NON-ENTERICS (Aeromonas, Plesiomonas, Campylobacter, Helicobacter).docx

***AEROMONAS spp.*** - Ubiquitous - Oxidase (+) - Glucose-fermenters - Widely distributed in: [freshwater, estuarine, marine] environment - Isolated from: [retail produce sources & animal meat products] - Only 8 spp., 2 biovars within *A. veronii* are frequently isolated...

***AEROMONAS spp.*** - Ubiquitous - Oxidase (+) - Glucose-fermenters - Widely distributed in: [freshwater, estuarine, marine] environment - Isolated from: [retail produce sources & animal meat products] - Only 8 spp., 2 biovars within *A. veronii* are frequently isolated from clinical specimens - Have **seasonal pattern** -- increased in warmer months \@US - Straight rods - Classified into two groups: - **Mesophilic group** (optimum growth \@37degC) - **Psychrophilic group** (optimum growth @ 22degC) - **Mesophilic group has 3 diff. complexes:** **-**grow well at 37degC **-**motile (monotrichous flagella) - **[*A. hydrophila* complex]** - *A. hydrophila* - *A. bestiarum* - *A. salmonicida* (motile strains) - **[*A. veronii* complex]** - *A. veronii* biovar **sobria** - *A. veronii* biovar **veronii** - *A. jandaei* - *A. trota* - *A. schubertii* - **[*A. caviae* complex]** - *A. caviae* - *A. media* - *A. eucrenophila* - **Psychrophilic group** - *A. salmonicida* -- fish pathogen, - NOT human pathogens - Grows at 22-25degC **[Clinical Manifestations:]** **Intestinal Manifestations** - Recognized as [enteric pathogens**,**]but not in the same manner as the more common enteric pathogens - Level &pattern of virulence is more like of *E.coli* subgroups associated with enteric diaease (ETEC, EHEC) - **5 diarrheal presentations observed:** 1. **Acute, secretory diarrhea** - With vomiting 2. **Acute, dysentery diarrhea** - Similar to shigellosis - With blood and mucus 3. **Chronic diarrhea** - Last for \>10 days 4. **Cholera-like disease** - Rice water stools 5. **Nebulous syndrome** - Traveler's diarrhea (Similar to ETEC) - Most cases are self-limiting - GIT infections - ***A. caviae*** - most frequently associated - Especially in neonates, pediatric population - Associated with inflammatory bowel disease - Other spp. associated with diarrhea: - *A. hydrophila* - *A. veronii* biovar **sobria** - *A. veronii* biovar **veronii** - More serious complications from include: - HUS - Kidney disease **Extraintestinal Infections** - Septicemia - Meningitis most common - Wound infections - Implicated in cases of: - Osteomyelitis; Pelvic abscess - Keratitis (contact lens wear); Endophthalmitis - **Wound infections** - Recent traumatic aquatic exposure - Occurs on [extremities] - **[Cellulitis]** -- most common presentation - Most common isolates: - *A. hydrophila* - *A. veronii* biovar **sobria** - *A. schubertii* -- almost exclusive from aquatic wound and bloodstream infections - Most common cause of skin & tissue infections among survivors of 2004 tsunami in Thailand. - *A. veronii* biovar **veronii** associated with surgical wound infections - Uses leeches *(Hirudo medicinalis)* for medicinal therapy to relieve venous congestion - **Aeromonad sepsis** - Most invasive type of infection - Strong association with: - *A. veronii* biovar **veronii** - *A. jandaei* - *A. hydrophila* [**Culture and Colony Morphology**:] - Grow readily in most media used for routine and stool cultures - 24-hr incubation: [large, round, raised, opaque, w/ entire edge, smooth, often mucoid surface] - There is an **extremely strong odor** present - Pigmentation: translucent to white to buff-colored - SBA -- hemolysis is variable - *A. veronii* biovar **sobria** - *A. jandaei* - *A. hydrophila* beta-hemolysis - *A. caviae* - Grow on almost enteric media - Often overlooked on MAC (stool cultures), because some aeromonads **ferment lactose** (*A.caviae)* - [Yields highest recovery] of aeromonads - SBA w/o ampicillin - Ampicillin inhibit some *A. caviae* - As well as *A. trota* -- hallmark: universally susceptible - Oxidase test performed in this colony = separates Ox+ *aeromonads* & Ox- *yersinia enterocoliica* - Modified CIN II - 4 ug of cefsulodin - Pink-centered colonies (**ferment mannitol**) **Biochemical tests:** - Important screening procedures, especially on suspicious colonies on SBA, with beta-hemolytic colonies: - **Oxidase test** - Oxidase-positive - Differentiates aeromonads from enterics - **Spot indole test** - Indole-positive (most clinically significant) - **String test** -- distinguish aeromonads from *Vibrio spp.* - **Negative** - **O/129** -- resistant - **Growth in NaC**l - distinguishes aeromonads & Plesiomonas from Vibrio) - **Nutrient broth w/ 0% NaCl --**P&A grow well - **Nutrient broth w/ 6% NaCl --** vibrio thrives - **Inositol fermentation** - Differentiate aeromonads and plesiomonads - Aeromonads -- **Negative** - Plesiomonads -- positive - **Glucose fermentation w/ or w/o gas production** - Distinguish aeromonas from oxidase-positive, non-fermenting Pseudomonas isolates. - **Esculin hydrolysis test** - Requires agar-based medium w/ or w/o bile - NEVER a standard broth or miniaturized cupule broth version - **POSITIVE:** - *A. hydrophila* - *A. veronii* biovar *veronii* - *A. caviae* - **Species-related disease syndromes** - **Aquatic wounds** - *A. hydrophila* - *A. schubertii* - **Septicemia, Meningitis, Medicinal leech therapy** - *A. veronii* biovar *sobria* - **Pediatric diarrhea** - *A. caviae* - **Antimicrobial susceptibility studies** - Should [always be determined by Kirby-Bauer disk diffusion method] - Because of possible serious discrepancies in beta-lactamase detection by rapid minimal inhibitory concentration methods that would directly affect the proper treatment for aeromonad infections - **Almost uniformly RESISTANT TO:** - Penicillin - Ampicillin *(except for susceptibility of A. trota and A. caviae to ampicillin)* - Carbenicillin **PLESIOMONAS spp.** - Oxidase-positive (differentiates it from Enterics) - Glucose fermenter - Facultative Anaerobe - Most strains **ferment lactose** - Closely related to *Enterobacteriaceae, **but** unlike the enterics, **Plesiomonas do not have the ability to produce gas from glucose*** - Susceptible to O/129 (differentiates it from Aeromonas) - ***P. shigelloides** --* only species in this genus - Straight rods, occur [singly, in pairs, in short chains] - Motile (monotrichous flagella or 2-5 lophotrichous flagella) - ***Plesiomonas & Shigella*** share biochemical and antigenic features. - ***Plesiomonads** often cross-agglutinte with S. sonnei, S. dysenteriae, S. boydii* - *Hence the spp is called shigelloides* - ***P. shigelloides*** has lower virulence potential \> Shigella - No spores or capsules - Can be serotyped by: **somatic O Ag and H Ag** - Intolerant to increased NaCl and minimum growth temp. of 8degC - Isolates found generally in fresh & estuarine waters in tropical and subtropical climates - Widely distributed among warm- and cold-blooded animals **Clinical Manifestations:** **[Gastroenteritis]** - Has **three** well-documented outbreaks of diarrheal disease - **MOT:** ingestion of contaminated water or food, particularly raw/undercooked seafood - **Virulence factors:** - **LT, ST** - **3 Major clinical types of gastroenteritis:** - **Watery/Secretory diarrhea** - More common - **Subacute / Chronic diarrhea** - Lasts 14 days to 2-3 months - **Dysenteric form** - Invasive - Resembles colitis - Single most common symptom: [abdominal pain] - Most cases are self-limiting **[Extraintestinal infections:]** - Source of infection can be from **occupational exposure** (e.g: veterinarians, zookeepers, fish handlers) - **More serious infections:** - Occur usually only in severely immunocompromised patients / neonates - **Bacteremia** - **Meningitis** - **Continuous ambulatory** - **Peritoneal dialysis-associated peritonitis** - **Biliary tract disease** -- possible risk factor for bacteremia **Culture and Colony Morphology** - Grow readily on most routine media - 18-24 hrs incubation @ 35 degC - **Shiny, opaque, non-hemolytic colonies, slightly raised center, smooth and entire edge** - Most are LF = delayed - **Oxidase test** - Easiest screening procedure - Performed on colonies from **nonselective media** (SBA/ CHOC agar) - **Specialized medium NOT RECOMMENDED** - Certain strains are inhibited in MAC/EMB - **Will not grow on** **TCBS agar** - **Will thrive on:** - **Inositol brilliant green bile agar** - Enhances isolation of plesiomonads - *Plesiomonas* colonies are **white -- pink** - *Coliform* colonies are green/pink - **CIN** - Selective agar for *Yersinia spp.* - Colonies are: opaque w/ opaque apron - Opaque (NMF) - Must be distinguished from other Oxidase-Pos organisms which will grow on CIN (e,g: *Aeromonas, Pseudomonas)* - **Any agar should NOT CONTAIN** **AMPICILLIN** - They are SUSCEPTIBLE **Glucose** **Oxidase** **Inositol** **Mannitol** **0% NaCl** **6.5% NaCl** TCBS O/129 ------------- ------------- -------------- -------------- ------------- --------------- ------ ------- \+ \+ \+ \- \+ \- \- \- **CAMPYLOBACTER & HELICOBACTER spp.** -has morphologic similarities -inability to recover these organisms using routine lab media for primary isolation -motile -with few exceptions, most of these bacteria are **microaerophilic** (5-10% O~2~ requirement) **[CAMPYLOBACTER]** - Slow, fastidious - Asaccharolytic - Oxidase-Positive - Known to cause **abortion in domestic animals** - Primarily **zoonotic organisms** - ***[C. jejuni] =* Most common cause of bacterial gastroenteritis worldwide** - **MOT:** - direct contact with animals and handling pet - indirectly: consumption of contaminated water & dairy products & improperly cooked poultry - Person to person, some are spp. are sexually transmitted - **[4^th^ most common cause] of foodborne gastrointestinal illness** - **[Common human pathogens:]** - ***C. jejuni*** - ***C. coli*** - Other *Campylobacter spp.* that cause GI disease: - *C. coli* - *C. lari* - ***C. fetus subsp. fetus*** -- isolated frequently from [blood cultures], rarely associated with GI disease - Does NOT multiply in food (unlike other foodborne agents) **Clinical Manifestations:** - *C. fetus has **two subspecies:*** - *C. fetus subsp. **fetus*** - *C.* fetus subsp. **venerealis** - **GI disease caused by *[C. jejuni:]*** - Diarrheal disease - Mild abdominal pain (2-10 days) - Cramps & bloody diarrhea - Illness is mostly self-limiting (resolves 2-6 days) - *Campylobacter spp.* produce **3 syndromes ^(bailey's)^** - **Febrile SYSTEMIC disease** - **Periodontal disease** - **Gastroenteritis --** most common - *Campylobacter* **plays a role** in **Guillain-Barre Syndrome** - **GBS --** [autoimmune disorder] caused by **[damage to peripheral nervous system]** and is characterized by acute paralysis - **Ab** produced during *Campylobacter* infection **bind to gangliosides** found on peripheral nerves. **Cross-reactivity with this nerve cells** in an autoimmune response = resp. for debilitating nerve disorder - Many patients with GBS test positive for Ab to *Campylobacter* **Specimen Collection & Transport:** - *Campylobacter* that can cause enteric illness - Stool specimen / rectal swab - Delay of more \>2 hours, placed in transport media: - **Cary-Blair** medium (4 degC) - **Modified Stuart medium** - **Campy thio** (thioglycolate broth base w/ 0.16% agar and certain antibiotics) - **Buffer glycerol saline** - TOXIC to enteric *campylobacters* **Culture Media** +-----------------------+-----------------------+-----------------------+ | | **Base** | **Antimicrobials** | +=======================+=======================+=======================+ | **Campy-BAP** | Brucella agar base | Vancomycin | | | | | | *(enriched selective | 10% **Sheep** RBCs | Trimethoprim | | medium)* | | | | | | Polymyxin B | | | | | | | | Amphotericin B | | | | | | | | Cephalothin | +-----------------------+-----------------------+-----------------------+ | **Skirrow's** | Oxoid BA base | Vancomycin | | | | | | | lysed, defibrinated | Trimethoprim | | | **horse** RBC | | | | | Polymyxin B | +-----------------------+-----------------------+-----------------------+ | **Butzler** | Thioglycolate fluid | Bacitracin | | | w/ agar added | | | | | Novobiocin | | | 10% **Sheep** RBCs | | | | | Actidione | | | | | | | | Colistin | | | | | | | | Cefazolin | +-----------------------+-----------------------+-----------------------+ | **Medium V** | \*modification of | Cefoperazone | | | Butzler, seems to | | | | **inhibit** normal | Amphotericin B | | | colon microbiota | | | | **better** than the | Rifampin | | | orig. formulation | | | | | Colistin | +-----------------------+-----------------------+-----------------------+ | **Campy-CVA** | \*provide **better | \*CVA -- | | | suppression of fecal | cefoperazone-vancomyc | | | biota** when | in-ampthothericin | | | incubated 37deg C | B | +-----------------------+-----------------------+-----------------------+ | **CCD Agar** | **\***blood-free | **Cefoperazone** | | | medium | | | | | Amphotericin B | | | Nutrient agar | | | | | | | | ** Charcoal** | | | | (replaces the blood) | | | | | | | | Sodium | | | | **deoxycholate** | | +-----------------------+-----------------------+-----------------------+ **Incubation & Cultivation:** - 42 degC (stool cultures) to recover *C. jejuni* - DOUBLE PURPOSE: - Grows optimally at 42 degC - Growth of colon microbiota is inhibited - *C. fetus subsp. fetus* is suppressed at 42deg C, incubated at 37degC - Requires a **microaerophilic & capnophilic envi.** - 5% O2 - 10% CO2 - 85% N2 - Except for ***C. curvus, C, rectus***, obligate anaerobic envi. DOES NOT SUPPORT growth of most *Campylobacters* - Recommended inoculation of **two selective agars** to increase recovery of organisms - **Filtration method** (0..65 um cellulose-acetate) used in conjunction w/ nonselective medium to enhance recovery **Identification:** - **Morphology:** - gram-neg rods - Comma, S, "gull wing" shapes - Motile (monotrichous) - **Darting motility** on hanging drop prep. or under microscope - To observe: **suspend in *Brucella/tryptic soy broth*** - Distilled water & saline seem to INHIBIT motility - Non-spore forming - Stains poorly in gram-stain - **Carbol fuschin** is recommended as a counterstain - **Safranin** is used, extend for 2-3 mins. - **Colony Morphology:** *C. jejuni & enteric campylobacters* - Moist, runny looking and spreading - Non-hemolytic - Some are round, raised or flat - *C. fetus subsp. fetus* - Smooth, convex, translucent - Gray-yellow gray-pink - Some exhibit tailing effect along streak line **Biochemical Tests: (*C. jejuni & significant spp.)*** +-----------------------+-----------------------+-----------------------+ | Oxidase | \+ | | +=======================+=======================+=======================+ | Catalase | \+ | | +-----------------------+-----------------------+-----------------------+ | Nitrate reduction | \+ | | +-----------------------+-----------------------+-----------------------+ | Urease | 0 | | +-----------------------+-----------------------+-----------------------+ | H2S production | 0 | | +-----------------------+-----------------------+-----------------------+ | Hippurate Hydrolysis | **+** | | | | | | | | only *C. jejuni* | | +-----------------------+-----------------------+-----------------------+ | Indoxyl acetate | **+** | | | hydrolysis | | | | | only *C. jejuni, C. | | | | coli* | | +-----------------------+-----------------------+-----------------------+ | **Susceptibility | *C. jejuni* | *C. fetus* | | to:** | | | | | *C. coli* | | +-----------------------+-----------------------+-----------------------+ | Nalidixic acid | **S** | **R** | +-----------------------+-----------------------+-----------------------+ | Cephalothin | **R** | **S** | +-----------------------+-----------------------+-----------------------+ | Lysis by complement | **S** | **R** | +-----------------------+-----------------------+-----------------------+ **Antimicrobial Susceptibility:** - *C. jejuni & C. coli* are susceptible to many antimicrobial agents - Macrolides - Tetracyclines - Aminoglycosides - Quinolones - DOC: Erythromycin (severe gastroenteritis) - **Agar dilution testing** - (CLSI) method of choice for *Campylobacter* susceptibility testing *p* **[HELICOBACTER spp.]** - Primary linked to **gastric infections** - Can cause low-grade inflam. process, producing chronic superficial gastritis - DOES NOT INVADE gastric epithelium, but infection is recognized by immune system - **MOT:** feco-oral or oral-oral transmission - **Virulence Factors:** **CLINICAL MANIFESTATIONS:** - **Major cause of type B gastritis** - Long term infection = chronic gastritis = risk factor for gastric carcinoma - Spectrum of disease: - Gastritis - Peptic ulcer disease - Gastric cancer **LAB DIAGNOSIS:** - Recovered from: gastric biopsy - **Transport medium:** Stuart medium - Tissue samples: - Cysteine-Brucella broth w/ 20% glycerol - Frozen at -70 degC - **Diagnosis:** - **Gastric biopsy spx**. Preserved in 10% formaldehyde **CULTURE MEDIA & INCUBATION & CULTIVATION:** - Non-selective medium: - **CHOC agar** - **Brucella agar w/ 5% horse RBCs** - **Brain Heart Infusion Agar^(baileys)^** - **Wilkins Chalgren Agar^(baileys)^** - **Trypticase soy Agar^(baileys)^** - Selective medium: - **Skirrow's agar** - **MTM^(baileys)^** - Inoculated medium should be fresh and moist - Cultured at **microaerophilic environment, increased humidity** - Required environment: - 5-10% O2 - 5-12% CO2 **IDENTIFICATION** - **Morphology:** - Resembles *Campylobacter* - But has multiple flagella at one pole (lophotrichous) unlike the monotrichous flagella of *Campylobacter* - **Colony Morphology:** - May require 4-7 days of incubation before some colonies are observed - Small, translucent, circular colonies - Plates should be reviewed daily for min. 10 days before reported as negative **BIOCHEMICAL TESTS:** Oxidase \+ ---------------------------- ----------------- Catalase \+ Nitrate reduction V Urease **+** H2S production 0 Hippurate Hydrolysis 0 Indoxyl acetate hydrolysis 0 **Susceptibility to:** Nalidixic acid **Resistant** Cephalothin **Susceptible** - Grows at 42 degC - **Serodiagnosis:** - **IgM\>IgG\>IgA**