Gram-Negative Enteric Rods and Escherichia Coli

Gram-Negative Enteric Rods

• Non-fastidious organisms
• Aerobic and facultative anaerobic bacteria
• All species belonging to this family are glucose fermenters, catalase positive, and oxidase negative
• Major pathogens include Salmonella spp., Shigella spp., and Yersinia spp.
• Other representatives of the family are members of the intestinal normal flora and are opportunistic pathogens

Escherichia Coli

• Specimens for intestinal infections due to enteric pathotypes of E. coli include stool and bile
• Specimens for extraintestinal infections depend on the site of infection, such as urine, pus, cerebrospinal fluid, and blood
• Direct examination of the specimen under microscopy reveals Gram-negative rods with rounded ends
• E. coli can be cultivated on blood agar plates
• MacConkey agar is also used for E. coli cultivation
• E. coli can grow on lactose agar
• E. coli can be cultivated on Levine Medium
• S-colonies appear on colonie morphology
• Some strains are beta-hemolytic on blood-agar plates
• Lactose-containing media reveals lactose-positive colonies
• Red colonies are formed on MacConkey agar
• Yellow colonies appear on lactose agar
• In SIM agar, H2S is negative, motility is positive, and indole is positive
• In TSI agar, glucose is positive, H2S is negative, and lactose and sucrose are positive
• No growth appears on Simmons agar
• Urease is negative in urea medium
• Agglutination reaction identifies specific pathotypes of Antigenic structure E. coli Enterohemorrhagic E. coli (ЕНЕС) is O157:H7
• Important resistance phenotypes: ESBL (extended-spectrum β-lactamase) producer strains in antibiotic susceptibility testing

Klebsiella Genus

• Medically important representatives include K. pneumoniae and K. oxytoca
• Specimens include urine, sputum, pus, cerebrospinal fluid, and blood
• Direct examination via Gram stain shows Gram-negative cocobacilli in pairs
• The negative stain reveals a capsule
• Blood agar plates are cultivation media
• Lactose agar is a cultivation media
• MacConkey agar is a cultivation media
• Identifying characteristics are morphology and colonies, specifically M colonies
• Lactose-containing media are lactose-positive
• MacConkey colonies appear lactose positive (red)
• colonies lactose agar are lactose positive (yellow)
• In SIM agar, H2S is negative, motility is negative, and indole is variable
• TSI agar shows glucose positive, H2S negative, and lactose and sucrose positive
• There is growth on Simmons agar
• Urease is positive in urea agar
• Quellung reaction identifies capsular antigens
• Antibiotic susceptibility testing identifies ESBL (extended spectrum β-lactamase) production
• KPC (Klebsiella pneumoniae carbapenemase)

Proteus Genus

• Medically important species are P. vulgaris, P. mirabilis, and P. penneri
• Specimens include urine, sputum, pus, cerebrospinal fluid, and blood Direct examination in microscopy reveals Gram-negative rods
• They can be cultivated on blood agar plates
• Used cultivation media: Lactose agar
• MacConkey agar is used
• Morphology is seen microscopy for identification
• Colonies are present on culture media without selective agents (without bile salts), swarming colonies are present
• Colonies are present on culture media with selective agents, S colonies
• Lactose-containing media yields lactose-negative colonies
• Black centered colonies contains iron salts
• SIM agar Biochemical patterns H2S is positive, motility is positive, indole is variable
• TSI agar Biochemical patterns glucose is positive, H2S is positive, lactose and sucrose negative
• Simmons agar - Biochemical patterns growth
• Urea agar - Biochemical patterns urease positive
• Antibiotic susceptibility testing should be performed

Shigella

• Shigella spp. cause dysenteria
• Shigella dysenteriae, flexneri, boydii, sonnei
• Specimens: stool, food (source detection)
• Direct examination: visual inspection reveals blood, pus, and mucus in stool; microscopy shows Gram-negative rods
• MacConkey and SS agar for cultivation
• Morphology is assessed for identification.
• Colonies and biochemical patterns can be viewed
• S lactose negative colonies
• SIM:
• H2S negative
• Motility negative
• Indole variable
• TSI:
• Glucose positive
• H2S negative
• Lactose, sucrose negative
• Simmons agar has no growth (green)
• Urea agar: urea negative
• Antigenic structure allows species identification (agglutination)
• Pathogenicity tests
• Serény test – eye infection in guinea pig
• Tests on cell cultures
• Strains that are resistant to aminoglycosides, 1st and 2nd generation cephalosporins should not be tested nor used in therapy

Salmonella Genus

• The species Salmonella Typhi and Salmonella Paratyphi A and B are associated with typhoid fever
• Salmonella Enteritidis, Typhimurium, etc. are associated with acute gastroenteritis
• The specimens for typhoid fever include blood, intestinal secretions (vomitus or duodenal fluid)
• Stool samples have 85-90% patients 1st week, 20-30% later
• Culture of urine may be positive, timing unpredictable
• Bone marrow aspirate has the highest sensitivity of detection
• Stool samples identify carriage
• Stool samples identify gastroenteritis
• Blood identifies disseminated infection
• Microscopic direct examination: Gram-negative rods
• Enrichment media for cultivation
• Lactose agar for cultivations is used
• MacConkey and SS are used for cultivations
• MacConkey: lactose-negative (transparent) S colonies during identification
• SS agar colonies - lactose-negative (transparent) S, black center upon identification
• SIM agar: H2S positive, motility positive, indole negative (Biochemical Patterns)
• TSI agar: glucose positive, H2S positive, lactose sucrose negative (Biochemical Patterns)
• Simmons agar: growth
• Urea agar: urease negative
• Agglutination reactions with O, H, and Vi antigens identify the antigenic structure
• O antigen serogroups are established by agglutination reactions with O, H, and Vi antigens
• Serotypes are determined according to Η anitgens.
• Vi antigens are surface antigens in S. Typhi and Paratyphi serovars, and they mask O antigens.
• The Widal reaction is a classic diagnostic method for typhoid fever.
• It is used to detect antibodies against O and H antigens using a quantitative agglutination test
• Some Salmonella are resistant to aminoglycosides and 1st and 2nd generation cephalosporins and should not be tested to be used in therapy

Yersinia Enterocolitica

• Specimens: stool sample (gastroenteritis), lymph node tissue (mesenteric adenitis, mesenteric ileitis, and acute pseudoappendicitis), and blood (sepsis)
• Microscopic direct examination: Gram-negative rods
• MacConkey used for cultivation
• Lactose agar used for cultivation
• CIN agar: selective culture medium for Yersinia enterocolitica cultivation
• Identification and morphology include small, transparent lactose-negative S colonies
• Identification and morphology include CIN agar: bull's eye colonies (red centered)
• SIM agar and biochemical patterns indole negative
• TSI agar and biochemical patterns glucose positive, H2S negative, and lactose negative which ferments sucrose
• Simmons agar: no growth
• Urea agar: positive
• O3 and O9 are the most frequently encountered serogroups in Europe
• An antibiogram should be performed for Yersinia spp. isolates during antibiotic testing.

Colony Morphology - Differential Characteristics

Vibrio Cholerae

• Vibrio cholerae is an aerobic, oxidase positive, glucose fermenter Gram-negative curved rod, found in aquatic environments
• Serogroups O1 and O139 are the causative agents of cholera
• Stool specimens, rectal swab, water and food samples
• Visual examination of the stool samples a watery stool with flakes of mucus and epithelial cells ("rice-water stool")
• Gram stain – Gram-negative, thin, curved, comma-shaped bacillus with one polar flagellum
• Wet mount examined in darkfield: motile bacteria
• ELISA: Detection of bacterial antigens (01, 0139) from the stool samples
• Non-fastidious organisms, which grow on alkaline pH.
• Alkaline peptone water for cultivation.
• Blood agar for cultivation.
• Special media (e.g. BSA - bile, salt, agar, TCBS -Thiosulphate Citrate Bile Salts Sucrose)
• Morphology (see direct methods) for identification
• Colony morphology for identification
• Rapid growth on liquid media (pellicle formation) – in 4 hours for identification
• Solid media – S colonies, watery appearance
• It is Oxidase and Glucose and Sucrose positive for identification structure
• Identification: antigen-antibody reactions to identify serogroups (01, 0139)
• Automated/Semiautomated Identification Systems and Molecular typing during Automated structure structures
• Suspected strains are referred to reference laboratories for confirmation