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Bah. Pathogenic Micro sucks.
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Escherichia coli
MOST COMMON CAUSE OF URINARY TRACT INFECTIONS
strain O157 : H7 - causes hemorrhagic colitis

VIRULENCE
certain O, H and K antigens are associated with infections

NOSOCOMIAL INFECTIONS COMMON! Ecoli is the most common hospital infection

PLATE MEDIA!
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BAP - gray, large, hemolysis varies
MacConkey agar- brick red colonies with acrid odor
Eosin Methylene blue (EMB) agar- dark colonies, green metalic sheen
INHIBITED ON XLD, HE AND SS

TSI - A/AG or A/A
IMViC ++--
urease -
usually motile, LDC +, ONPG +




Citrobacter freundii
some C. freundii may be associated with diarrhea
Bethesda Ballerup strains of C. freundii may have Vi antigen
C. freundii can look like an E. coli on plate media

BUT!!!!! H2S + on TSI slant (A/A with hydrogen sulfide)


Citrobacter diversus
MAY CAUSE BRAIN ABCESSES OR MENINGITIS IN BABIES!
less common pathogenic.
resembles a citrate + E. coli



Klebsiella pneumoniae
member of normal gut flora, opportunist, coliform
3% of acute bacterial pneumonia
Virulence factors--large mucoid polysaccharide capsule is antiphagocytic and can cause chronic abcess.

BAP - large mucoid colony, slimy,
Mac - large pink mucoid colony, yeasty smell
EMB - large pink mucoid colony with purple center


Tubed media

TSI - A/AG
IMViC (--++)
NONMOTILE NONMOTILE NONMOTILE NONMOTILE, ODC -, LDC+

K. Pneumoniae is INDOLE NEGATIVE!!!!!

Klebsiella oxytoca
exactly the same as pneumoniae BUT!!!

OXYTOCA IS INDOLE POSITIVE!!!


Enterobacter cloacae

look similar Klebsiella, but smaller less mucoid

may be a bit less pink than most Klebsiella
TSI - A/AG
IMViC (--++)
motile, ODC +

Enterobacter aerogenes
look similar Klebsiella, but smaller less mucoid

may be a bit less pink than most Klebsiella
TSI - A/AG
IMViC (--++)
motile, ODC +


Pantoeaea agglomerans
plants
similar to Enterobacter, but is negative for all common decarboxylases
may produce light yellow pigment


Serratia marcescens
nosocomial opportunist, may be invasive
RESISTANT TO TREATMENT!!
ASSOCIATED WITH RES DISEASES!!!
produces lipase and DNAse

*IS THE ONLY ENTERIC BACTERIA TO PRODUCE GELATINASE*
looks like enteric on BAP
may produce red prodigiosin pigment (RT); most are nonpigmented
May appear as nonlactose fermenter on Mac or EMB because it is a DELAYED LACTOSE FERMENTER!.
may be colorless on XLD, HE, SS or severely inhibited


TSI --> K/AG or A/AG (ONPG +)
IMViC --> --++

DNase +, gelatinase +
looks like a DNAse + E. aerogenes


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NON COLIFORMS
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Proteus mirabilis
opportunistic.
P. mirabilis much more common
Gray SWARMING COLONIES!!! SWARMING SWARMING SWARMING!!!

TSI ---> K/A and hydrogen sulfide gas causes blackening
Urease + (rapidly)
deaminase +
H2S +

Can look like salmonella!
P. mirabilis/// indole -, ODC +
P. vulgaris/// indole +, ODC -


Proteus vulgaris

SAME AS MIRABILIS!!! but remember--

P. mirabilis/// indole -, ODC +
P. vulgaris/// indole +, ODC -


Proteae
SWARMING SWARMING SWARMING!

Providencia
looks and smells like other members of the Proteae tribe
no swarming, no hydrogen sulfide
most are urease negative, deaminase +

Morganella
ooks and smells like Proteus, but never swarms
Hydrogen sulfide negative, ODC +, Indole +, TSI K/A
deaminase +,Urease +


Salmonella enteritidis
Gastroenetritis
BAP - nearly all Enterobacteriaceae produce grayish large colonies on BAP
SS is far more selective --> colorless with black or completely black.
XLD is also more selective --> colorless with black or completely black.
ONPG neg
Tubed media


TSI --> K/A +H2S
S. typhi has small ring of H2S)
S. paratyphi has no H2S

urease -, deaminase
ONPG neg
Selenite F or GN broth or Tetrathionate are three common enrichment broths.
all species are routinely serotyped for O and Vi antigens


Salmonella typhi
Enteric fever
Do blood cultures in first week of symptoms!!!
Vi antigen (an envelope antigen)
BAP - nearly all Enterobacteriaceae produce grayish large colonies on BAP
SS is far more selective --> colorless with black or completely black.
XLD is also more selective --> colorless with black or completely black.
Tubed media


TSI --> K/A +H2S
S. typhi has small ring of H2S)
S. paratyphi has no H2S

urease -, deaminase

ONPG neg
Selenite F or GN broth or Tetrathionate are three common enrichment broths.
all species are routinely serotyped for O and Vi antigens



Salmonella cholera-suis
. Septicemia
BAP - nearly all Enterobacteriaceae produce grayish large colonies on BAP
SS is far more selective --> colorless with black or completely black.
XLD is also more selective --> colorless with black or completely black.
Tubed media


TSI --> K/A +H2S
S. typhi has small ring of H2S)
S. paratyphi has no H2S

urease -, deaminase
ONPG neg
Selenite F or GN broth or Tetrathionate are three common enrichment broths.
all species are routinely serotyped for O and Vi antigens



Salmonella arizonae (Arizona hinshawii)
BAP - nearly all Enterobacteriaceae produce grayish large colonies on BAP
SS is far more selective --> colorless with black or completely black.
XLD is also more selective --> colorless with black or completely black.

Tubed media


TSI --> K/A +H2S
S. typhi has small ring of H2S)
S. paratyphi has no H2S

urease -, deaminase

IS ONPG POSITIVE!!!!!
Selenite F or GN broth or Tetrathionate are three common enrichment broths.
all species are routinely serotyped for O and Vi antigens


Shigella sonnei
GROUP D SHIGELLA
Mildest
Shigella has ability to survive gastric acidity
Shigellosis; bacillary dysentary
Invade mucosa, ulcers
BAP - nearly all Enterobacteriaceae look similar on BAP.... grayish, large

EMB and Mac are mild selective --> colorless colonies
HE, XLD, SS are more selective --> colorless colonies
All Shigella are:

TSI --> K/A no gas, no H2S
nonmotile, LDC negative

S. sonnei is ODC +, often ONPG+

all species are confirmed with antisera
S. dysentariae = group A Shigella
S. flexneri = group B Shigella
S. boydii = group C Shigella
S. sonnei = group D Shigella






Shigella dysentariae
GROUP A SHIGELLA
S. dysentariae is most severe
Shigella has ability to survive gastric acidity
Shigellosis; bacillary dysentary
Invade mucosa, ulcers
BAP - nearly all Enterobacteriaceae look similar on BAP.... grayish, large

EMB and Mac are mild selective --> colorless colonies
HE, XLD, SS are more selective --> colorless colonies

All Shigella are:

TSI --> K/A no gas, no H2S
nonmotile, LDC negative


Shigella boydii
GROUP C SHIGELLA
Shigella has ability to survive gastric acidity
Shigellosis; bacillary dysentary

Invade mucosa, ulcers
BAP - nearly all Enterobacteriaceae look similar on BAP.... grayish, large

EMB and Mac are mild selective --> colorless colonies
HE, XLD, SS are more selective --> colorless colonies
All Shigella are:

TSI --> K/A no gas, no H2S
nonmotile, LDC negative

Shigella flexneri
GROUP B SHIGELA
Shigella has ability to survive gastric acidity
Shigellosis; bacillary dysentary
Invade mucosa, ulcers
BAP - nearly all Enterobacteriaceae look similar on BAP.... grayish, large

EMB and Mac are mild selective --> colorless colonies
HE, XLD, SS are more selective --> colorless colonies
All Shigella are:

TSI --> K/A no gas, no H2S
nonmotile, LDC negative



Yersina pestis
causes cyanosis

1.black or bubonic plague
Enter flea bite site--> lymphatics --> bubo (swollen lymph nodes)
bubos become hemorrhagic, cause DIC
maybe conjunctivitis

2.septicemic form
If not filtered by nodes spreads to spleen, liver, lungs, meninges also causing DIC

3.pneumonic form
Septic emboli spread to lung or inhale droplets -->pneumonia. Fatal within 2 days

PESTIS HAS V and YOP antigens --- V=virulence; YOP = Yersinia outer membrane proteins;

RESISTS PHAGOCYTOSIS

Envelope antigen (fraction 1) --- capsular polysaccharide; antiphagocytic?; antibodies to it are protective

pesticin 1 ---a bacteriocin with coagulase and fibrinolysin activity

NEGATIVE FOR ALL DECARBOXYLASES!!!
sylvatic or wild plague ---among wild rodents
urban or domestic ---among rats in cities
human ---contact other cycles or infected humans

pinpoint on BAP;; beaten copper color on BAP

Mac colorless, tiny at 24 hours

TSI weak acid production at best



Yersinia enterocolitica
Clue: Grows to small pinpoint colony on most media at 24 hours
nonhemolytic on BAP, small at 24 hours
generally colorless on Mac, EMB and SS
salmon colored on HE, yellow on XLD
cold enrichment enhances recovery


Cefsulodin irgasan novobiocin (CIN) is highly selective
CIN agar --> bulls eye appearance

AVOID EMB due to sucrose

motile at Room Temperature, nonmotile at 35 degrees

VP + at room temperature, negative at 35 degrees

ONPG +
TSI = A/A for enterocolitica
LDC -
ODC +
may use KIA instead of TSI due to sucrose content

KIA = K/A

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Coliform/ noncoliform
Coliforms ferment lactose rapidly-- resemble E.Coli
Noncoliforms dont ferment lactose, or ferment it slowly.

What defines an Enterobacteriaceae?
ALL ENTEROBACTERIACEAE ARE CYTOCHROME OXIDASE NEGATIVE!!!
All enterobacteriaceae reduce nitrate to nitrite!!!!
Very common, fermenters oxidase NEGATIVE

MOTILE VIA PERITRICIOUS FLAGELLA!!!

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Pseudomonas aeruginosa
GROUP 1
FLUORESCES UNDER UV LIGHT
this is the most common NFB
usually a harmless saprophyte
prefers compromised hosts
burns, drugs, inhalation therapy equipment, cystic fibrosis
infects any site... swimmers ear, UTI , wounds, transplants, lung...

rarely infects healthy host, look for a compromise, ... as simple as antibiotic caused overgrowth


produces green pus, grape like odor
Critical Tests

Mac+, oxidase +, TSI=K/K
oxidizes glucose in OF medium,
grows at 42 degrees Celcius
fruity grapelike odor from most strains
pyocyanin is green pigment seen on most media
pyoverdin is white to blue green fluorescent pigment




Stenotrophomonas maltophilia
GROUP 5
second most common NFB
nosocomial infections, transplants
opportunistic, pulmonary
May be yellowish colony on clear media, lavender green on BAP
brownish discoloration of BAP, ammonia odor

TSI=K/K, OF maltose oxidizer, slow glucose oxidizer
Grows well on MacConkey

ONPG +, LDC+, DNase +
oxidase -, motile



Burkholderia cepacia
GROUP 2
IV bottles
common in Cystic fibrosis patients
Inoculate selective media for B. cepacia

Burkholderia pseudomallei
GROUP 2
melliodosis
Lab associated infections ... handle with great care
putrid odor, then earthy odor

confirm with reference lab



Burkholderia mallei
GROUP 2
the only true animal parasite in the Pseudomonas
glanders, an animal infection of the lymphatics
multiple abscesses, septicemia
rarely transmitted to man, but can be as fatal in man
Lab associated infections in man


nonmotile,

Acinetobacter baumanii
among most common NFB
Grows good on MacConkey with cornflower blue tinted colonies
Oxidative (glucose and lactose)
cytochrome oxidase -
nonmotile
catalase +



What defines a nonfermenter?
OXIDIZERS!!! ASSACHAROLYTIC!
DO NOT FERMENT SUGAR!!!!
associated with water mostly
obligate aerobes
TSI or KIA = K/K = red throughout slant and butt
OF medium - either oxidize sugars or are assacharolytic in OF medium
common in water,

quite drug resisitant

Most prefer compromised hosts
cystic fibrosis
burn patients
COPD
patient’s on antibiotics, chemotherapy...


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VIBRIO
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Vibrio cholerae
cholera with > 50 % fatality
RICE WATER STOOLS (ew)
enterotoxin causes fluid, electrolyte loss
water or food contamination by human feces
BAP --> looks like other GNR;
classical is gamma hemolytic
El Tor biotype is beta hemolytic

TCBS medium --> yellow colonies

Thiosulfate citrate bile salt sucrose highly selective for Vibrios...

V. cholerae ferments sucrose to cause yellow colonies due to acid production.

can enrich with alkaline peptone water (pH = 8.6)

use polyvalent 01 antisera
Most epidemic stains are 01 positive

+ string test
mix colonies with 0.5% sodium desoxycholate
place loop in mixture and pull away--> string

darting motility, curved bacilli
polar monotrichous flagella
NG in 7 or 11% salt; grows in 0% salt




Vibrio parahemolyticus
seafood
Kanagawa phenomenon
TCBS--> blue green colonies,
oxidase +, tolerant of 7% salt, NG in 11% salt or 0%



Vibrio vulnificus
septicemia from ingestion of seafood; esp. raw oysters
extremely virulent, high fatality rate
especially dangerous in compromised patients
lactose +, ONPG +, TSI A/A
tolerate 7% salt, not 0 or 11% salt
LACTOSE POSITIVE!!!

Aeromonas hydrophila
can look like an E. coli or Enterobacter
oxidase positive, TSI A/A


What defines Vibrionaceae?
Less common, fermenters, oxidase negative. CURVED!!!


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CAMPYLOBACTER
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Campylobacter jejuni
GI illness with PMN’s in stool
linked to poultry, milk, water
obligate microaerophile
Examine stool for WBC’s, curved S shaped or gull winged rods
If +, inoculate Selective “Campy” BAP or chocolate

Inoculate Campy thio in refrigerator overnight, incubate 42 degrees
Colonies are .3 -.5 mm at 24 hr, run along streak lines

oxidase +, catalase +, hippurate +, urease -
CF = resistant ......Nalidixic acid = susceptible

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Helicobacter
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Helicobacter pylori
gastrititis; stomach ulcers
urease +, oxidase +, catalase +, hippurate -
inhibited by Cephalothin, susceptible to Nalidixic acid
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Serologic tests

            O antigen

 O antigen is the somatic or body antigen that is on the surface polysaccharides of the cell wall.
O antigen typing is performed on ALL salmonella and shigella species.
O antigens are also heat stable since they are made up of lipopolysaccharide cell wall components.

            H antigen

The H antigen is a FLAGELLAR antigen, but serological tests for this antigen are RARELY PERFORMED!

K antigen

 K antigen is an envelope antigen (may be capsular)
K antigen may mask O antigen.. But K antigen is heat labile, so if you boil it for a few minutes and repeat the O antigen typing, the masking effect of K is destroyed.


Vi antigen

Vi antigen is a type of K or envelope antigen ; it is known as the virulence antigen

Salmonella typhi has an envelope with Vi. Vi can also mask the O antigen , but just like K, it is ALSO heat labile. Boil it to get rid of it and retest for O.

 

What if get negative serologic test even though it should be positive?

Envelope antigens like K and Vi are frequently associated with virulence, but they can mask O cell wall antigens when you are performing serotypes on certain bacteria. In order to destroy K or Vi so they CANT mask the O cell wall antigen, just boil the  suspension of bacteria and repeat the O antigen test. K and Vi are easily destroyed by heat.

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Reagents for tests

p-dimethyl aminobenzaldehyde (Kovacs)-- used to test for indole in VP. If positive, fuschia color develops.

Alpha naphthol and KOH-- used in VP to test for the neutral end products of glucose fermentation. If positive, turns a red color.

Alpha naphtholamine and sulfanilic acid-- reagents fr nitrate reduction that cause a red reaction if nitrite is present.

Add zinc dust to be SURE nitrate was reduced if you get a negative reaction.

Tetra-methyl phenylenene diamine (Oxidase reagent)-- a colorless substrate that becomes colored if the cytochrome oxidase enzyme is present.  turns blue

Ferric chloride (for phenylalanine or tryptophane deaminase tests)

                                                  If phenylalanine is present, turns green if ferric chloride is added.

                                                  If tryptophane is present, turns brown if ferric chloride is added.

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Media

PLATE MEDIA

MacConkey agar

Macconkey agar is used to detect lactose fermentation and helps to differentiate between the major groupings of the enterobacteriaceae.

Other sugars can be used in place of lactose though.

Macconkey agar is a PINK COLOR before it is inoculated, but contains a pH indicator to detect acid produced by sugar fermentation.

MACCONKEY PREVENTS THE GROWTH OF COCCI AND GRAM POSITIVE BACILLI!

Enteric bacteria often produce typical colonies and odors on macconkey.

MACCONKEY IS A DIFFERENTIAL MEDIUM THAT SELECTS FOR ONLY GRAM NEGATIVE BACILLI and helps to differentiate between those gram negative bacilli.

 

Eosin Methylene blue agar

EMB agar is virtually the same thing as Macconkey, and performs the same functions.

EMB is PURPLE before it is inoculated.

MACCONKEY AND EMB ARE NEVER USED AT THE SAME TIME, since they are basically the same thing. Some labs prefer EMB, some labs prefer Macconkey, since they both work fine for growing enterobacteriaceae.

HOWEVER-- EMB is SLIGHTLY LESS SELECTIVE  for gram negative bacilli.

 

SS agar

SS agar is SALMONELLA AND SHIGELLA AGAR.

SS agar is ONLY ONLY ONLY used when fecal samples are processed.

It is PINK before it is inoculated.

MUCH MORE SELECTIVE FOR THE PATHOGENIC ENTERIC BACTERIA LIKE SALMONELLA AND SHIGELLA!

SS is SIMILAR to macconkey, but is much more inhibitory to nonpathogens.

ALSO DETECTS HYDROGEN SULFIDE PRODUCTION

Lactose fermenters are detected on this medium by their acid production.

LACTOSE FERMENTERS/ ACID PRODUCERS TURN THE COLONIES PINK TO RED!!

NON LACTOSE FERMENTERS have CLEAR colonies, since they dont produce acid from lactose.

IF IT PRODUCES HYDROGEN SULFIDE-- CLEAR COLONY WITH BLACK CENTER or an ENTIRELY BLACK COLONY!

is a highly selective media, but also differentiates those colonies that are not inhibited.


XLD agar

XLD is another highly selective and differential medium that is used only for fecal sample inoculation.

XLD is RED before it is inoculated!

XLD DETECTS XYLOSE FERMENTATION which produces an acid and changes the pH of the medium causing it to turn yellow!

***Bacteria that can decarboxylate lysine can change the pH back to red again!

Also-- if the colony produces HYDROGEN SULFIDE, BLACK COLONIES!

***some non-fecal pathogens like the PROTEAE can look like pathogens on XLD!

 

CIN agar

Cefsulodin irgasan novobiocin (CIN) is highly selective
CIN agar --> bulls eye appearance for YERSINIA ENTEROCOLITICA!

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SF broth/GN broth


TCBS agar


Campy agar


TSI or KIA reactions (coliforms and non coliforms) (Fermenters and nonfermenters)

If glucose is fermented, butt turns yellow.

If lactose or sucrose are fermented ENTIRE TUBE turns yellow.

If Hydrogen sulfide is produced,  iron in media turns black as iron sulfide.

If gas is produced-- bubbles or cracks

Coliforms-- A/A (but serratia might be K/A)

Non Coliforms-- K/A

Fermenters

OF medium (Fermenters, oxidizers, nonfermenter-nonoxidizer)

Fermenters-- K/A or A/A

Nonfermenters-- K/K


Cytochrome oxidase

Only obligate aerobes and some facultative anaerobes posess this enzyme.  Detects the terminal cytochrome oxidase in electron transport system which catalyzes removal of final hydrogen from cytochrome.

produces water or hydrogen peroxide waste product

IMViC reactions for E. coli and KES group

E. Coli is IMViC ++--  *****

Klebsiella is  --++

Enterobacter cloacae and aerogenes are also --++

Serratia Marcescens is also --++

Deaminase reaction to differentiate Proteae from Salmonella or Shigella

In an LIA tube, R/A is deaminase positive, which means its a PROTEAE!

salmonella and shigella are deaminase NEGATIVE.

            Lysine deaminase

turns red in the slant if lysine is deaminated

            Phenylalanine deaminase

reduced to phenpyruvic acid and turns green with ferric chloride

            Tryptophane deaminase

reduced to indole and turns brown with ferric chloride

Motility differentiates Klebsiella from other KES group

KLEBSIELLA AnD SHIGELLA ALWAYS NONMOTILE!

Motility differentiates Shigella from motile bacteria

Shigella and Klebsiella ALWAYS NONMOTILE

Motility differentiates Yersinia enterocolitica from … how?

Yersinia enterocolitica is motile at room temperature, yet NONMOTILE at 35*.

Yersinia enterocolitica is also VP+ at room temp, yet VP - at 35*

Positive and negative test reactions for decarboxylase tests and why.


Urease for differentiating Proteus and …


Neutral endproducts of fermentation

the conversion of glucose to acetoin and diacetyl (which are neutral endproducts)can be measured using VP

Quality control tests on media and reagents

Quality control is much more difficult and time consuming to prepare and run on conventional methods.

            ONPG

 

DNAse

Serrratia produce DNAse while Enterobacter do not.

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