CLINICAL BACTERIOLOGY (LECTURE)

Some produces H2S gas

- Name Change: In 2020, a taxonomy change was (blackening of the colony or medium)
Triple Sugar Iron Agar (TSI)
of a new scientific order. o Salmonella
- are now a family within o Proteus
o Arizona
Erwinaceae, Pectobacteriaceae, Yersiniaceae, o Citrobacter
Hafniaceae, Morganellaceae, and Budvicaceae o Edwardsiella
- Taxonomic ranks or levels in ascending order Lysine Iron Agar (LIA)
Rank or Level Example o Salmonella
Species E. coli o Arizona
Genus Escherichia o Citrobacter
Family Enterobacteriaceae o Edwardsiella
Order Enterobacterales
Class -Proteobacteria
Phylum Proteobacteria
Domain Bacteria
- Often referred to as enterics
- Gram-negative bacilli and coccobacilli
- Oxidase negative
- Do not produce cytochrome oxidase except for
Plesiomonas
- All ferment glucose (Reactions: A/A or K/A)
- Reduce nitrate to nitrite except for
Photorhabdus and Xenorhabdus
- Motile at body temperatures except for
Klebsiella, Shigella and Yersinia
- Appear large, moist, and gray on sheep blood
agar (SBA), chocolate (CHOC) agar, and most
nonselective media
- Generally aerogenic except Shigella Some produces Urease Enzyme
Rapid Urease Producers
The following may or may not be aerogenic: o Proteus
Salmonella o Providencia
Serratia o Morganella
Proteus Slow Urease Producers
Providencia o Citrobacter
Klebsiella o Klebsiella
o Enterobacter except E. gergoviae
Can be differentiated based on lactose o Yersinia
o Serratia
fermentation:
Rapid Lactose Fermenter
Color Pink
Reaction A/A
o Escherichia
o Enterobacter (can have K/A reaction)
o Klebsiella
Late Lactose Fermenter
Color Slightly colorless or pigmented
Reaction A/A or K/A
o Hafnia
o Serratia (reddish top) Some are Deaminase producing:
o Citrobacter Proteus
o Salmonella arizonae Providencia
o Shigella sonnei Morganella
o Yersinia enterocolitica
Non-lactose Fermenter Can be differentiated based on Lysine
Color Colorless
Reaction K/A
Decarboxylation (LDC)
o All Salmonella except S. arizonae LDC positive (+)
o All Shigella except S. sonnei Color Reaction Purple
o All Yersinia except enterocolitica o Klebsiella
o Proteus o Escherichia
o Providencia o Edwardsiella
o Morganella o Serratia
o Edwardsiella o Salmonella except S. paratyphi A.
o Hafnia
 CLINICAL BACTERIOLOGY (LECTURE)
LDC negative (-)
Color Reaction Yellow
o Proteus
o Providencia
o Morganella
o Citrobacter
o Yersinia
o Enterobacter except E. aerogenes (now
known as Klebsiella aerogenes) and E.
gergoviae (now known as Pluralibacter
gergoviae)
o Shigella
ANTIGENS
O antigen or Somatic antigen This is a heat-
stable antigen located on the cell wall
H antigen or Flagellar antigen This is a heat-
labile antigen found on the surface of flagella,
structures responsible for motility
K antigen or Capsular antigen - This is a heat-
labile polysaccharide found only in certain
encapsulated species
o E.g., K1 antigen of E. coli and the Vi
antigen of Salmonella enterica subsp.
enterica serotype typhi
PLASMIDS
- Can provide antimicrobial resistance genes
- E. coli, K. pneumoniae, and Klebsiella oxytoca
produce plasmid-mediated extended spectrum
B-lactamases (ESBLs) including carbapenemases
cephalosporinases, or metllo-B-lactamases
which can inactivate extended-spectrum
cephalosporins (e.g., cefotaxime), penicillins,
and aztreonam
- Strains harboring these plasmids have been
found in healthy volunteers and hospitalized
patients
- Resides in the gastrointestinal tract (GIT)
- They are often commensals, causing no harm,
and yet they can be responsible for a large
number of opportunistic infections when
introduced into inappropriate body sites
- 2 Categories:
o Opportunistic pathogens
o Primary pathogens includes
Salmonella spp., Shigella spp., and
Yersinia spp.
Escherichia coli (IMVC ++--)
- Colon bacillus/ golden bacillus
- Indole (+), Methyl Red (+), Vogues-Proskauer (-),
Citrate (-)
- Used as a primary marker of fecal contamination
in water quality testing
- Motile and generally possess adhesive fimbriae
and sex pili and O, H and K antigen
- MAC Agar: It usually appears as a lactose-
positive (pink) colony with a surrounding area of
precipitated bile salts
- EMB Agar: it has a green metallic sheen
- E. coli is associated with the following properties:
o Fermentation of glucose, lactose,
trehalose and xylose
o Production of indole from tryptophan
o Glucose fermentation by the mixed acid
pathway: Methyl Red (+) positive and
Voges-Proskauer (-)
o Does not produce H2S DNase, urease or
phenylalanine deaminase
o Cannot use citrate as a sole carbon
source
Uropathogenic Escherichia coli
- Most common cause of UTIs in humans
Gastrointestinal Pathogens
Enterovirulent E. coli or diarrheogenic E. coli:
1. Enterotoxigenic Escherichia coli (ETEC)
- Associated with diarrhea of adults and especially
children in tropical and subtropical climates,
especially in developing countries, where it is
one of the major causes of infant bacterial
diarrhea
- Most common cause of a diarrheal disease
sometimes referred to as
- Produces heat-labile enterotoxin
- Spread commonly via consumption of
contaminated food or water
2. Enteropathogenic Escherichia coli (EPEC)
- Known to cause infantile diarrhea
- O serogroups: cause of diarrhea
- H antigenic: intestinal infections
- Stool: watery with mucus but without blood
 CLINICAL BACTERIOLOGY (LECTURE)
3. Enteroinvasive Escherichia coli (EIEC)
- Produce dysentery with direct penetration,
invasion and destruction of the intestinal
mucosa
- Produces Shiga-like toxin hence causing Shigella-
like diarrhea
- Stool: watery with blood, pus, and mucus
4. Enterohemorrhagic Escherichia coli (EHEC)
- Also known as Verotoxigenic E. coli (VTEC)
- O157:H7 has since been associated with
hemorrhagic diarrhea, colitis, and hemolytic
uremic syndrome (HUS)
- Watery diarrhea that progresses to bloody
diarrhea with abdominal cramps and low-grade
fever or an absence of fever
- Stool: watery with excessive blood
5. Enteroadherent Escherichia coli
- Diarrheal syndromes and UTIs
- 2 Types:
o Diffusely Adherent Escherichia coli
(DAEC) May be associated with UTIs
and diarrheal diseases. Uropathogenic
DAEC strains are closely associated with
cystitis in children and acute
pyelonephritis in pregnant women
o Enteroaggregative Escherichia coli
(EAEC) Causes diarrhea by adhering to
the surface of the intestinal mucosa.
These strains are found to adhere to
Hep2 cells, packed in an aggregative
- pattern on the cells and
between the cells by means of fimbriae
- Extraintestinal Infections
o Most common causes of septicemia and
meningitis among neonates, accounting
for about 40% of the cases of gram-
negative meningitis
o Capsular antigen K1: most documented
virulence factor associated with
neonatal meningeal infections
Other Escherichia Species
Escherichia hermannii
o A yellow-pigmented organism that has
been isolated from cerebrospinal fluid
(CSF), wounds and blood
Escherichia vulneris
o Has been isolated from humans with
infected wounds
o More than half of the strains of E.
vulneris also produce yellow-pigmented
colonies
Escherichia albertii
o Associated with diarrheal disease in
children
Klebsiella and Raoutella (IMVC --++)
- Usually found on the intestinal tract of humans
and animals or free-living in soil, water, and on
plants
- Associated with various opportunistic and
hospital-acquired infections, particularly
pneumonia, wound infections and UTIs
- Biochemical Reactions:
o Most grow on Simmons citrate and in
potassium cyanide broth
o None produce H2S
o A few hydrolyze urea slowly
o All give a negative reaction with the
methyl red test and a positive reaction
with the Voges-Proskauer test
o With a few exceptions, no indole is
produced from tryptophan
o Motility is variable
Klebsiella pneumoniae
- Most commonly isolated species and has the
distinct feature of possessing a large
polysaccharide capsule
- The capsule offers the organism protection
against phagocytosis and antimicrobial
absorption, contributing to its virulence
- The capsule is also responsible for the moist,
mucoid colonies characteristic of K. pneumoniae
- Capsulated: mucoid colonies or tend to
Klebsiella oxytoca
- The only indole positive Klebsiella species
- Has been isolated in stool and blood cultures
- Isolates have been linked to antibiotic-
associated hemorrhagic colitis
Klebsiella pneumoniae subsp. Ozaenae
- Highly associated with the presence of plasmid-
mediated ESBLs, contributing to the large
numbers of antimicrobial-resistant hospital
acquired infections seen today
- VP negative
Klebsiella pneumoniae subsp. Rhinoscleromatis
- Has been isolated from patients with
rhinoscleroma
Raoutella (Klebsiella) ornithinolytica
- Indole and ornithine decarboxylase-positive
Raoutella (Klebsiella) planticola
- Have been isolated from the urine, respiratory
tracts and blood of humans
Klebsiella variicola
- Has been isolated from primarily sterile sites
 CLINICAL BACTERIOLOGY (LECTURE)
Enterobacter, Cronobacter,
and Pantoea (IMVC --++)
- Motile
- Resembles Klebsiella when growing on MAC agar
- grow on Simmons citrate medium and in
potassium cyanide broth
- The methyl red test is negative, and Voges-
Proskauer test is positive.
- Usually produce ornithine decarboxylase
- Lysine decarboxylase is produced by most
species but not by E. gergoviae or E. cloacae
Enterobacter cloacae
- Predominant clinical isolate
- Associated with bacteremia, respiratory
tract infections, UTI, and wound infection in
burn patients
- Also contaminated IV fluids and other
hospital instrumentation
Enterobacter. Cancerogenus
(Enterobacter. taylorae)
- Associated with osteomyelitis after
traumatic wounds
- Unique member because it is lactose
negative but ONPG positive
Cronobacter (Enterobacter) sakazakii
- Typically produces a yellow pigment
- Has been documented as a pathogen in
neonates causing meningitis and
bacteremia, often coming from powdered
infant formula
- Isolated from cultures taken from brain
abscesses and respiratory and wound
infections
Enterobacter (Pluralibacter) gergoviae
- Resembles Enterobacter aerogenes
- Rapid Urease Producer (RUP)
- Causes infection of the urinary tract, and
blood
Pantoea (Enterobacter) agglomerans
- Gained notoriety with a nationwide
outbreak of septicemia resulting form
contaminated intravenous fluids
- Lysine-, ornithine-, and arginine- negative or
Enterobacter hormaechei
- Isolated from human sources such as blood,
wounds, and sputum
Enterobacter asburiae
- Similar biochemically to E. cloacae
- Has been isolated from blood, urine, feces,
sputum, and wounds
Enterobacter. dissolvens and
Enterobacter. nimipressuralis
- Newly recognized species with unknown
clinical significance
Serratia (IMVC -V++)
- Opportunistic pathogens associated with
outbreaks in health care settings
- Ferment lactose slowly and are positive for
the o-nitrophenyl-B-D-galactopyranoside
(ONPG) test except S. fonticola
- Ability to produce extracellular DNase
- S. marcescens, S. rubidaea, S. plymuthica
o Often producing a characteristic
pink-to-red pigment, prodigiosin,
especially when the cultures are
incubated at room temperature
o Pigment production is typically a
characteristic in those strains of
environmental origin
Serratia odorifera
- Dirty, musty odor resembling that of rotten
potatoes
- Two biogroups:
o S. odorifera biogroup 1 Isolated
predominantly from the respiratory
tract and is positive for sucrose,
raffinose, and ornithine. In addition,
biogroup 1 may be indole-positive (60%)
o S. odorifera biogroup 2 negative for
sucrose, raffinose, and ornithine and has
been isolated from blood and CSF.
Biogroup 2 may also be indole-positive
(50%)
Serratia liquefaciens
- Very similar to S. marscesens but differentiated
to S. marscesens by its ability to ferment
arabinose
 CLINICAL BACTERIOLOGY (LECTURE)

Serratia rubidaea Proteus penneri

- Also produces red pigment prodigiosin - Also swarm on nonselective media
- Has been isolated from patients with diarrhea
Hafnia (IMVC -V+-)
- Has been linked to gastroenteritis and is
occasionally isolated from stool cultures
- A delayed positive citrate reaction is a major
characteristic of Hafnia
- Has been isolated from many anatomic sites in
humans and in the environment
- All Klebsiella, Enterobacter, Serratia, and Hafnia
are biochemically similar.
o Voges-Proskauer (VP) positive (+)
o Produce large amount of gas in the TSI
butt portion
Morganella (IMVC ++--)
Hafnia alvei - Motile but does not swarm
- Representative organism of this genus; formerly - Lactose, citrate, H2S and LDC negative (-)
Enterobacter alvei - Urease and Deaminase, ODC positive (+)
- Although it has been recovered from stool and - M. morganii is the only known species in this
wound specimens, its clinical significance is still genus
questionable - Infections include nosocomial infections of UTI
- Resembles Enterobacter. To differentiate: and wound
o Late Lactose Fermenter - Proteus, Providencia, and Morganella
o Citrate Negative o All are motile
- To differentiate from Serratia: o Methyl red (MR) positive
o DNAse negative o Deaminase positive
o Lipase negative o LIA-R/A (red/yellow)
o Gelatinase negative
Providencia (IMVC ++-+)
Proteus Providencia rettgeri
- Widely disseminated in the environment, are - Is a documented pathogen of the urinary tract
normal intestinal microbiota and are recognized and has caused occasional outbreaks in health
as opportunistic pathogens care settings
- Ability to deaminate the amino acid - It has also been implicated in diarrheal disease
phenylalanine among travelers
- Did not ferment lactose
- Unique Characteristics: Providencia stuartii
o Rapid Urease Producers (RUP-RXN - Has been implicated in outbreaks in burn units
within 4 hours) and has been isolated from urine cultures
o Typical swarming motility (growth in
waves or swarms) Providencia alcalifaciens
o H2S producing in TSI but not in LIA
- Most commonly found in the feces of children
with diarrhea
P. mirabilis and P. vulgaris
- Can produce swarming Providencia rustigianii
- Colonies on nonselective media such as SBA
- Rarely isolated, and its pathogenicity also
- burnt chocolate
remains unproven
- Thought to play a role in the ascending nature of
Proteus associated UTIs
- P. mirabilis: indole (-) and ornithine (+) Providencia heimbachae
- P. vulgaris: indole (+) and ornithine (-) - Has yet to be isolated from any clinical
o Ferment sucrose and gives an acid/acid specimens
reaction in triple sugar iron (TSI) agar
 CLINICAL BACTERIOLOGY (LECTURE)
Edwardsiella (IMVC ++--)
- Negative for urea and positive for lysine
decarboxylase, H2S, and indole and do not grow
on Simmons citrate
Edwardsiella tarda
- An opportunistic, causing bacteremia and wound
infections
Edwardsiella hoshinae
- Has been isolated from snakes, birds, and water
Edwardsiella ictaluri
- Causes enteric septicemia in fish
Erwinia and Pectobacterium
- Plant pathogens and are not significant in human
infections
- Erwinia organisms grow poorly at 37C and fail to
grow on selective media, such as EMB and MAC,
and other differential media typically used for
the isolation of enterics
Citrobacter
- Resembles Salmonella but differs because it is:
o ONPG positive
o LDC negative
o Slow Urease Producer (SUP)
- Important human pathogen which are
commonly associated with UTIs and respiratory
tract infections
Salmonella (IMVC -+--)
- gram-negative, facultatively anaerobic bacilli
- Produce clear, colorless, non-lactose fermenting
colonies
- Colonies with black centers are seen if the media
(e.g., HE or XLD) contain indicators for H2S
production
- Biochemical Features:
o Do not ferment lactose
o They are negative for indole, Voges-
Proskauer test, phenylalanine
deaminase and urease
o Most produce H2S; a major exception is
Salmonella paratyphi A, which does not
produce H2S
o Does not grow in medium containing
potassium cyanide
- Pathogenic for humans and animals
- Anima sources includes:
o Poultry (chicken, ducks and turkeys)
o Pets (dogs, cat, hamsters, turtles)
o Others (pigs, sheep, cows, donkeys,
snakes, parrots)
- Causes enteric/typhoid fever S. typhi (most
common cause of typhoid fever) S. paratyphi A/B
- S. cholerasuis (most common cause of
bacteremia)
- S. typhimurium (most common cause of
enterocolitis and gastroenteritis)
Salmonella bongori
- A rarely isolated species that is named after the
town of Bongor in Chad, Africa, and was initially
isolated in 1966 from a lizard.
- It is usually isolated from cold-blooded animals
and the environment
Virulence Factors
Fimbriae
Enterotoxin
o Produced by certain Salmonella strains
that cause gastroenteritis
Antigenic Structures
Somatic O Antigens
o Heat stable
o Located on the outer membrane of the
cell wall
Flagellar H Antigens
o Heat labile
Capsular K Antigens
o Heat-labile Vi
o Plays a significant role in preventing
phagocytosis of the organism
o Blocks the O antigen during serologic
typing but may be removed by heating
Clinical Infections
- Acute gastroenteritis or food poisoning
characterized by vomiting and diarrhea
- Typhoid fever, the most severe form of enteric
fever, caused by Salmonella serotype Typhi, and
enteric fevers caused by other Salmonella
serotypes (e.g., Salmonella paratyphi and
choleraesuis)
- Nontyphoidal bacteremia
Shigella (IMVC ++--)
- Cause bacillary dysentery
- Inert bacilli (yields negative reaction to most
tests)
o They are nonmotile
o Except for certain types of S. flexneri,
they do not produce gas from glucose
o They do not hydrolyze urea
o They do not produce H2S
o They do not decarboxylate lysine
 CLINICAL BACTERIOLOGY (LECTURE)
Antigenic Structures
- All Shigella spp. posses O antigens, and certain
strains can possess K antigens
- Nonmotile; therefore, they lack H antigens
Clinical Infections
Shigellosis/ Bacillary Dysentery
o Vary from asymptomatic to severe forms
of the disease
o 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
o Early Stage
Incubation period for 1-7 days
Fever, abdominal cramping and
pain, diarrhea
o Diarrheic Stage
Watery diarrhea for 3 days
o Dysenteric Stage
Frequent stools with blood, puss
and mucus
Bacteria had invaded the lining
of the GIT
Yersinia
- Small coccobacilli that produce small pinpoint
colonies on MAC
- Y. enterocolitica LLF, ONPG positive, weakly
fermentative, requires cold enrichment
- Normal fecal flora, hence use a selective media:
Cefsulodin, Irgasan, Novobiocin (contains:
mannitol, peptones, yeast, neutral red indicator,
crystal violet)
- of Y. enterocolitica
- Selective also for Aeromonas spp. and
Plesiomonas shigelloides
- 3 forms:
o Bubonic or Glandular Form
Most common, usually results
from an infected flea
Symptoms appear 2 to 5 days
after infection
high fever with painful regional
lymph nodes known as buboes
(swollen lymph nodes)
o Septicemic Form
Occurs when the bacteria
spread to bloodstream
o Pneumonic Form
Occurs secondary to bubonic
plague or septicemic form when
organisms proliferate in the
bloodstream and respiratory
tract
Can be a primary infection if the
bacteria are inhaled
Fatality rate is high essentially
100% in untreated patients
Yersinia pestis
- Gram-negative, short, plump bacillus
- bipolar staining appearance
- Although it grows at 37°C, it has a preferential
growth temperature of 25°C to 30°C
Yersinia enterocolitica
- Found in a wide variety of animals, including
domestic swine, cats, and dogs
- Several Forms
o Acute enteritis
o Appendicitis-like syndrome
o Arthritis
o Erythema nodosum (inflammation of fat
cells under the skin, tender red nodules)
Yersinia pseudotuberculosis
- Pathogen primarily of rodents, particularly
guinea pigs
- Causes a disease characterized by caseous
swellings called pseudotubercles
- Appears as a typical-looking plague bacillus
- It can be differentiated from Y. pestis by its
motility at 18°C to 22°C, production of urease
and ability to ferment rhamnose
Budivicia
- Usually found in water, however, they
occasionally occur in clinical specimens
Buttiauxella
- Only B. agrestis and B. noackiae have been
isolated from human specimens
- Biochemically, these organisms are similar to
both Citrobacter and Kluyvera species, but DNA
hybridization distinctly differentiates
Buttiauxella from both genera
 CLINICAL BACTERIOLOGY (LECTURE)

Cedecea
- Composed of five species: C. davisae, C. lapagei,
C. neteri and Cedecea species types 3 and 5
- Most have been recovered from sputum, blood,
and wounds
Ewingella
- Ewingella americana is the only species of the
genus Ewingella
- Most isolates have come from human blood
cultures or respiratory specimens and exhibit
resistance to multiple antimicrobial agents
Trabulsiella
- isolated from vacuum-cleaner contents on the
island of Guam when environmental indoor dirt
samples were being collected
Yokenella
- They are biochemically similar to Hafnia but
differ primarily by yielding negative Voges-
Proskauer test results

Kluyvera
- Three closely related species: K. ascorbata (the
most common clinical isolate), K. cryocrescens,
and K. georgiana
- found in respiratory, urine and blood cultures
- Most strains are nonpigmented but occasional
isolates may produce a reddish-blue or violet
pigment

Leclercia
- L. adecarboxylata, which can have a yellow
pigment but only on initial isolation
- Similar IMViC reactions to E. coli
- negative for lysine and ornithine decarboxylase
and arginine dihydrolase

Leminorella
- Was proposed as a genus with two species: L.
grimontii and L. richardii
- Produce H2S and have shown weak reactions
with Salmonella antisera

Moellerella
- The genus Moellerella contains one species, M.
wisconsensis
- isolated from feces in two cases of diarrhea,
infected gallbladders and a bronchial-aspirates

Obesumbacterium
- Fastidious, slow-growing organisms at 37°C and
have not been found in human specimens

Photorhabdus
- Three species:
o P. luminescens
o P. asymbiotica
o P. temperate
- Their natural habitat is the lumen of
entomopathogenic nematodes, but strains have
occasionally been isolated from human
specimens

Rahnella
- Rahnella aquatilis is the name given to a group
of water bateria that are psychrotolerant,
growing at 4°C