Evaluation of media for isolation of Campylobacter fetus subsp. jejuni from fecal specimens.

Isolation rates of Campylobacter fetus subsp. jejuni from human fecal specimens were equivalent after broth enrichment (thioglycolate medium containing antibiotics) and direct inoculation on two brucella blood agar media containing ferrous sulfate, sodium metabisulfite, and sodium pyruvate, identical concentrations of vancomycin and trimethoprim, and different concentrations of polymyxin B and cephalothin. Studies with clinical isolates of C. fetus subsp. jejuni demonstrated temperature-dependent activity of polymyxin E (colistin) and substantial inhibition of growth on Thayer-Martin and Martin-Lewis media.     

Evaluation of transport media for Campylobacter jejuni in human fecal specimens

It is not always possible to culture feces immediately, and appropriate methods for transport of human specimens, unlike those from animals, have not been fully evaluated. Therefore, we took serial subcultures in two phases from six transport media inoculated with human diarrheal stools known to be positive for Campylobacter jejuni. In phase 1, Cary-Blair medium and buffered glycerol saline did not preserve C. jejuni as well as did alkaline peptone-water (APW), modified Cary-Blair medium, thioglycolate broth (Thio), and Campy-Thio. The four best media (APW, Cary-Blair medium, Thio, and Campy-Thio) preserved 20 fecal samples with C. jejuni better at 4 degrees C (90% survival for 5 to 8 days) than at 25 degrees C (90% survival for 1.7 to 2 days). In phase 2, APW and Thio, along with four modifications of the best media in phase 1, were tested with 23 positive strains. The ranges of survival times with modified media at 25 degrees C were 1.3 to 2.2 days (90%) and 4.7 to 6.8 days (50%). APW with reducing agents preserved C. jejuni better than did APW alone, Thio plus ox bile, or Campy-Thio plus ox bile (P less than 0.05). Thio at pH 8.5 was better at preserving C. jejuni than was APW or Thio plus ox bile (P less than 0.05). If human fecal specimens cannot be refrigerated during transport or storage, we recommend the use of Thio at pH 8.5 or APW with reducing agents for preservation of C. jejuni at 25 degrees C.     

Characterization of species B adenoviruses isolated from fecal specimens taken from poliomyelitis-suspected cases.

BACKGROUND: Human adenoviruses are classified into six species, A-F, and 51 serotypes are recognized. Adenoviruses can cause a broad range of diseases. Serotypes 3, 7 and 21 are most commonly associated with CNS disease. Serotype 21 (specie B) was isolated from brain tissue and CSF of patients with acute flaccid paralysis (AFP) in Malaysia. OBJECTIVES: Characterize, by molecular methods, species B adenoviruses isolated from poliomyelitis-suspected cases and investigate the possible etiological role of adenoviruses in acute flaccid paralysis (AFP). STUDY DESIGN: 622 virus isolates, including Sabin-related polioviruses, non-polio enteroviruses (NPEV) and adenoviruses, were recovered from fecal specimens in our laboratory during the period of 1997-2002 from AFP cases occurring in Brazil, Peru and Bolivia. Negative controls consisted of 528 fecal specimens collected from healthy children <==5 of age. Of these, 478 were contacts of AFP negative cases and 50 were from a day-care center. RESULTS: Sixty-four adenovirus strains isolated in HEp2 (human laryngeal tumor cells) cells were confirmed as such by an adenovirus-group specific PCR. Nucleotide sequencing identified the following adenovirus species: A (3 isolates), B (20 isolates), C (38 isolates), D (2 isolates) and E (1 isolate). The following serotypes belonging to the species B were identified: Ad3 (1 strain), Ad7 (17 strains) and, Ad16 (2 strains). CONCLUSION: Other viral agents became more recognized in association with CNS diseases in areas where wild polioviruses have been eradicated. The possible role of species B adenoviruses in the etiology of AFP cases similar to that caused by wild poliovirus is discussed.     

Frequency of preclumped virus in routine fecal specimens from patients with acute nonbacterial gastroenteritis

A low-speed centrifugation technique for the preparation of grids after minimal purification of fecal extracts is described for examination of viruses by direct electron microscopy using negative staining. Results showed that adenovirus, astrovirus, rotavirus, and "small round" viruses were frequently shed into the gastrointestinal tract in clumps of variable size. Differential centrifugation study showed that a substantial proportion of the virus in the sample was lost in the initial pellet at the first step of clarification; this finding casts doubt on the validity of immune electron microscopy for direct typing of strains of these viruses from stools. In addition, particle counts based on conventional specimen processing are likely to grossly underestimate the true value.     

Use of pooled formalin-preserved fecal specimens to detect Giardia lamblia.

Three formalin-preserved fecal specimens from the same child attending a child-care center were pooled and compared with the three separate individual specimens by a single microscopic examination of concentration sediment for Giardia lamblia. The sensitivity of the pooled system was 100% when two or more individual specimens were positive and 88% when only one individual specimen was positive. The organism density in a single specimen was not a factor of whether the pool of specimens was positive or negative. Nearly half of the pools that contained positive specimens had only one of three specimens with positive results, reinforcing the need for multiple stool examinations when diagnosing G. lamblia infections.     

DNA probe culture confirmation assay for identification of thermophilic Campylobacter species.

We studied the ability of a new DNA probe-based assay system to correctly identify isolates of the thermophilic campylobacters Campylobacter jejuni, C. coli, and C. laridis grown in vitro. We examined 424 organisms, including 214 Campylobacter isolates and 210 other aerobic and anaerobic isolates. The probe assay, which uses a new homogeneous system in which all reactions take place within a single tube, demonstrated 100% accuracy, producing neither false-positive nor false-negative results. The assay does not, however, distinguish among C. jejuni, C. coli, and C. laridis.     

Detection of pathogenic Campylobacter species in blood culture systems.

Because differences in recognition of Campylobacter fetus and Campylobacter jejuni in systemic infections may be due partially to differences in the ability to cultivate these organisms, we studied their growth characteristics in two widely used blood culture systems. In the Roche Septi-Chek system (Hoffman-La Roche, Inc., Nutley, N.J.), over a broad range of inocula all strains were detected in broth within 2 days and on paddles within 3 days. In the BACTEC 6B aerobic bottles (Johnston Laboratories, Inc., Towson, Md.), C. jejuni and C. fetus took a median of 5 and 3 days, respectively, to reach the growth index threshold. However, in the BACTEC 7D anaerobic bottles, C. fetus required a median of 2 days to reach the growth index threshold, whereas for C. jejuni the median was greater than 10 days. The poor performance of C. jejuni in both BACTEC systems may have been due to unfavorable incubation atmospheres and may partially explain why C. jejuni bacteremia is so infrequently detected. Overall, the Roche Septi-Chek system was excellent for detecting Campylobacter strains in blood cultures.     

Semisolid selective-motility enrichment medium for isolation of salmonellae from fecal specimens.

A semisolid selective-motility enrichment medium for the isolation of salmonellae from fecal specimens was developed which was based on Rappaport enrichment broth. During a 7-year period more than 30,000 stool samples were tested. The medium showed a high specificity (95.1%) and sensitivity (80.3%) when compared with MacConkey agar, SS agar, and brilliant green agar (after Selenite-F Enrichment [BBL Microbiology Systems]). Furthermore, our isolation rate of Salmonella species from fecal samples showed an increase of 22.3% when this semisolid medium was added to the routine culture media. Growth could easily be interpreted. The medium has a bias toward the isolation of Salmonella paratyphi B, but it is unsatisfactory for detecting the nonmotile strains Salmonella typhi and S. paratyphi A.     

Use of Sentinel blood culture system for analysis of specimens from potentially infected prosthetic joints.

The Sentinel blood culture system was used for the analysis of 657 specimens from infected prosthetic joints and blood cultures (83 from prosthetic joints and 574 from standard blood cultures). The positivity rate was similar for specimens from prosthetic joints and blood cultures (18% compared with 14%). However, there was an unacceptable rate of false positive results with specimens from prosthetic joints (58% compared with 8%). This high false positivity rate was due to (i) prolonged incubation and (ii) the lack of blood in these specimens. It is therefore recommended that the Sentinel system should only be used for the initial seven days of incubation of specimens taken from prosthetic joints. Further incubation should take place in a standard incubator and a terminal subculture performed after 21 days.     

Use of colony morphology to distinguish different enterococcal strains and species in mixed culture from clinical specimens

Colony morphology on kanamycin esculin azide agar was investigated as a means of selecting different species and strains of enterococci from clinical specimens. Four representative colonies of each morphotype were indistinguishable by pulsed-field gel electrophoresis, biotype, and antibiogram analysis. The optimum time for identification of different colony morphotypes was 72 h.     

Evaluation of culture methods and a DNA probe-based PCR assay for detection of Campylobacter species in clinical specimens of feces

Campylobacter species are the leading agents of bacterial gastroenteritis in developed countries. In this study 320 specimens of feces from patients with symptoms of acute gastroenteritis were cultured for Campylobacter species by direct plating on modified charcoal cefoperazone deoxycholate agar and by enrichment in modified Preston broth, with or without blood added, for 48 h at 37 degrees C prior to plating. A 16S/23S PCR/DNA probe membrane-based colorimetric detection assay was evaluated on a subset of the feces (n = 127), including 18 culture-positive and 109 culture-negative specimens. DNA was extracted directly from the fecal specimens by using the QIAamp DNA stool Minikit for the DNA probe-based PCR assay (PCR/DNA probe assay). A second PCR/DNA probe assay based on the 16S rRNA gene in Campylobacter spp. was applied to all specimens that were culture negative, PCR/DNA positive on initial analysis. Campylobacter species were cultured in 20 of the 320 specimens. The 16S/23S PCR/DNA probe assay detected campylobacter DNA in 17 of 18 (94% sensitivity) culture-positive specimens and in 41 (38%) culture-negative specimens. The presence of campylobacter DNA in 35 of 41 culture-negative specimens was confirmed by the 16S PCR/DNA probe assay. DNA sequence analysis of seven 16S/23S PCR products and five 16S PCR products amplified from a selection of these specimens confirmed the presence of campylobacter DNA and more specifically Campylobacter jejuni, C. concisus, C. curvus, and C. gracilis DNA in these specimens. The molecular assays described in this study are rapid methods for the detection and identification of Campylobacter species in fecal specimens. The finding of Campylobacter spp. DNA in a large number of specimens of feces from patients with no other identified cause of diarrhea may suggest that Campylobacter spp. other than C. jejuni and C. coli may account for a proportion of cases of acute gastroenteritis in which no etiological agent is currently identified.     

Use of a semiselective medium to culture Legionella pneumophila from contaminated lung specimens.

Legionella pneumophila was successfully isolated, using a semiselective medium, from two of three lung specimens heavily contaminated with other organisms. This medium is composed of charcoal yeast extract agar, supplemented with vancomycin and polymyxin B. L. pneumophila was observed at 8 days on plates containing less than or equal to 40 units of polymyxin B and less than or equal to 1 microgram of vancomycin per ml.     

Use of a selective enrichment broth for isolation of Campylobacter species from human feces

Isolation of Campylobacter species from 1126 fecal specimens from patients with diarrhea was compared using direct plating and selective enrichment broth. The use of the enrichment broth did not increase the isolation rate, which was 4.2%. While a selective enrichment broth may have advantages where there is delay in transit to the laboratory, or where small numbers of organisms are sought, we do not recommend its use for clinical specimens from patients with diarrhea.     

Evaluation of broth media for routine culture of cerebrospinal and joint fluid specimens

Broth cultures of cerebrospinal and joint fluids are important components in the culture detection of meningitis and septic arthritis. The authors examined 121 strains of bacteria isolated from clinical specimens representing 13 species or groups that cause meningitis and arthritis for growth in supplemented Thioglycolate broth (THIO), Supplemented Peptone Broth (SPB), and minced beef heart (MBH) media each alone or with added IsoVitaleX. Both SPB and MBH with IsoVitaleX performed better as broth culture media than the media without IsoVitaleX or THIO with or without IsoVitaleX.     

Comparison of six media, including a semisolid agar, for the isolation of various Campylobacter species from stool specimens.

A recently described semisolid blood-free selective motility medium (SSM) (J. Goossens, L. Vlaes, I. Galand, C. Van den Borre, and J. P. Butzler, J. Clin. Microbiol. 27:1077-1080, 1989) was compared with two charcoal-based selective media (charcoal-based selective medium [CSM] and modified charcoal cefoperazone deoxycholate agar [CCDA]), two blood-based media (Skirrow medium [SKM] and CampyBAP), and a passive, 0.65-microns-pore-size cellulose acetate membrane filter technique for the recovery of campylobacters from stools of patients with diarrhea. A total of 1,980 specimens were tested, 161 of which were found to be positive for campylobacters. Campylobacter jejuni was isolated in 148 specimens (91.9%), C. coli was isolated in 27 (7.5%), and "C. upsaliensis" was isolated in 1 (0.6%). After 72 h of incubation with a single medium, the cumulative percentages of Campylobacter-positive specimens isolated on CSM, CCDA, SKM, and SSM were 87, 83, 80, and 72%, respectively. The filter method alone enabled us to recover 61% of all campylobacters. The "C. upsaliensis" strain was isolated by this method only. The highest isolation rates were observed when two media, including CSM, were combined. The combination of CSM and SSM yielded the highest rates (96%), but these were not statistically different from the rates observed with combinations of CSM and SKM (94%) or of CSM and the filter method (91%). Extending the incubation time from 48 to 72 h led to an increase in the isolation rate regardless of the medium used (P less than 0.001). CSM and CCDA were the most selective media. SKM and CampyBAP appeared to be the most inhibitory media for the isolation of C. coli.     

Use of culture, PCR analysis, and DNA microarrays for detection of Campylobacter jejuni and Campylobacter coli from chicken feces

A DNA microarray for detection of Campylobacter spp. was recently developed and applied to detect Campylobacter spp. directly from chicken feces. Sixty-five pooled chicken cloacal swab samples from 650 individual broiler chickens were included in the study. The results of Campylobacter sp. detection obtained with DNA microarrays were compared to those obtained by conventional culture and gel electrophoresis. By conventional culture, 60% of the samples were positive for either Campylobacter jejuni or Campylobacter coli. By PCR and capillary electrophoresis, 95% of the samples were positive for Campylobacter spp., whereas with DNA microarrays all samples were positive for Campylobacter spp. By application of DNA microarray analysis, the isolates in 4 samples (6%) could not be identified to the species level, whereas by PCR-capillary electrophoresis, the isolates in 12 samples (19%) remained unidentified. Interestingly, PCR-capillary electrophoresis analysis revealed that two (3%) of the samples were positive for both C. jejuni and C. coli, while DNA microarray analysis revealed that nine (14%) of the samples were positive for both species. Of 65 samples, 2 samples were identified to contain C. coli by conventional culture but were positive for C. jejuni by both PCR-capillary electrophoresis and DNA microarray analysis. The discrepancy between the methods is discussed.