BACTERIOLOGY SEMINAR 1 MIDTERMS

03/10/2023
LACTOSE FERMENTERS
- Contains lactose, red or pink in color
FAMILY ENTEROBACTERIACEAE - Converts glucose
PRIMARY PATHOGENS (capable of causing disease) YEKSS
NON-LACTOSE FERMENTERS
- Yersinia spp. - Does not contain lactose, yellow in color
- Escherichia coli - Does not convert glucose
- Klebsiella pneumoniae
- Salmonella spp. STRUCTURE OF BACTERIA
- Shigella spp.

OPPORTUNISTIC PATHOGENS (can only cause disease under

certain conditions or its host)
- Klebsiella pneumoniae
- Escherichia coli
- Enterobacter spp.
- Proteus
- Serratia
- Morganella
- Providencia

NON-PATHOGENIC (cannot cause harm/disease) CHEEK

- Citrobacter spp.
- Hafnia spp.
GRAM STAIN:
- Edwardsiella spp. - Used to identify if the bacteria is Gram Negative (Pink) or
- Ewingella spp. Gram Positive (Purple)
- Khyvera spp. - Note: Purple and G. positive kasi makapal ang cell wall nila, na
aabsorb and stain. while and G. negative pink kasi na
MATERIAL USED dedecolorize yung stain kasi manipis ang cell wall, nag lighten
- Agar plate ang color.
- This must be released first to identify what type of bacteria is
present, for the physician to provide antibiotics for the patient.
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- Edwardsielleae
CULTURE AND SENSITIVITY TURNAROUNT TIME: - Salmonelleae
- URINE CULTURE: 2 – 3 days - Citrobacteriaceae
- SPUTUM CULTURE: 2 – 3 days - Klebsielleae
- ASPIRATES, WOUNDS: 3 – 5 days o Klebsiella
- BLOOD CULTURE: 5 – 7 days o Enterobacter
o Pantoea
UTI – prolonged UTI can cause SEPSIS (presence of bacteria in the o Cronobacter
blood). o Hafnia
SUSCEPTIBLE – drugs that can be used by patient against bacteria o Serratia
INTERMEDIATE – this can be effective or not, nag aadjust and
- Proteeae
dosage
o Proteus
RESISTANT – Drugs cannot be used for specific type of bacteria.
o Morganella
o Providencia
ENTEROBACTERIACEAE ANTIBIOTIC INTERMEDIA
SENSITIVE RESISTANT
S TE
DOMAIN: Bacteria Amoxycillin 20 23 55
PHYLUM: Proteobacteria Amikacin 33 38 27
CLASS: Gamma Proteobacteria Aztreonam 19 17 62
ORDER: Enterobacteriales Carbenicillin 17 23 58
FAMILY: Enterobacteriaceae Ceftazidime 24 20 54
Ciprofloxacin 23 37 38
CASE ANALYSIS TO BE CONSIDERED: Cotrimazole 22 21 55
- Sample, General Characteristics (Microscopic, Macroscopic,
Biochemical Test rxn, Age, Gender, Culture Media Used, Reasons for different types of antibiotics: because every
IMVIC rxn, LIA rxn, Motility. antibiotic has different mode of actions.

TRIBES AND GENUS OF FAMILY ENTEROBACTERIACEAE CLASSIFICATION OF ENTEROBACTERIACEAE:

More than 15 different genera
- Escherichieae
o Escherichia Escherichia
o Shigella Edwardsiella
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Enterobacter - All glucose fermenters
Shigella - Motile except: SKY
Salmonella o Shigella
Serratia o Klebsiella
Citrobacter o Yersinia
Klebsiella
- Klebsiella and Enterobacter exhibits mucoid colonies on plated
Hafnia
media
CHARACTERISTICS OF ENTEROBACTERIACEAE - Proteus exhibits swarming motility on plated media
- E. coli are Beta hemolytic in Sheep Blood Agar (SBA)
- All Enterobacteriaceae - Stool enrichment protocols and use of differential and selective
o Gram-negative rods (PINK) media.
o Ferment glucose with acid production
o Reduce nitrates into nitrites CLINICAL SIGNIFICANCE OF ENTEROBACTERIACEAE
o Oxidase negative
- Ubiquitous in nature (seen everywhere)
- Facultative anaerobic
- Commonly found in the GI tract except for SSY
o (Can grow with or without oxygen)
o Salmonella
- Motile except Shigella and Klebsiella
o Shigella
- Non-Capsulated except Klebsiella
o Yersinia
- Non-fastidious
- Commensals
o Mabilis tumubo
- Opportunistic pathogens (intestinal and extra intestinal)
o Fastidious: matagal patubuin
- Primary intestinal pathogens
- Grow on bile containing media (MacConkey agar)

GENERAL CHARACTERISTICS
- Major clinical isolates:
o E. coli, K. pneumoniae, P. mirabilis
- Enterics TIPS FOR POSITIVE AND NEGATVE REACTIONS
- All are oxidase negative except Plesiomonas
- Biochemically similar with Vibrio and Aeromonas - If you cannot remember both, remember 1
- Gram-negative - It is better to remember positive reactions
- Non-spore forming
- Facultative anaerobic bacilli/coccobacilli
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- Associate biochemical reactions (positives) to attribute o Edwardsielleae
profiles of a person or an object or a concept. o Citrobacteriaceae
o Example 1: o Salmonelleae
 Klebsiella o Proteeae
 Klebsiellarge - UREASE (+)
 Large mucoid o Citrobacteriaceae
 Lactose fermenting colonies o Klebsielleae
 MR (+) Methyl Red o Proteeae
 Citrate (+) o Yersinieae
A large man with pink mucus wearing a red coat and - INDOLE (+)
a blue cap o Escherichieae
 Large: mucoid o Edwardsielleae
 Red: positive o Citrobacteriaceae
 Blue: color of citrate when positive, but o Proteeae
green when negative o Yersinieae
- METHY RED/MR (-)
o Example 2:
o Klebsielleae
 Proteus
- VOGUES-PROSKAUER (+)
 Proteuswarming
o Klebsielleae
 Swarming colonies
- PHENYLALANINE DEAMINASE (+)
 Methyl Red (+)
o Proteeae
 H2S (+)
- MANNITOL (+)
 Indole (+)
o Edwardsielleae
 Motility (+)
 Citrate (+) INDOLE: seen in Sulfur, Indole, Motility (SIM) agar using Ehrlich
A rainbow (because the biochemical testing panel is
Reagent (colorless: negative), (Positive: Pink).
very colorful)
: sulfide – blackening. Indole – taas ng tube.
BIOCHEMICAL CHARACTERISTICS OF TRIBES OF
OPPORTUNISTIC PATHOGENS OF THE FAMILY
ENTEROBACTERIACEAE
ENTEROBACTERIACEAE

- H2S (+) Hydrogen sulfide

Escherichia
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Klebsiella and Raoutella PRIMARY INTESTINAL PATHOGENS OF THE FAMILY
Enterobacter, Cronobacter, Pantoea ENTEROBACTERIACEAE
Serratia
Hafnia - Vomiting, causes pathogen inside our intestine
Proteus - Salmonella, Shigella Agar (SSA) selective media for
Morganella salmonella and shigella only.
Providencia - Cefsulodin-Irgasan-Novobiocin agar (CIN) selective media,
Edwardsiella
Agar useful for Yersinia enterolytica, cultivation 24hrs at 37
Erwinia and Pectobacterium
degrees Celsius.
Citrobacter
SALMONELLA SHIGELLA YERSINIA
Always LACTOSE FERMENTING (LF) in MacConkey agar
plate (MAC)
- Escherichia
- Citrobacter
- Enterobacter
- Serratia

LACTOSE FERMENTERS
Pink colonies, converts glucose, contains lactose Blackening
SSA SSA CIN
NON-LACTOSE FERMENTERS
Yellow in color, cream/white colonies
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BACTERIAL SPECIES AND INFECTIONS THEY COMMONLY Yersinia
- Yersinia pestis Plague
PRODUCED
- Yersinia
BACTERIAL SPECIES DISEASES pseudotuberculosis Mesenteric adenitis, diarrhea
Escherichia coli Bacteriuria, septicemia, neonatal - Yersinia enterolitica
sepsis, meningitis, diarrheal Erwinia spp. Wounds contaminated with soil
syndrome Pectobacterium spp. or vegetation
Shigella spp. Diarrhea, dysentery
Edwardsiella spp. Diarrhea, wound infection, Escherichia coli: Most common Enterobacteriaceae clinical isolate;
septicemia, meningitis, ecteric indicator of water quality (coliform count)
fever
Salmonella spp. Septicemia, enteric fever, Bacteriuria: Bacteria in urine
diarrhea
Citrobacter spp. Opportunistic and hospital Meningitis: inflammation of meninges
acquired infections (wound, Septicemia: Bacteria in Blood
urinary)
Klebsiella spp. Bacteriuria, pneumonia, Dysentery: Bloody diarrhea
septicemia
Opportunistic and hospital acquired infections (wound, urinary):
Enterobacter spp. Opportunistic and hospital
acquired infection, wound LF in MAC, H2S (-).
Serratia spp.
infections, septicemia, Klebsiella spp, Enterobacter spp: Mucoid LF colonies
bacteriuria
Proteus spp. Bacteriuria, wound infection, Serratia spp: Red (Prodigiosin) pigmented colonies
septicemia
Proteus spp: Swarming motility in plated media
Providencia spp. Opportunistic and hospital-
acquired infection, wound Wounds contaminated with soil or vegetation: Environmental-
infections, septicemia, human transmission (anthropogenic activities)
bacteriuria
Morganella spp. Opportunistic and hospital- Prodigiosin: Pigment given by Serratia
acquired infections
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ESCHERICHIA COLI complement
Anti-phagocytosis protects
- E. coli is the most important isolates causing; Escherichia coli from
Capsule
o UTI phagocytosis. Protect the
o Diarrhea organism from serum killing.
The capability of Escherichia
o CNS infection Sequestration of growth
coli to compete nutrients in host
- Primary marker of fecal contamination in water quality factors
cells
testing
- Mostly LACTOSE FERMENTER in MAC
- Exhibits GREENISH-METALLIC SHEEN in EMB
- Presumptive ID:
o A/A in TSI (Yellow/Yellow)
o H2S (-)
 H2S positive (blackening)
 H2S negative (no blackening)
o Indole (+)
 SIM Ehrlich reagent
o Citrate (-)
 Color green (negative)
 Color blue (positive)
VIRULENCE FACTORS OF ESCHERICHIA COLI
Virulence Factors Biological functions
Adherence of bacteria to
gastrointestinal tract;
Of importance in urinary tract
Fimbriae
infections;
And cause mannose-resistant
hemagglutination.
Endotoxin Systemic manifestations of
endotoxic shock, and protect the
bacillus from phagocytosis and
from bactericidal effects of
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Adhesins facilitate firm adhesion TSI: A/A
of Escherichia coli to the MR (+)
gastrointestinal or urinary tract VP (-)
Adhesions mucosa, thereby preventing the SULFIDE (-)
bacteria from being eliminated INDOLE (+)
by the flushing action of voided MOTILITY (+)
urine or intestinal motility. CITRATE (-)
Important in the pathogenesis of
Hemolysins (HlyA) disease caused by Uropathogenic ATCC: American Type Culture Collection
strains of Escherichia coli. TSI REACTIONS:
Enterotoxins
- Yellow: A
Shiga toxins Cytotoxins
- Red: K
Causes increased secretion of
Heat stable toxin - A/A: Yellow/Yellow
fluids
Watery diarrhea due to - A/K: Yellow/Red
Heat labile toxin hypersecretion of fluid into the - K/A: Red/Yellow
lumen of the gut - K/K: Red/Red
Removes iron from mammalian
Siderophores iron transport proteins like
transferring and lactoferrin
Virulence factor: pathogenicity, what causes the signs and symptoms VIRULENT STRAINS OF ESCHERICHIA COLI
of disease. Enterovirulent/Diarrheogenic*

UTI, catheters, pyelonephritis,

Uropathogenic E. coli dominant colon resident, pili
(UPEC) (adherence), cytolysins,
Aerobactin
Enterotoxigenic E. coli Traveler’s diarrhea,
(ETEC) achlorhydria, Heat-labile toxin
Enteroinvasive E. coli
Dysentery
(EIEC)
Enteropathogenic E. coli
Infantile diarrhea
(EPEC)
Enterohemorrhagic E. coli Hemolytic uremic syndrome
(EPEC) (HUS)
 BACTERIOLOGY SEMINAR 1 MIDTERMS
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(EAEC)

CELL CULTURE
ASSAY
- HeLa cell
adherence
assay
- DNA probes
BIOCHEMICAL
ASSAY
- MUG
MOLECULAR
ASSAY
- DNA probes
SEROLOGIC
ASSAY
- Immunoassay
s for LT or ST

Entero/Diffusely adherent E.
coli UTI, diarrhea
(EAEC/DAEC)
Enteraggregative E. coli xxx
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Biochemical reaction against E.

S.No. Result
coli
1 Lactose fermentation +
2 Sucrose fermentation +
3 Dextrose fermentation +
4 Indole production +
5 Mannitol fermentation +
6 Voges Proskauer test +
7 Methyl Red Test +
Gas +
Triple sugar H2S -
8
iron test Slant Yellow
Butt Yellow
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GRAM NEGATIVE BIOCHEMICAL TEST (IMVIC)

Gram Negative Bacteria Indole MR VP Citrate

Escherichia coli + + - -
Enterobacter aerogenes - - + +
Enterobacter cloacae - - + +
Klebsiella pneumoniae - -/+ - +
Klebsiella oxytoca + - + +
-
Citrobacter freundii + - +
H2S Klebsiella pneumoniae
Proteus vulgaris + + - + TSI: A/A
Proteus mirabilis - - - + SULFIDE: (-)
Morganella morganii + + - - INDOLE: (-)
MOTILITY: (-)
ESCHERICHIA COLI MR: (+)
VP: (-)
I M V C CITRATE: (+)
+ + - -

KLEBSIELLA Dozens of bacteria BIOCHEMICAL TESTS- TSI, SIM, Urease and

Citrate
E.
- Opportunistic, hospital acquired/nosocomial infections. coli/Klebsiella/Pseudomonas/Acinetobacter/Shigella/proteus/Serrati
- Pneumonia, wound infections, and UTI a/Morganella and more…
- Presumptive ID
o Currant jelly sputum
o Large, Mucoid (Capsule+), LF in MAC
o Non-motile
o Citrate (+)
o Urease (+)
o Indole (-) K. pneumoniae
o Indole (+) K. oxytoca

Escherichia coli Klebsiella pneumoniae

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TSI: SIM: Urease: Citrate: TSI: SIM: Urease: Citrate: - Hospital acquired UTI, pneumonia, intra-abdominal abscess,
A/A (+) (-) (-) A/A (-) (+) (+) endocarditis in IV drug users
- Citrobacter koseri: nursery outbreaks of neonatal meningitis
Motility : spread of haziness (+) and brain abscess
Citrate positive : Blue - Can be mistaken for Salmonella biochemically
Citrate negative : Green - Presumptive ID:
o Only LF Enterobacteriaceae that is H2S (+)
Indole positive : Pink/Red ibabaw o Citrate (+)
Indole negative : Yellow o MRVP (+, -)
Urease positive : Pink o Urease (+) C. freundii
- Black color: indicates the deposition of hydrogen sulfide,
Urease negative : Yellowish
(H2S) under alkaline conditions
H2S positive : blackening
H2S negative : no blackening

TSI SIM CIT GLU UREASE

ENTEROBACTER -
A/A + + fermenters +
+
- Colonial morphology resembles Klebsiella
- Enterobacter cloacae and Enterobacter aerogenes (most
common species in clinical isolates)
- Wounds, urine, blood, and CSF
- Presumptive ID
o Mucoid, LF in MAC
o Citrate (+)
o MRVP (+, -) among Enterobacteriaceae along with
Cronobacter

CITROBACTER
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SERRATIA

- Opportunistic
- Outbreaks in health care settings
- Highly antibiotic resistant PROTEUS
- Pink to red pigment
o PRODIGIOSIN - environmental and intestinal bacteria
o Environmental strains - Proteus mirabilis (most common clinical isolate)
 S. marcescens - Presumptive ID:
 S. rubidea
o Swarming, NLF in MAC
 S. plymuthica
o LIA deamination (+)
- Presumptive ID:
o H2S (+)
o ONPG (+)
o Urease (+)
o DNAse (+)
o Gelatinase (+)
o Indole (+): Proteus vulgaris

BIOCHEMICAL TEST
ID BIOCHEMIC P. P.
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AL TEST MIRABILIS VULGARIS
1 Oxidase - -
2 Catalase + +
3 Indole - +
Citrate
4 v v
Utilization
Triple Sugar
5 K/A++ K/A++
Iron Agar
6 Urease + +
7 Methyl Red + +
Vogas-
8 - -
Proskauer
+ positive, - negative, v variable, K alkaline, A acidic

BACTERIOLOGICAL TEST
Swarming on
1 + +
Blood Agar
Hemolytic
2 Non hemolysis Non hemolysis
Activity
3 Lactose Non lactose Non lactose
fermentation on fermenter fermenter
MacConkey
agar

CATALASE TEST: uses H2O2 + (bubble formation)

- Is used to determine the ability of bacteria to convert

tryptophan into indole
- Proteus Mirabilis can be differentiated from Proteus vulgaris
by indole test (same with pneumoniae and oxytoca)
- Proteus mirabilis (negative) yellow
- Proteus vulgaris (positive) pink/red
IMViC TEST 1-indole TEST
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STOOL: MAC, BAP, SSA, Vibrio SS agar. Colonies with black
URINE: MAC, BAP center colony of salmonella SI M V CI
TSI GLU
spp. And colorless colony of M R P T
shigella spp.
SALMONELLA A/
+
NLF
+ + - +
A -
+
- Acute gastroenteritis causes
food poisoning usually seen - Type: Bacteria
in poultry meat. - Diseases: Enteric fever or typhoid fever
- Typhoid fever, Salmonella - UV dose: 15.20 mJ/cm2
carrier state
- Contamination with human
excreta SHIGELLA
- Presumptive ID:
o NLF in MAC
- Closely related to Escherichia
o Black colonies (SSA, HE, XLD)
- Shigellosis (bacillary dysentery)
o H2S (+)
- Humans are the only known reservoir
o Motility (+)
- Person-to-person transmission (fecal-oral)
- Symptoms
- Presumptive ID:
o Diarrhea (sometimes bloody)
o NLF in MAC (24hrs)
o Stomach pains or cramps
o Colorless in SSA
o Fever
o H2S (-)
o Nausea and vomiting
o Motility (-)
o Headache
o ONPG (+)
- Laboratory Test: Typhi dot
- SSA: selective
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BIOCHEMICAL TESTS FOR IDENTIFICATION

TSI: A/A BIOCHEMICAL SHIGELLA SALMONELL

MR: + TESTS A
VP: - Catalase Test + +
S: - Oxidase Test - -
I: - Motility Test -
M: - Methyl Red Test + +
CIT: - Voges Proskauer Test - -
GLU: NLF – Indole Production Test + -
Simmon Citrate Test - +
Urea Hydrolysis Test - -
Sugar Fermentation
SHIGELLA INFECTION F F
Test
- An intestinal disease caused by a family of bacteria known Dextrose Fermentation
as shigella +
Test
Dulcitol Fermentation
SYMPTOMS OF SHIGELLA INFECTION -
Test
- Diarrhea Maltose Fermentation
+
- Abdominal pain or cramps Test
- Fever Mannitol Fermentation
+ +
Test
Sorbitol Fermentation
COMPLICATIONS +
Test
- Dehydration
Xylose Fermentation
-
- Seizures Test
- Rectal prolapse TSIA SHIGELLA SALMONELLA
- Hemolytic uremic syndrome Glucose + +
Gas from Glucose - +
- Toxic megacolon Sucrose - -
- Reactive arthritis Lactose - -
H2S - +
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YERSINIA PESTIS

- EPIDEMIC: Plague, Black death

- Class A Bioterrorism agent
- Primarily a disease of rodents
- Bites from fleas
- Bubonic/Glandular form (buboes)
- Septicemic/pneumonic form (inhalation)
- Bipolar staining (safety pin appearance)
- Presumptive ID:
o Mainly dependent of patient history, signs and
symptoms, and frequency of cases
- Morphology:
o Short rods or coccobacilli
o Retain staining at the ends of the cells “safety pin”
appearance
o Bipolar staining

YERSINIA ENTEROLITICA
- From animals and pets
- Clinical form mimics appendicitis
o Abdominal pain concentrated in the right lower
quadrant
- Presumptive ID:
o Bacilli (safety pin appearance)
o Cold enrichment (25 centigrade)
o NLF in MAC
o Cefsulodin-Irgasan Novobiocin (CIN) agar
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GUIDE:
DIAGNOSTIC

MICROBIOLOGY
CONNIE MAHON
 BACTERIOLOGY SEMINAR 1 MIDTERMS
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2019