Modern methods for species identification of thermophilic bacteria of Campylobacter species

Emergent thermophilic Campylobacter hepaticus is the causative agent of Spotty Liver Disease (SLD) in laying hens. C. hepaticus is difficult to cultivate because commercial media for the isolation and cultivation of Campylobacter contain cefoperazone, which inhibits many isolates of the C. hepaticus species. Campylobacter was isolated using modified Preston broth, incubated at 37 °C under microaerophilic conditions for 7 days and then subcultured onto selective Preston agar, erythritol agar with Oxoid selective additives and 5–7% defibrinated horse blood. Commercial test systems (API Campy) were used for identification. The use of the classical bacteriological diagnostic method, which is considered the 'gold' standard, is limited due to the difficulties of cultivation. The identification of new Campylobacter species requires revision of phenotypic identification algorithms. Specific primers for the identification of new Campylobacter species also need to be developed. In our studies, using the KAM-BAC kit, we detected Campylobacter jejuni DNA in clinically healthy birds. Consequently, the carriage of Campylobacter is massive. 30 samples of test material were examined using the molecular-biological method, and 60 samples using the bacteriological method. Analyzing the results of Campylobacter detection, it should be noted that thermophilic Campylobacteria were isolated from 60 clinical samples by the bacteriological method in 5,0% (3 Campylobacter cultures), and from 30 samples by the molecular-biological method in 27,0% (8 positive samples). Based on the analysis of the study results, it is necessary to conduct an in-depth study of the natural sources of Campylobacter hepaticus distribution, virulence factors, pathogenesis and mechanisms of infections caused by these emergent pathogens. The most promising research in the study of the causative agents of Campylobacteriosis in birds will be based on the application of innovative genomic technologies based on multiplex polymerase chain reactions and genome sequencing of Campylobacter hepaticus.

Investigation of some gastric Helicobacter species in saliva and dental plaque of stray cats by cultural and PCR methods

The purpose of this study was to explore the presence of gastric Helicobacter species in the oral cavity of stray cats in the Kars region. Saliva and dental plaque samples collected from 100 stray cats were evaluated by culture and PCR methods in terms of gastric Helicobacter species. For culture, samples were plated on 5% defibrinated-horse blood and 5% defibrinated-sheep blood enriched selective agar plates supplemented with Vancomycin (6 μg/ml), Polymyxine B (2.500 IU/l), Trimethoprim (20 μg/ml) and Amphotericin B (2.5 μg/ml). Molecular methods were also included to study by using the PCR targeting amplification of the 16S rRNA gene sequence for Helicobactergenus and urease B gene sequence for each Helicobacter species. As the results of cultural examination, Helicobacter spp. were isolated from 10 (10%) cats (10 saliva and 5 dental plaque samples) and these were further identified as H. heilmannii by PCR. Direct analysis of samples by genus-specific PCR revealed that a total of 70 (50 saliva and 20 dental plaques) samples from 65 cats were positive in terms of Helicobacter DNA. As the results of species – specific PCR analysis of these samples 34 (48.57%) (24 saliva and 10 dental plaque samples) were identified as H. heilmannii, while the remaining 36 (51.42%) were found to be negative in terms of related species (H. heilmannii, H. pylori and H. felis). It has been concluded that these bacteria, identified in the oral cavity of the cats, may play a role in transmission of infection to humans.

Development and Validation of a New Protocol for Detecting and Recovering Clostridium difficile from Meat Samples

ABSTRACT There is no recommended protocol for detecting and isolating Clostridium difficile present in food samples. Here, we have evaluated the recovery of C. difficile in meat samples after incubating them in various enrichment broths. The media were as follows: cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme; cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme; and cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, C. difficile moxalactam norfloxacin selective supplement, and lysozyme. Samples were inoculated with various strains and quantities of C. difficile and then enriched in the different broths for 1, 4, and 7 days. C. difficile was isolated on agar plates and detected with quantitative real-time PCR (qPCR). The procedure using enrichment in cycloserine-cefoxitin fructose broth supplemented with taurocholic acid, d-cycloserine, cefoxitin, and lysozyme and incubation for 4 days for qPCR detection and 7 days for isolation (plating on C. difficile agar base with added C. difficile selective supplement and 7% [v/v] defibrinated horse blood after alcoholic shock and centrifugation) was validated. Samples of different kinds of meat and meat preparation were contaminated and used for validation of the chosen protocol. The sensitivity of detection with qPCR was 100%, and the sensitivity of the isolation method was 96%.

Ultimate pH values and bacteriological condition of meat and stress metabolites in blood of transported reindeer bulls

Twenty-three reindeer bulls, aged 2-3 years, fed during two winter months at the Vuolda reindeer research station in Arjeplog, Sweden, were used in the study. The first group of eight reindeer was moved from their feeding corral to a selection corral, captured by lasso and stunned with a captive bolt outside the selection corral. The second group of seven reindeer was moved to the selection corral, captured by lasso and restrained, after which they were loaded onto a lorry- and transported for 1 hour and then slaughtered. The third group of eight reindeer was moved to the selection corral and herded directly onto the lorry, without any manual handling. They were transported for 5 h and then slaughtered. In both transport groups, four reindeer were fitted with pre-programmed automatic blood sampling equipment (ABSE). ABSE sampled blood at predetermined times via a jugular vein catheter. Ultimate pH-values in three muscles (Mm. longissimus, triceps brachii and biceps femoris) were significantly lower in the group carefully handled and transported for 5 h compared with the other two groups. The physiological mechanisms behind these results are discussed. Samples from M. semimembranosus were collected at slaughter and after 2, 6 and 10 days of refrigerated storage (+4 °C). The samples were analysed for total counts of aerobic bacteria (pour-plated in Tryptone Glucose Extract Agar, Difco, incubated at 20 °C and 30 °C, respectively for 72 h), coliform bacteria 37 °C (pour-plated in Violet Red Bile Agar, Oxoid, incubated at 37 °C for 24 h), Enterococci (surface-plated onto Slantez and Bartley Agar, Oxoid, incubated at 44 °C for 48 h) and Bacillus cereus (surface-plated onto Blood Agar Plates (Blood Agar Base, Difco, supplemented with 5% defibrinated horse blood) 30 °C for 24 h). All samples fell in the range 'fit for consumption'. At slaughter, there was no difference in ASAT activity, urea and Cortisol concentrations between the two transported groups. However, the plasma ASAT activity and urea concentrations at slaughter were significantly lower in the non-transported group. In both transport groups, the plasma Cortisol concentrations increased during loading onto and unloading from the lorry. Abomasal lesions were observed in all treatment groups. It was concluded that reindeer showed an acute stress response to manual handling and transport.

Characterization of a Culturable “Gastrospirillum hominis” (Helicobacter heilmannii) Strain Isolated from Human Gastric Mucosa

Spiral organisms were isolated from an antral gastric mucosal biopsy specimen from a dyspeptic patient with gastritis. Only corkscrew-shaped organisms resembling “Gastrospirillum hominis” (“Helicobacter heilmannii”) but noHelicobacter pylori-like organisms were seen in histological sections. H. pylori was not cultured from specimens from this patient. On the basis of biochemical reactions, morphology, ultrastructure, and 16S DNA sequencing, the isolated “G. hominis” was shown to be a trueHelicobacter sp. very similar to Helicobacter felis and the “Gastrospirillum” but was separate from H. pylori. “G. hominis” is a pleomorphic gram-negative cork-screw-shaped, motile rod with 3 to 8 coils and a wavelength of about 1 μm. In contrast toH. pylori, it has up to 14 sheathed flagellar uni- or bipolar fibrils but no periplasmic fibrils. “G. hominis” grows under microaerobic conditions at 36 and 41°C on 7% lysed, defibrinated horse blood agar plates within 3 to 7 days and can be subcultured under microaerobic but not under anaerobic conditions on media similar to those used for H. pylori and H. felis. The small translucent colonies were, in contrast to those of H. felis, indistinguishable from those of H. pylori. “G. hominis” is, like H. pylori and H. felis, motile, is oxidase, catalase, nitrite, nitrate, and urease positive, and produces alkaline phosphatase and arginine arylamidase. Like H. pylori and H. felis, it is sensitive to cephalothin (30-μg disc), resistant to nalidixic acid (30-μg disc), and sensitive to most other antibiotics. The 16S DNA sequence clusters “G. hominis” together with “Gastrospirillum,” H. felis,Helicobacter bizzozeronii, Helicobacter salmonii, Helicobacter nemestrinae, Helicobacter acinonychis, and H. pylori.

Examination of Poultry Giblets, Raw Milk and Meat for Campylobacter fetus subsp. jejuni

An MPN procedure was used to determine the presence of Campylobacter fetus subsp. jejuni in poultry giblets. This procedure consists of (a) subculturing a sample in Brucella broth supplemented with 0.15% agar, 0.05% sodium pyruvate and the following antimicrobial agents per liter: vancomycin 10 mg, trimethoprim 5 mg, polymyxin B sulfate 2,500 IU, amphotericin B 2 mg and cephalothin 15 mg and (b) subsequent streaking of a loopful of Brucella broth held at 42 C for 48 h on plates of Brucella agar supplemented with 10% defibrinated horse blood and the concentrations of antimicrobial agents identified above. C. fetus subsp. jejuni was present in 85% of the chicken livers and in 89% of the chicken gizzards obtained immediately after evisceration. The organism was not recovered from samples treated with chlorinated water. C. fetus subsp. jejuni was not recovered from raw milk (bulk tank samples or individual cow samples) or from beef (infraspinatus or biceps femoris muscles).

Effects of Preenrichment Media and Their Incubation Conditions on Isolating Salmonellae from Fish Meal

Various combinations of preenrichment media, their incubation times and temperatures and atmospheres were examined for their efficacy in recovering salmonellae naturally occurring in fish meal. Variations included three preenrichment media (lactose broth, lactose broth supplemented with 10% defibrinated horse blood, and lactose broth with 10% egg yolk), three (25, 37 and 43 C) incubation temperatures, two (24 and 48 h) incubation times, and two (aerobic and anaerobic) incubation atmospheres. Three hundred and twenty samples (50 g each) of various consignments of fish meal known to be contaminated with salmonellae were examined. The lowest number (282) of isolations of salmonellae was obtained using lactose broth for preenrichment. Lactose broth with addition of blood gave 321 isolations, and lactose broth with egg yolk 357 isolations. In general, advantage was observed for preenrichment for 48 h (504) over preenrichment for 24 h (456). This was particularly evident at 25 C with 201 and 144 isolations, respectively. However. at 43 C the results were reverse (135 isolations after 48 h. and 156 after 24 h). At 37 C not so significant differences were found (168 - 48 h, and 156 - 24 h). Salmonellae were isolated 345 times with preenrichment at 25 C. 324 times at 37 C and 291 times at 43 C. Anaerobic preenrichment gave more (510) positives as compared with aerobic preenrichment (450 positives).

Selective medium for isolation of Bacteroides nodosus

The sensitivity of Bacteroides nodosus, the causative agent of sheep foot rot, to 24 selected antimicrobial agents was tested. Many contaminants ordinarily associated with foot rot lesions were sensitive to lincomycin, whereas B. nodosus demonstrated resistance to this antibiotic. A concentration of 1 microgram of lincomycin per ml of basal medium optimally inhibited contaminants while allowing growth of B. nodosus. The basal medium was Eugon agar with 0.2% yeast extract and 10.0% defibrinated horse blood. Parallel inoculations of 31 foot rot lesion specimens onto basal medium and basal medium containing lincomycin (selective medium) were performed. B. nodosus was isolated from 16 of the specimens cultured on the selective medium and from only 3 of the specimens cultured on the basal medium. The concentration of agar in the standard Eugon medium was found to influence the growth of B. nodosus in the presence of lincomycin. Trimethoprim also exhibited potential selectivity for B. nodosus.

Enrichment medium for the isolation of Bordetella

The development of a specimen collection and transport medium outfit for the rapid laboratory diagnosis of whoping cough is described. The transport medium consisted of a semisolid agar containing charcoal, cephalexin, and defibrinated horse blood. It was also found to be an excellent enrichment medium for the selective isolation of Bordetella pertussis and B. parapertussis from scantily populated specimens. The investigation of 3,237 specimens that yielded 1,419 positive isolates of Bordetella, including 86 B. parapertussis, during a 20-month period is presented. A total of 3,076 specimens were processed in the laboratory by using the enrichment medium in addition to the routine procedure. Of these specimens, 757 were submitted in our medium, from which 137 (18%) were positive. Of the 567 specimens received in Amies transport medium, 290 (51%) positive cultures were obtained by the enrichment method only and not by primary culture.