CHAPTER 19- Micro1 Lab

Enterobacteriaceae
● Commonly referred to as enterics
● Gram negative, non spore forming, facultative anaerobic bacilli/coccobacilli
● All are catalase positive
● All are oxidase negative except for Pleisomonas
● Biochemically similar to Vibrio and Aeromonas
● All are glucose fermenters
● All are motile except klebsiella, shigella, and yersinia (KYS)
● Klebsiella and enterobacter exhibit mucoid colonies on plated media (Large pink mucoid
colonies on MAC)
● Proteus exhibits swarming motility on plated media
● EMB, MAC, HEAL XLD- media for presumptive identification
○ Check the book for meaning
● Box 19-1 (READ)

Selective media for Enterobacteriaceae

● MacConkey Agar
○ Selects gram (-) bacteria and also differentiates lactose fermenters from non-lactose
fermenters
◆ Bile salts and crystal violet— Inhibit gram (+) bacteria
○ Lactose— Carbohydrate source
○ Neutral red— Indicator
● Fermentation will produce COLOR CHANGE
● Positive= Pink= Fermenter
● Negative= Colorless= Non-fermenter

● Eosin methylene blue

○ Selects gram (-) bacteria and also differentiates lactose fermenters from non-lactose
fermenters
◆ Eosin and Methylene blue
◇ Inhibits gram (+) bacteria
◆ Lactose and sucrose- for differentiation on fermentation
○ Lactose negative= Colorless
○ Lactose positive= Violet colonies
○ E. coli= Metallic green sheen (lactose positive)

● Hektoen Enteric agar

○ Inhibits normal flora coliforms and also differentiates lactose fermenters from non-
lactose fermenters
◆ Bile salts— Inihibit gram (+) bacteria and gram (-)
◆ Lactose, sucrose, and salicin— Carbohydrate source
 ◆ Bromthymol blue— Indicator
◆ Sodium thiosulfate— sulfur source
◆ Ferric ammonium citrate- detect H2S production
◇ Blackening in the media
○ Most nonpathogens ferment lactose and sucrose show ORANGE COLOR (low pH)
◆ Green to Orange (NON PATHOGENIC)
○ Pathogens (salmonella and shigella)— Green to blue green color with H2S gas creating
a blsck precipitate (NO FERMENTATION)

● XLD AGAR
○ Selects for stool pathogens and differentiates xylose fermenters (yellow) from non-
xylose fermenters (red); selective for salmonella and shigella from stool specimens
◆ Sodium deoxycholate— Inhibit gram (+) cocci, gram (-) coliforms, swarming of
Proteus
◆ Lysine— To detect decarboxylation
◆ Xylose— Carb source
◆ Phenol red- Indicator
◆ Sodium thiosulfate- Sulfur source
◆ Ferric ammonium citate- Detects H2S production
● Salmonella-Shigella Agar
○ Selects for stool pathogens and differentiates lactose fermenters (pink-red) from non-
lactose fermenters (clear)
◆ Bile salts and Brilliant green agar— Inhibit gram (+), gram (-) coliforms
◆ Lactose- Carbs source
◆ Neutral red— Indicator
◆ Sodium thiosulfate— Sulfur source
◆ Ferric ammonium citrate- detection of H2S production
Groups Lactose Colonies on Examples
fermentation MacConkey Agar
Lactose fermenters Ferment lactose Produce pink Escherichia,
(LF)— All are producing acid colored colonies Klebsiella,
coliform bacilli Citrobacter
Nonlactose Do not ferment Produce pale or Salmonella,
fermenters lactose colorless colonies Shigella, Proteus,
Morganella,
Providencia, and
Yersinia
Late lactose Ferment lactose At 24 hrs incubation Shigella sonnei
fermenters (LLF or after 2-8 days of — produce pale or
previously called as incubation colorless colonies,
paracolon bacilli) After 2 days
 Ferment lactose At 24 hrs incubation Shigella sonnei
fermenters (LLF or after 2-8 days of — produce pale or
previously called as incubation colorless colonies,
paracolon bacilli) After 2 days
produces pink color
colonies

Virulence factors
● Adherence: Prevent from being removed from tissue
● Toxins: Food poisoning
● Invasive enzymes: Spreading factors
● Antibiotic resistsnce
○ Extended spectrum B-lactamase (ESBL)
● Serology
○ O antigen (Somatic antigen)
◆ Heat stable antigen located in the cell wall (LPS)
○ H antigen
◆ Flagellar antigen; heat labile
○ K antigen
◆ Capsular antigen; heat labile
◆ Meningitis and septicemia— K1 antigen of E. coli
◆ Vi antigen of Salmonella typhi
● Clinical significance
○ Ubiquitous in nature
○ Commonly found in the GI tract except for Salmonella, Shigella, and Yersinia
○ Commensals
● Opportunistic pathogens
○ Normally part of the intestinal flora that may produce infection outside the intestine
● Primary Intestinal pathogens
○ Salmonella, Shigella, Yersinia spp. (YSS)
○ True pathogens

Selective media for Y. enterocolitica— Cefsulodin-Irgasan Novobiocin agarp

● Escherichia coli
○ E. coli is the most important (UTI, diarrhea. CNS infection)
○ #1 cause of Urinary Tract Infection
○ Primary of fecal contamination in water quality testing)
○ Meningitis and septicemia on neonates
○ Mostly Lactose Fermenters in MAC
○ Exhibits greenish-metallic sheen in EMB
 ○ Presumptive Identification
◆ A/A in TSI, H2S (-)
◆ Indole (+)
◆ Citrate (-)
● TSI A/A, MR (+), VP (-), Sulfide (-), Indole (+). Motility (+), Citrate (+)
● UTI’s and Septicemia
● Most strains have K1 capsule serotypes
● Diarrhea and gastroenteritis disease syndromes
○ Uropathogenic E. coli (UPEC) — UTI
○ Enterotoxic E. coli (ETEC)
○ Enteroinvasive E. coli(EIEC)
○ Enterpathogenic E. coli (EPEC)
○ Enterohemorrhagic E. coli (EHEC)
○ Enteroaggragative E. coli (EAEC)

VIRULENCE FACTORS AND DISEASE

. Enterohemorrhagic E. coli (EHEC)

● Also known as Verotoxic E. coli (VTEC)
○ Virulence factor
◆ Hemorrhagic diarrhea, colitis, HUS
◆ WBCs absent in stool
◆ Frequently associated with E. coli O157:H7 and O157:NM (SEROTYPES)
◇ The only strain that is SORBITOL NEGATIVE
○ Disease
◆ Hemorrhagic colitis
◆ Hemolytic uremic syndrome- A condition results from the damage of small blood
vessels in kidneys. This causes diarrhea, abdominal pain, vomiting and fever

. Entertocigenic E. coli (ETEC)

○ Also known as “Montezuma’s revenge” or “turista” which is TRAVELERS DIARRHEA
● Virulence factor
○ Pili
○ Toxins: Heat labole and heat stable enterotocins
● Disease
○ Mild, watery, diarrhea
○ Travelers and childhopd diarrhea (epidemic diarrhera)
● Only grows in blood agar

. Enteroinvasive E. coli
○ Shigella like infection
● Virulence factor
○ Invades enterocytes (intestinal epithelium) lining the large intestine causing a shigella
like infection
 ○

like infection
● Disease
○ Dysentery and Bloody stools (RBC, NEUTS, AND MUCUS)
● Other characteristics
○ Most strains are non-motile and late or non-lactose fermenters

. Shiga toxin producing E. coli

● Shiga toxin 2 (Stx1 and 2 pero 2 lang nasa ppt ni miss)- Similar to the toxin produced by S.
dysenteriae type 1
◆ Also known as Verotoxi
● Disease
○ Watery diarrhea that progresses to bloody diarrhea with abdominal cramps and fever

. Enteropathogenic E. coli (EPEC)

● Virulence factor
○ Bundle forming pilus and other factors for attachment of cells of the small bowel
○ Non invasive and does not produce toxins
● Disease
○ Diarrhea in infants (watery with mucus but no blood)
● Other characteristics
○ May be isolated in enteric media
. Enteroaggregative E. coli (EAEC)
● Virulence factor
○ Actual pathogenic mechanism not known
○ Virulence genes associated with aggregative regulator gene/ AGGR
○ Stick together when cultured in monolayers, produces STACKED BRICK PATTERN
● Disease
○ Watery diarrhea and do not contain WBCs

Table 19-4
Extraintestinal infections
● Most common in neonates and very young children
● Gain infection just before or during delivery and when infections involve the amniotic fluid
● Capsule antigen K1
○ Predisposition for meningitis
Escherichia hermanii
● Yellow pigmented
● Isolated from CSF, wounds, and blood
● Isolated from foodstuffs such as raw milk and beef
E. vulneris
● Newest member
● wound infections
Klebsiella, Enterobacter, Serratia, Fantoea, Cronobacter, and Hafnia
● Tribe Klebsielleae
● Opportunistic and nosocomial infections
● General characteristics
○ Simmons citrate positive
○ Potassium cyanide broth negative
○ NO H2s production
○ No deamination of phenylalanine
○ Generally urease negative
○ Indole negative; MR negative
○ VP positive
○ Thus MVC: nega, negs, posi,mposi
Klebsiella
● Opportunistic, hospital-acquired/nosocomial infections
● Pneumonia, wound infections, UTI
● Mucoid, pink—> capsulated
● Virulence factor: polysaccharide capsule
● Presumptive ID:
○ Large mucoid (capsule +), Lactose fermenter in MAC
○ non motile
○ Citrate (+)
○ Urease (+)
○ Indole (-) K. pneumonia
○ Indole (+) K. oxytoca
● TSI A/A, Sulfide (-), Indole (-), Motility (-), MR (+), VP (-), Citrate (+)

K.oxytoca
● Similar to K. pneumoniae except indole positive
● Affects similar sites
K. ozaenae
● Isolated from nasal secretions and cerebral abscesses
○ Plasmid mediaed ESBLS
K. pneumoniae subspecies rhinoscleromatis
● Rhinoscleroma
○ Intense swelling and malformation of the entire face and neck

Enterobacter
● Colonial morphology resembles Klebsiella
● E. cloacae and E. aerogenes (most common species in clinical isolates)
● Wounds, urine, blood, and CSF
● Presumptive ID:
○ Mucoid, Lactose Fermenter in MAC
 ○ Citrate (+)
○ MRVP (+,-) among Enterobacteriaceae along with Chronobacter
● Beige to yellow colonies
● Pink mucoid colonies on MAC
● Gaining resistance with antibiotics

Pantoea agglomerans
● Formerly Enterobacter agglomerans
● Similar to K. pneumoniae, so need to differentiate
● Primarily a plant pathogen
Cronobacter sakazakii
● Meningitis and bacteremia in neonates from powdered infant formula

Citrobacter
● Hospital acquired UTI
● Pneumonia
● Intrabdominal ahscess
● Endocarditis in IV drug users
● C. koser: Nursery outbreaks of neonatal meningitis and brain abcess
● Can be mistaken for Salmonella biochemically
● Presumptive ID
○ Only LF enterbacteriaceae that is H2s+
○ Citrate (+)
○ MRVP (+,-)
○ Urea (+), C. freundii
◆ Yellow colored colonies on XLD
Serratia
● Opportunistic
● Outbreaks in health care settings
● Organisms possess Prodigiosin
● highly antibiotic resistant
● Pink to red pigment (Prodigiosin)
● Environmental strains
○ S. marcescens, S. rubidea, S. plymuthica
● Presumptive ID
○ ONPG+
○ Dnase +

Proteus
● Environmental and intestinal bacteria
● P. mirabilis (most common isolate)
● Presumptive ID
 SWARMING, NLF IN MAC
○
○ LIA deamination +
○ H2s+
○ Urease+
○ Gelatinase+
○ Indole+: P. vulgaris
● Swarming because of Peritrichous flagella

Providencia species
● Five species
○ 2 major pathofens
◆ P. alcalifaciens may also be a pathogen
○ P. rettgeri
◆ Urinary tract pathogen
◆ Occasionally nosocomial outbreaks
○ P. stuartii
◆ Nosocomial burn unit outbreaks
◆ Also isolated in urine cultures
◆ No swarming
○ HIGHLY RESISTANT TO ANTIBIOTICS

Salmonella
● Virulence Factors and disease— Gastroenteritis/food poisoning
● Disease (Type of Salmonella infection)
○ Gastroenteritis and diarrhea
◆ Associated with S. typhimurium and S. enteritidis
○ Bacteremia and extra intestinal infections
◆ S. choleraesius and S, dublin
○ Enteric fever (typhoid fever, or typhoid)
◆ Associated with S. typhi and S. paratyphi
● Salmonella= Black centered colonies in SSA

Shigella
● Closely-related to Escherichia
● Causative agent of Shigellosis (bacillary dysentery)
● Humans are the only known reservoir
● Person to person transmission (fecal-oral)
● Presumptive ID:
● NLF in MAC (24 hours)
● Colorless in SSA
● H2S-
● Motility -
 ● ONPG +: S. sonnei (LLF in MAC)

Classification
● S. dysenteriae (Group A)— Most severe
● S. flexneri (Group B)
● S. boydii (Group C)
● S. sonnei (Group D)— Most common

Virulence factors and disease

● Virulence factor
○ SEVERAL FACTORS
◆ Mediates adherence and invasion odds mucosal cells, escapemfrom phagocytic
vesicle, intracellular spread
◆ Shiga toxin 1

Yersinia pestis
● Epidemic: Plague, Black death
● Class A bioterrorism agent
● Primarily a disease of rodents
● Bites from fleas
● Bubonic/Glandular form (buboes)
● Septicimic/pneumonic form (inhalation)
● Bipolar staining (safety pin appearance)
● Presumptive ID:
○ Mainly dependent of patient history, symptoms, and cases
● Culture
○ Exhibit a rough, cauliflower appearance at 48 hrs in SBA
○ Exhibit a stalactite pattern in broth culture

Yersinia enterocolitica
● Isolated from animals and pets
● Clinical dorm mimics appendicitis: Abdominal pain concentrated in the right quadrant
● Presumptive ID
○ Bacilli (safety pin appearance)
○ Cold enrichment (25 centrigrade)
○ NLF in MAC
○ CIN agar
● Culture
○ Bull’s eye colonies in CIN agar after 48 hours incubation for Y. enterocolitica
○ Optimal growth= Room TEmp; Cold enrichment