Because no special considerations are required for isolation of these genera from extraintestinal sources, However, stool specimens suspected of containing Vibrio spp. should be collected and transported only in Cary-Blair medium. Buffered glycerol saline is not acceptable, because glycerol is toxic for vibrios. Feces is preferable, but rectal swabs are acceptable during the acute phase of diarrheal illness.

DIRECT DETECTION METHODS

 V. cholerae toxin can be detected in stool using an enzyme linked immunosorbent assay (ELISA) or a commercially available latex agglutination test (Oxoid, Inc., Odgensburg, New York), but these tests are not widely used in the United States.

Microscopically, vibrios are gram-negative, straight or slightly curved rods (Figure1). When stool specimens from patients with cholera are examined using dark- field microscopy, the bacilli exhibit characteristic rapid darting or shooting-star motility. However, direct microscopic examination of stools by any method is not commonly used for laboratory diagnosis of enteric bacterial infections.

Fig1. Gram stain of Vibrio parahaemolyticus.

Aeromonas spp. are gram-negative, straight rods with rounded ends or coccobacilli. No molecular or serologic methods are available for direct detection of Aeromonas spp. Cells of C. violaceum are slightly curved, medium to long, gram-negative rods with rounded ends. A polymerase chain reaction (PCR) amplification assay has been developed for identification of C. violaceum.

CULTIVATION

Media of Choice

Stool cultures for Vibrio spp. are plated on the selective medium thiosulfate citrate bile salts sucrose (TCBS) agar. TCBS contains 1% sodium chloride, bile salts that inhibit the growth of gram-positive organisms, and sucrose for the differentiation of the various Vibrio spp. Bromothymol blue and thymol blue pH indicators are added to the medium. The high pH of the medium (8.6) inhibits the growth of other intestinal flora. Although some Vibrio spp. grow very poorly on this medium, those that grow well produce either yellow or green colonies, depending on whether they are able to ferment sucrose (which pro duces yellow colonies). Alkaline peptone water (pH 8.4) may be used as an enrichment broth for obtaining growth of vibrios from stool. After inoculation, the broth is incubated for 5 to 8 hours at 35°C and then subcultured to TCBS.

Chromogenic Vibrio agar, which was developed for the recovery of Vibrio parahaemolyticus from seafood, supports the growth of other Vibrio spp. Colonies on this agar range from white to pale blue and violet.

Aeromonas spp. are indistinguishable from Yersinia enterocolitica on modified cefsulodin-irgasan-novobiocin (CIN) agar (4 µg/mL of cefsulodin); therefore, it is important to perform an oxidase test to differentiate the two genera. Aeromonas agar is a relatively new alternative medium that uses D-xylose as a differential characteristic. These organisms typically grow on a variety of differential and selective agars used for the identification of enteric pathogens. They are also beta-hemolytic on blood agar.

C. violaceum grows on most routine laboratory media. The colonies may be beta-hemolytic and have an almond like odor. Most strains produce violacein, an ethanol soluble violet pigment.

All of the genera considered in this chapter grow well on 5% sheep blood, chocolate, and MacConkey agars. They also grow well in the broth of blood culture systems and in thioglycollate or brain-heart infusion broths.

Incubation Conditions and Duration

These organisms produce detectable growth on 5% sheep blood and chocolate agars when incubated at 35°C in carbon dioxide or ambient air for a minimum of 24 hours. MacConkey and TCBS agars only should be incubated at 35°C in ambient air. The typical violet pigment of C. violaceum colonies (Figure 2) is optimally produced when cultures are incubated at room temperature (22°C).

Fig2. Colonies of Chromobacterium violaceum on DNase  agar. Note violet pigment. 

Colonial Appearance

Table 1 describes the colonial appearance and other distinguishing characteristics (e.g., hemolysis and odor) of each genus on 5% sheep blood and MacConkey agars. The appearance of Vibrio spp. on TCBS is described in Table 2 and shown in Figure 3.

Table1. Colonial Appearance and Characteristics

Table2. Key Biochemical and Physiologic Characteristics of Vibrio spp. and Grimontia hollisae

Fig3. Colonies of Vibrio cholerae (A) and V. parahaemolyticus (B) on TCBS agar. 

APPROACH TO IDENTIFICATION

The colonies of these genera resemble those of the family Enterobacteriaceae but can be distinguished notably by their positive oxidase test result (except for V. metschniko vii, which is oxidase negative). The oxidase test must be performed from 5% sheep blood or another medium without a fermentable sugar (e.g., lactose in MacConkey agar or sucrose in TCBS), because fermentation of a carbohydrate results in acidification of the medium, and a false-negative result may occur if the surrounding pH is below 5.1. Likewise, if the violet pigment of a suspected C. violaceum isolate interferes with performance of the oxidase test, the organism should be grown under anaerobic conditions (where it cannot produce pigment) and retested.

The reliability of commercial identification systems has not been widely validated for identification of these organisms, although most are listed in the databases of several systems. The API 20E system (bioMérieux, St. Louis, Missouri) is one of the best for vibrios. Because the inoculum is prepared in 0.85% saline, the amount of salt often is enough to allow growth of the halophilic (salt-loving) organism.

The ability of most commercial identification systems to accurately identify Aeromonas organisms to the species level is limited and uncertain, and with some kits, difficulty arises in separating Aeromonas spp. from Vibrio spp. Therefore, identification of potential pathogens should be confirmed using conventional biochemical tests or serotyping. Tables 2 and 3 show several characteristics that can be used to presumptively group Vibrio spp., Aeromonas spp., and C. violaceum.

Table3. Key Biochemical and Physiologic Characteristics of Aeromonas spp., P. shigelloides, and C. violaceum

Comments Regarding Specific Organisms

 V. cholerae and Vibrio mimicus are the only Vibrio spp. that do not require salt for growth. Therefore, a key test for distinguishing the halophilic species from V. cholerae, V. mimicus, and Aeromonas spp. is growth in nutrient broth with 6% salt. Furthermore, the addition of 1% NaCl to conventional biochemical tests is recommended to allow growth of halophilic species.

The string test can be used to differentiate Vibrio spp. from Aeromonas spp. In this test, organisms are emulsified in 0.5% sodium deoxycholate, which lyses Vibrio cells, but not those of Aeromonas spp. Cell lysis releases DNA, which can be pulled up into a string with an inoculating loop (Figure 4).

Fig4. String test used to differentiate Vibrio spp. (positive)  from Aeromonas spp. and P. shigelloides (negative). 

A Vibrio static test using 0/129 (2,4-diamino-6, 7-diisopropylpteridine)–impregnated disks also has been used to separate vibrios (susceptible) from other oxidase positive glucose fermenters (resistant) and to differentiate V. cholerae O1 and non-O1 (susceptible) from other Vibrio spp. (resistant). However, recent strains of V. cholerae O139 have demonstrated resistance, so the depend ability of this test is questionable.

Serotyping should be performed immediately to further characterize V. cholerae isolates. Toxigenic strains of serogroup O1 and O139 can be involved in cholera epidemics. Strains that do not type in either antiserum are identified as non-O1. Although typing sera are commercially available, isolates of V. cholerae are usually sent to a reference laboratory for serotyping.

Identification of V. cholerae or V. vulnificus should be reported immediately because of the life-threatening nature of these organisms.

Aeromonas spp. and C. violaceum can be identified using the characteristics shown in Table 3. Aeromonas spp. identified in clinical specimens should be identified as A. hydrophilia, A. caviae complex, or A. veronii complex.

Pigmented strains of C. violaceum are so distinctive that a presumptive identification can be made based on colonial appearance, oxidase, and Gram staining. Nonpigmented strains (approximately 9% of isolates) may be differentiated from Pseudomonas, Burkholderia, Brevundimonas, and Ralstonia organisms based on glucose fer mentation and a positive test result for indole. Negative lysine and ornithine reactions are useful criteria for distinguishing C. violaceum from Plesiomonas shigelloides. In addition to the characteristics listed in Table 3, failure to ferment either maltose or mannitol also differentiates C. violaceum from Aeromonas spp.

SERODIAGNOSIS

Agglutination, vibriocidal, or antitoxin tests are available for diagnosing cholera using acute and convalescent sera. However, these methods are most commonly used for epidemiologic purposes. Serodiagnostic techniques are not generally used for laboratory diagnosis of infections caused by the other organisms discussed in this chapter.

اخر الاخبار

اخبار العتبة العباسية المقدسة

قسم الشؤون الدينية يضاعف نشاطه الإرشادي والعقائدي في شهر صفر

شعبة فاطمة بنت أسد تسلّم نتائج امتحانات الفصل الدراسي الثاني لطالبات المدرسة القرآنية

العتبة العباسية المقدسة تواصل أعمال تهيئة مجموعة مواكب أم البنين (عليها السلام) لخدمة زائري الأربعين

إذاعة الكفيل النسوية تطلق دورة تدريبية بعنوان (سلامة اللغة العربية)

المزيد

الآخبار الصحية

"شعور لا مفر منه" في الحياة يهدد حياة الملايين بالموت المبكر!

أعراض للخرف قد تظهر في المشي.. كيف نكتشفها؟

ماذا يحدث لسكر الدم عند شرب القهوة؟

سرطان القلب.. لماذا يعد من أندر أنواع السرطانات؟

المزيد

الآخبار التكنلوجية

صهر أردوغان يطلق منصة تواصل جديدة.. تنافس فيسبوك ؟!

غوغل تطلق "مرشد الويب" لتنظيم نتائج البحث بالذكاء الاصطناعي

بعد 20 عاما.. رصد أصغر أفعى في العالم مجددا في بربادوس !

ذكر أم أنثى؟.. دراسة تنسف الاعتقاد الشائع حول جنس المولود

المزيد

مواضيع ذات صلة

Bacterial Virulence Factors : Toxins

Pathogenesis and Spectrum of Disease of Vibrio, Aeromonas, Chromobacterium, and Related Organisms

Laboratory Diagnosis of Alcaligenes, Bordetella (Non-pertussis), Comamonas, and Similar Organisms

Antimicrobial Susceptibility Testing and Therapy of Rhizobium, Ochrobactrum, and Similar Organisms

Laboratory Diagnosis of Rhizobium, Ochrobactrum, and Similar Organisms

Antimicrobial Susceptibility Testing and Therapy of Pseudomonas, Burkholderia, and Similar Organisms

Chlamydia trachomatis

Mycoplasma Pneumonia

Leptospira interrogans

Laboratory Diagnosis of Pseudomonas, Burkholderia, and Similar Organisms

Pathogenesis and Spectrum of Disease of Pseudomonas, Burkholderia, and Similar Organisms

Antimicrobial Susceptibility Testing and Therapy of Acinetobacter, Stenotrophomonas, and Similar Organisms