Enterobacterales

What are the general characteristics of enterobacterales (9) (mention exceptions)

• Gram-negative rods

• Aerobic/ FA

• Non-encapsulated EXCEPT Klebsiella

• Non-spore formers

• Motile with peritrichous flagella EXCEPT: Shigella and klebsiella - Non-motile, and Yersinia enterocolitica - motile at 22C but not at 37C (SKY)

• Grow on MacConkey (Bile salt + Crystal violet →Selective against G+ | Lactose + phenol red → Differentiate based on lactose fermentation)

• Grow between 10-40 (optimal 37)

• Oxidase -, Catalase + (S. dysenteriae is Cat -), Nitratase + (nitrate reductase), Glc + (with exceptions

• High rates of conjugation and transduction

Differentiate between Enterobacterales based on their biochemical characteristics

Very important to separate enterobacterales into lactose fermenters and non-fermenter

Note: In the image, disregard Oxidase, they’re all Oxidase negative

What are the culture media used for enterics

• MacConkey agar

• EMB agar

• Salmonella Shigella agar (SS)

• Triple Sugar Iron agar (TSI)

How does Enterobacterales serotyping work?

Based on 3 antigens:

• K - Capsular Ag (Majority don’t have it, and in the case of Salmonella, it’s called Vi)

• O - Somatic Ag

• H - Flagellar antigen

What are the main E.coli virulence factors

Structural virulence factors

• LPS surface O Ag - Septic shock + Protects from phagocytosis and complement system

• Envelope K Ag - Protects against phagocytosis → Can help it in causing pneumonia and neonatal meningitis

• Fimbriae (P Pili) - Help the bacteria colonize (GI colonization > Diarrhea/ UT colonization → UTI)

Exotoxins (Cause of diarrhea)

• Labile Toxin (Heat-labile) - LT

• Stable Toxin (Heat-stable) - ST

• Verocytotoxin or Shiga-like toxin - VT

Hemolysins (may be nephrotoxic)

Explain the mechanisms of LT and ST

LT → Adenylyl cyclase activation → cAMP → Secretion of Na⁺ and Cl^- → Water follows

ST → Guanylyl cyclase activation → cGMP → Inhibition of ionic uptake in enterocytes → Osmotic loss of water from cells

What clinical presentations can E. coli be associated with

• UTIs

• Diarrhea

• Septicemia → Septic shock

• Pyogenic infections

  • Wound infections (Especially after lower intestinal tract surgery)

  • Biliary tract infection

  • Neonatal meningitis

  • Peritonitis

What E. coli serotypes are responsible for UTIs

The same ones found in feces

How does E. coli cause UTIs?

2 routes:

→ Ascending = via urethra
→ Descending = Hematogenously

How do the kidneys and bladder get infected generally

Kidneys may become infected by urine backflow, while the bladder usually gets infected by incomplete urine emptying

What are the physiological cause of UTIs in women

The rectum and vagina are in close proximity to the urethra, which is significantly shorter than the urethra in males → Easy ascend

What are the physiological causes of UTIs in men

• The penis can easily come in contact with bacteria that can travel upwards the urethra (However urethra is quite long, so much less likely to get cystitis).

• Prostate enlargement in males may block urine flow and cause infections

What does the presence of nitrite in urine indicate

It indicates the presence of enterobacterales, since they are nitrate reductase +

UTI stuff

What kind of urine specimens are used for UTI assessment

• Mid-stream urine

• Catheter specimen urine

• Suprapubic aspiration

*Note: In case of urine retention, might need to directly remove urine from bladder

Review this slide later

Explain the Kuss semi-quantitative method

Diagnoses UTIs depending on bacterial count:

• <10000/mL = Contaminant unless there are symptoms

• 10000 to 100000 = Inconclusive results → Repeat test. If symptoms are present → Bacteriuria

• >100000/mL = Significant bacteriuria

Exceptions: If patient is taking antibiotics or diuretics, or if the pathogen is S. Aureus, then even small numbers can be significant

What mediums are used to culture E. coli

Blood agar, MacConkey

Biochemical identification of E. Coli

If we have an organism that’s G -, Indole +, Rod → 90% E. coli

What clinical conditions is EPEC associated with

Infantile diarrhea
institutional outbreaks.

Describe pathogenesis in EPEC

EPEC adhere via fimbria and disrupt the brush border microvilli → Diarrhea (Noninvasive; Nontoxigenic)

What are the symptoms that we see in EPEC

Fever, diarrhea, vomiting, nausea, non-bloody stools BUT with mucus

EPEC diagnosis

• Culture on BA/MA

• EPEC O antigen serotyping

What does ETEC cause

Traveler’s diarrhea

ETEC Pathogenesis

Production of plasmid-encoded toxins (LT/ST) - (Noninvasive, toxigenic)

Clinical features of ETEC

Watery diarrhea, vomiting, abdominal pain

ETEC diagnosis

Demonstration of the presence of enterotoxin

DNA probes for LT/ST

EIEC Pathogenesis

EIEC invade the epithelial cells by endocytosis and can spread laterally to adjacent cells → Tissue destruction and necrosis → Dysentery

Lab diagnosis of EIEC

Virulence marker antigen ELISA (VMA ELISA)

DNA probes

EHEC main virulence factor

What’s the most common EHEC serotype

EHEC produces verocytotoxin - VT, a Shiga-like toxin (SLT) (aka Verocytotoxigenic E. coli VTEC)

O157:H7

What the mechanism of action for Verocytotoxin?

It targets vascular endothelial cells and mucoal epithelial cells and inhibits protein synthesis → Cytotoxicity

Explain EHEC detailed pathogenesis

Here’s what maybe happens

1. EHEC attaches to gut mucosa epithelium

2. EHEC starts producing VT

3. VT moves to the lamina propria, either by Leakage or transcytosis → May then enter the circulation (HUS cause)

4. VT can only enter and disrupt protein synthesis in cells that express Gb3, this includes endothelial cells of the lamina propria, and those of kidney Glomeruli

   1. In the gut, necrosis of endothelial cells due to VT leads to ischemia → Indirect damage to enterocytes of the mucosa + leakage of blood from the lamina propria into the intestinal lumen (Hemorrhagic colitis)

   2. Damage to endothelial cells of kidney glomeruli + Widespread damage to RBCs leads to the formation of microthrombi that can block the already damaged kidneys → Triad of Acute Renal failure, hemolytic anemia, and thrombocytopenia

What are EHEC outbreaks associated with

Fast food - Hamburgers
Undercooked meat
Raw leafy vegetables (Especially vegetables grown by natural fertilizers since EHEC is a normal part of cattle microbiota)

How can we diagnose EHEC

By demonstration of bacilli or VT in stools

or

DNA probes for VT

or

Culture: On Sorbitol MacConkey agar or blood agar
→ Unlike other E. coli, they cannot ferment sorbitol → Colorles/pale colonies
→ EHEC is Hemolytic on blood

E. Coli treatment

No.1 = Fluid replacement

No.2 if severe and systemic (e.g. HUS) = Antibiotics

What is the reservoir for salmonella species

Intestinal tracts of warm and cold-blooded animals

What are the salmonella species

2 Species

• Salmonella enterica (Many subspecies + over 2500 serotypes), Serotypes include:

  • Typhoidal:
    → Typhi
    → Paratyphi

  • Non-Typhoidal:
    → Enteritidis
    → Typhimirium
    → Paratyphi
    → Choleraesuis

• Salmonella bangori

What are the 2 clinical manifestations that can be produced by salmonella species depending on serotype?
Explain the detailed pathogenesis in both cases

In typhoidal serotypes, we get Enteric fever (typhoid fever), and in non-typhoid, and:

Pathogenesis: Salmonella mainly invades the gut through M-cell mediated transcytosis, after which it is phagocytosed by the macrophages of the lamina propria.

• In typhoid serotypes: Phagocytosis is ineffective due to the Vi capsules of salmonella that prevent the fusion of lysosomes with phagosomes → Macrophages act as trojan horses and transport the bacteria systemically

• In non-typhoidal serotypes: Phagocytosis is eventually complete; however, inflammation is very severe at the level of the intestines due to the lack of immune evasion → Enteritis

  • However, they can still cause systemic diseases in old, young, or IC patients. AND, in sickle cell anemia, they’re also associated with osteomyelitis (Mostly non-typhoids)

How does salmonella keep being shed in feces for months

It uses cholesterol gallstones, which provide a solid surface to establish a biofilm → Chronic shedding
And if immunity wanes → Re-infection

Do typhoid and non-typhoid salmonellas have the same reservoirs

No,

Typhoid → Only humans
Non-typhoid → Humans and animals

Describe the documented symptoms of Typhoid fever

• Enteritis-like: Headache, malaise, anorexia, abdominal pain/discomfort coated tongue

• ONE LOOSE STOOL FOLLOWED BY CONSTIPATION

  • *Note: If someone is at the stage of constipation, we have to take a blood sample to test, not a stool sample

• Rose spots - Truncal maculopapular rash

• Flu-like: Prolonged fever, chills, bradycardia, dry cough, epistaxis (nose beeds)

• Bacteremia: Soft palpable spleen, hepatomegaly

• Chronic infection: Low WBC count, but Monocytosis (seen in chronic)
  

Compare enteritis and Typhoid fever

Slide 43

What are the methods for Salmonella isolation (Samples used)

Blood culture (Gold standard - 80-100% accuracy in detecting bacteremia)

Stool culture

Bone marrow (Rare)

Describe the non-cultural diagnostic techniques for salmonella

Widal test: Detection of antibodies (Inclonclusive cuz depends on stage of the disease)

Detection of antigens in blood and urine

When should we do blood culture vs stool culture

Should do blood cultures in the early stages of the disease, because bacteremia occurs in the early stages.

Stool culture is best in the later stages of the disease (Chronic shedding thing). 80% sensitivity in 3rd week patients

For how long do nontyphoidal strains keep being shed in stools?

for 4-5 weeks and rarely, for over a year - Slow clearance

Salmonella culture

• MCA/DCA → Pale (cuz NLF)

• Hektoen enteric agar (HEA) → Positive H2S → Cat eye colonies

  • Note: S. typhi i a weak H2S producer

• SS → Black colonies with silver/metallic sheen

• EMB → Colorless/Light purple (NLF)

• TSI → + H2S and Gas

Describe Salmonella’ environmental resilience

Survives on:

 55º C – 1 h

 60º C – 15 min

 Boiling, Chlorination, Pasteurization destroy the Bacilli.

Salmonella Gastroenteritis treatment

• Replace lost fluids orally and IV

• Antibiotics actually worsen things in uncomplicated cases:
  → Don’t shorten illness
  → Prolong carrier state due to dysbiosis
  → Increase risk of relapse
  * Give ABios if IC or severe disease or infants

Typhoid fever treatment for resistant and non-resistant strains

• Non-resistant:

  • Fluoroquinolones (Ciprofloxacin) PO or IV

  • Ceftriaxone or Cefepime

• Resistant:

  • Ceftriaxone IV

  • Azithromycin PO

  • High-dose Ciprofloxacin (Only if isolates were tested and found to be less susceptible)

Salmonella vaccine

*Both for typhoid, none for enteritis

Oral vaccine: Live-attenuated vaccine

IM Vaccine: Vi capsular polysaccharide

What are the shigella species

Shigella dysenteriae (Group A) - The only one which produces Stx
Shigella flexneri (Group B) - Developing countries
Shigella boydii (Group C)
Shigella sonnei (Group D) - Industrial world

What’s the major cause of bacillary dysentry

Shigellosis

What are the reservoirs for shigellas

Humans only

How high is the infectious dose for shigella

It’s quite low (10²-10⁴ CFU), cuz its acid-stable

Shigella incubation period

1-3 Days

What is Shigella’s epidemiological significance

 Common in Pediatric age group (1-10 yrs)

 Leading cause of infant diarrhea & mortality in developing countries

 Outbreaks in daycare centers, nurseries, institutions

Explain shigella pathogenesis and symptoms

Shigella enters through M cells → Phagocytosis by waiting macrophages → Shigella triggers macrophage pyroptosis which causes massive neutrophil recruitment which disrupt tight junctions and increase permeability, which appears as Watery diarrhea (May contain some blood & mucus)→ Shigella is released and enters enterocytes of the mucosa through their basolateral sides → Now shigella can manipulate actin filaments to spread from cell to cell laterally → This causes epithelial cell death + mucosal ulceration
Add intense inflammation + microvascular damage → blood + mucus + pus in stool (Dysentry) + Fever (If S. dysentariae cuz Stx is also neurotoxic)

What are the common shigella complications

HUS/ Reiter’s syndrome (arthritis)

What kind of specimens do we use for shigella

Stools or rectal swab from ulcer

Describe fecal microscopy in shigella vs amoebic dysentries

Shigella: RBC + lots of pus cells

Amoeba: RBC but few pus cell

Shigella treatment

Adults: Rehydration + Norfloxacin or Ciprofloxacin

Children: Rehydration + TMP-SMX or Ampicillin

Shigella precention

What is the pathogenesis for Y. pestis

S66

What is the pathogenesis for Y. enterolitica and Y. pseudotuberculosis

What are the clinical manifestations of Y. pestis

How can Y. enterolitica be acquired

Contaminated milk/pork/ contaminated market meat (by feces) and vacuum-packed beef

Y. enterolitica clinical manifestations

Gastroenteritis + fever

OR

Bloody diarrhea, pseudoappendicitis, reactive arthritis (adults)

Y. enterolitica lab stuff and treatment

List the klebsiella species

K. pneumoniae, K. oxytoca, K. ozaenae, K. rhinoscleromatis

What differentiates Klebsiella pneumoniae

It has a polysaccharide capsule that protects it against phagocytosis/antbiotics.

In the lab: The capsule causes the growth of moist-mucoid colonies with a yeasty odor

What are the clinical manifestations of Klebsiella pneumoniae

• Pulmonary infection/ Aspiration pneumonia:
  → Dark red “currant jelly” sputum + Abcesses in lungs and liver

• Meningitis & Enteritis in infants

• UTIs

• Septicemia

• Nosocomial infections (OXA48)

Pneumonia and drug abuse and alcohol

Drug abuse - think S.aureus

Alcoholism - Think klebsiella

Klebsiella lab stuff

Where are enterobacter, Serratia, and Citrobacter commonly found

Moist environments and hospitals

Name the enterobacters

Isolation specimen

Characteristic

→ E. aerogenes / E. cloacae
→ Isolated from wounds, urine, blood, CSF
→ Colonies resemble lebsiella, also MOTILE

Name the serratias

Only S. marcescens

What is the clinical significance of Serratia

Responsible for nosocomial infections of UT/RT.
Associated with nurseries and cardiac sergeries, and burn units

Serratia biochemical characteristics

Produce characteristic pink pigment, especially when cultures are left at room temperature
Slow LF

Citrobacter species

Citrobacter freundii

What clinical manifestations is citrobaccter associated with

Nosocomial infections and UTIs/ Pneumonia

Citrobacter biochem

 Ferments lactose

 Hydrolyzes urea slowly

 Citrate +

What are the characteristics of Morganella, Proteus, and Providencia (MPP)

All part of our normal flora, and all opportunistic pathogens
Phenylalanine demaminase (PDA) +
Lactose -

Why is proteus called proteus

Cuz it’s pleomorphic

Proteus species

P. mirabilis and P. vulgaris

Where can proteus be isolated

Urine - Associated with urinary catherization, wounds, ear, blood infections (bacteraemia)

*Note: In case of catheterization, urine pH can tell us about proteus infection

Proteus biochemistry

Urease +

PDA +

Gelatinase +

H2S+ on TSI (Look scheme in general characteristics)

Morganella morganii everything

Causes UTIs
Urease +
PDA +
H2S -

Providencia species and their clinical significance

Providencia rettgeri and stuartii

Cause UTIs and nosocomial outbreaks

H2S negative on TSI

What are ESBLs

Plasmid-encoded Extended-spectrum Beta-lactamases
→ Destroy Penicillins, Cephaloporins, Azteronam. ALSO, non Beta-lactamase antbiotics (FQs/AGs/TMP)
Produced by some E. coli and Klebsiella strains

Counter: Carbapenems (However, there are Carbapenemase producers now)