surrounding pH is below
5.1 may result a false-negative
lactose-positive colonies from selective-differential
media such as MacConkey or may give false positive
oxidase reactions
197
Oxidase test
N,N,N,N-Tetramethyl1,4phenylendiammonium
dichloride for oxidase
test
198
String test
Most vibrios also exhibit a positive
string test observed as a mucoid
"stringing" reaction after
emulsification of colonies in 0.5%
sodium desoxycholate
199
STRING TEST
 0.5% SODIUM DESOXYCHOLATE
 MIX WITH COLONY
 PUT LOOP IN MIXTURE AND RAISE UP
 V. CHOLERAE PRODUCES MUCOID
STRING
200
201
Vibrio
Most species are generally susceptible to the vibriostatic
compound 0/129 150-microgram (2,4-diamino-6,7diisopropylpteridine)
Exhibiting a zone of inhibition on either MuellerHinton or trypticase soy agar
 MHA 4% NaCL if no growth on MHA
202
Vibrio cholerae
 Negative for Gas from Glucose
 Positive for Growth in 6% NaCl
 Positive for Sucrose
 Negative for Lactose
 Positive for ODC & LDC
 Negative for ADH
203
204
205
Antigenic Structure
 Three major subgroups of V. cholerae are
 V. cholerae 01 /
 V. cholerae 0139/ Bangal
 Strains of V cholerae 01 and V. cholerae 0139 are associated
with epidemic cholera
 V.cholerae non-O1 / NAG
 02 – 138 NOT EPIDEMIC ASSOCIATED
 Strains that phenotypically resemble V. cholerae but fail to
agglutinate in 01 antisera are referred to as V. cholerae nonO1
206
V.cholerae serogroup 0139
The emergence of V.cholerae serogroup 0139,
originating in Madras, India
It is believed that resurgence of this strain
will be the cause of the eighth pandemic of cholera.
Some of these strains share cross-reacting antigens
with Aeromonas trota
207
Antigenic Structure
Based on the composition of the 0
antigen,V.cholerae 01 organisms are divided into
the following serotypes:
 Ogawa (A, B) , 1377 / IRAN
 Inaba (A, C) , 1384 -1387
 Hikojima (A, B, C)
208
Biogroups
V. cholerae 01 strains occur in two biogroups:
 Classic
 El Tor
EI Tor has been the predominant biogroup in the last
two pandemics
Recent studies in Bangladesh, however, indicate a
rapidly occurring re-emergence of the classic biogroup
209
Biogroups
The EI Tor biogroup differs from the classic in that :
EI Tor is Voges-Proskauer positive
Hemolyzes erythrocytes
Inhibited by polymyxin B (50 microgram)
210
211
Reporting
Vibrio cholorae serogroup O1 or
O139 or (non-O1 or non-O139 )
,serotype of O1 strains (Inaba
,Ogawa ,and Hikojima) ,biotype (El
Tor or Classic)
212
Serogrouping
It generally is considered sufficient for most clinical
laboratories below the level of reference laboratory
simply to screen their presumptive V.cholerae isolates
with commercially available polyvalent 01 antiserum
However, some non-O 1 V.cholerae misidentified
asV.cholerae O1 and vice versa have been reported
213
214
215
216
217
Treatment and management
The administration of antimicrobial agents can
shorten the duration of diarrhea and thereby
reduce fluid losses
Resistance to tetracycline and doxycycline has
been reported
218
Vibrio cholerae CLSI /NCCLS
Testing Conditions
Medium: Mueller-Hinton agar
Inoculum: Growth method or direct colony
suspension, equivalent to a 0.5 McFarland
standard
Incubation: 35 ± 2 °C; ambient air; 16 to 18
hours
219
Vibrio cholerae CLSI /NCCLS
 The results of disk diffusion tests for ampicillin, tetracycline,
trimethoprim-sulfamethoxazole, correlate well with results
determined by broth microdilution MIC
 Disk diffusion tests should not be used for erythromycin,
because there is a poor correlation with MIC results
 AST for the first 30-50 isolates at the beginning of an epidemic
then every 2-6 months
220
Vibrio cholerae CLSI /NCCLS
221
Vibrio cholerae CLSI /NCCLS
Antimicrobial agents for treating
cholera patients include
furazolidone > 18 mm &
Ciprofloxacin > 21 mm are also
effective
222
223
AEROMONAS
.
224
225
Aeromonas
Aeromonas spp. There are currently 14 valid
species in the genus
Aeromonas are currently recognized as human
pathogens causing a variety of clinical infections
including gastroenteritis, cellulitis, and
bacteremia
226
Aeromonas/ Intestinal Infections
Five diarrheal presentations
An acute, secretory diarrhea often accompanied by
vomiting
An acute, dysenteric form of diarrhea with blood and
mucus
A chronic diarrhea usually lasting more than 10 days
A cholera-like disease including rice-water stools
Traveler's diarrhea
227
Aeromonas/ Intestinal Infections
 Most cases are self-limiting,
 In the pediatric and geriatric populations, supportive therapy and
antimicrobials are often indicated
 Most frequently
 A. veronii biovars sobria
 A. caviae
• Pediatric diarrhea
 A. hydrophila
228
Aeromonas/ Intestinal Infections
A. veronii bv. sobria has been linked to choleralike disease characterized by abdominal pain,
fever, and nausea
Complications, usually from A. hydrophila and
A. veronii bv. sobria include hemolytic uremic
syndrome or kidney disease that may require a
kidney transplant
229
Aeromonas/Extraintestinal Infections
 Septicemia and wound infections are the most common
 A. veronii bv. Sobria in Septicemia
 Most aeromonas wound isolates are
 A. veronii bv. Sobria
 A. hydrophila
 A. schubertii :Exclusively from aquatic wound infections
and blood
 leech Hirudo medicinalis (leech therapy ) has a symbiotic relationship
with the aeromonads within its gut ; A. veronii bv. Sobria & A. hydrophila
230
Laboratory Diagnosis / Culture Media
 Aeromonas grow readily on most media used for both routine and stool
cultures.
 After 24-hour incubation at 35° C, aeromonas appear as large round,
raised, opaque colonies with an entire edge and a smooth, often mucoid
surface.
 Often an extremely strong odor is present
 Pigmentation ranges from translucent and white to buff
 Hemolysis is variable on SBA,with most species displaying B-hemolysis.
231
232
233
234
235
236
Aeromonas
 Oxidase positive
 Glucose-fermenting
 Gram-negative rods
 Growth on MacConkey
 Most are motile
Most are INDOLE +
CATALASE +
TCBS GROWTH - / +
237
Identification
 Oxidase Positive
 Oxidase test on B-hemolytic colonies
 Indole Positive
 Spot indole on B-hemolytic colonies
 Ability to ferment glucose distinguishes Aeromonas from
Pseudomonas
 Aeromonas are resistant to 0/129, whereas the Vibrio spp. are
sensitive
238
Aeromonas
 Negative for Growth in 6% NaCl
 Mostly Positive for Gas from Glucose ;
 except A.caviae
 Aeromonas ; mostly Negative for Growth on TCBS
 Aeromonas ; Negative for ODC
 Aeromonas ; Positive for ADH
 except A.veronii bv veronii
 Aeromonas ; Positive for LDC
 except A.caviae
239
240
241
242
243
244
245
246
Plesiomonas shigelloides
.
247
248
Plesiomonas /Epidemiology
Plesiomonas shigelloides is found in both soil and
aquatic environments
But because of intolerance to increased NaCl and a
minimum growth temperature of 8° C, they are
generally found only in the fresh waters of tropical and
subtropical climates
Like the genus Aeromonas, they are widely distributed
among both warm- and cold-blooded animals
249
Plesiomonas shigelloides
Potential cause of enteric disease in humans
Probably underreported because of the similarity
to Escherichia coli on most ordinary enteric
media
 Reported cases in mostly in adults
250
Plesiomonas /Gastroenteritis
 Three major clinical types of gastroenteritis
 More common watery or secretory diarrhea
 Subacute or chronic disease that lasts between 14 days and 2
to 3 months
 Invasive, dysenteric form that resembles colitis
 On average, 25% to 40% of all patients present with fever or vomiting or
both, and the single most common clinical symptom for all such patients is
abdominal pain
251
Plesiomonas
Because of phenotypic characteristics, the genus
Plesiomonas was formerly in the family
Vibrionaceae
Recent phylogenetic studies have presented
evidence that Plesiomonas is actually closely
related to members of the family
Enterobacteriaceae, particularly the genus
Proteus
252
Plesiomonas
Like the Vibrios and Aeromonas




Oxidase-positive
Glucose-fermenting
Facultatively anaerobic
Gram-negative rods
 P.shigellodes cells tend to be pleomorphic gram-negative
rods
 Motile
 Plesiomonas shigelloides is the only species in the genus
253
Plesiomonas shigelloides
 The genera Plesiomonas and Shigella share both biochemical
and antigenic features
 Plesiomonas often cross-agglutinate with
 Shigella sonnei
 S. dysenteriae
 S. boydii
 Hence the species name shigelloides
254
Plesiomonas / Laboratory Diagnosis
 Plesiomanas grows readily on most media routinely used in the
clinical laboratory
 After 18 to 24 hours incubation at 35° C, shiny, opaque,
nonhemolytic colonies appear, with a slightly raised center and
a smooth and entire edge
 Because most strains ferment lactose the easiest screening
procedure is an oxidase test performed on colonies from
nonselective media, such as SBA
255
Plesiomonas / Identification
Sensitivity to the vibriostatic agent 0/129 separates it
from Aeromanas
Ability to ferment Inositol separates it from all
Aerornanas and nearly all Vibria spp
Its unique profile of positive Ornithine and Lysine
decarboxylases and Arginine dihydrolase reactions,
combined with the fermentation of inositol
256
Plesiomonas / Laboratory Diagnosis
Certain of plesiomonads from
stool strains are inhibited on
eosin-methylene blue or
MacConkey agar
257
258
Plesiomonas /Gastroenteritis
 Most cases are self-limited,
 Antimicrobial therapy is indicated in severe and prolonged
cases
 Quinolones / Ciprofloxacin
 Cephalosporins
 Carbapenems
 Trimethoprim-sulfamethoxazole
259
Aeromonas /Gastroenteritis
Most cases of associated gastroenteritis are selflimited
Quinolones / Ciprofloxacin
Trimethoprim-sufamethoxazole
Aminoglycosides
Variable Susceptibility to piperacillin as well as
the cephalosporins
260
Aeromonas & Plesiomonas CLSI
2006 / M45-P
Aeromonas spp. are uniformly resistant to
ampicillin; however, susceptibility to amoxicillinclavulanic acid and cefazolin differs among
species
Aeromonas strains may possess multiple, distinct,
inducible beta-lactamases and like other genera
with inducible beta-lactamases, resistance may
emerge during therapy with a beta-lactam
261
Aeromonas & Plesiomonas CLSI
2006 / M45-P
Plesiomonas spp. are resistant to
penicillins due to the production of
penicillinases.
As a result, testing of P. shigelloides to
ampicillin is not recommended.
262
Aeromonas & Plesiomonas CLSI
2006 / M45-P
Testing Conditions
Mueller-Hinton agar for disk diffusion
testing
Inoculum: Direct colony suspension,
equivalent to a 0.5 McFarland standard
Incubation: 35 °C; ambient air; 16-20
hours
263
Aeromonas & Plesiomonas CLSI
2006 / M45-P
 Agents to Consider for Primary Testing
 Amoxicillin-clavulanic acid
 Fluoroquinolones
 Trimethoprim-sulfamethoxazole
 3rd- or 4th-generation cephalosporins
 Testing is often limited to isolates from extraintestinal sites.
 Perform AST on Aeromonas & Plesiomonas in fecal culture
& report. ASM
264
Aeromonas & Plesiomonas CLSI
2006 / M45-P
265
Aeromonas & Plesiomonas CLSI
2006 / M45-P
266
267
268