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Modern microbiological techniques using characterization of DNA are increasingly used in ecological microbiology to describe the diversity of microfloras. The benefit is that systems can be described with a much higher degree of reality. In dialysis, one application is the evaluation of fluid-system disinfection strategies.
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106 samples of chilled, cured canned hams and shoulders have been examined with a traditional plate count technique and with the Direct Epifluorescent Filter Technique (DEFT). The results have shown that the traditional bacteriological technique for enumeration of viable bacteria and DEFT do not correlate and further that DEFT, better than the traditional bacteriological technique, can predict microbiological problems for sliced products produced from chilled canned cured meat products.
Undefined Nurmi-type cultures (NTCs) have been used successfully to prevent salmonella colonisation in poultry for decades. Such cultures are derived from the caecal contents of specific-pathogen-free birds and are administered via drinking water or spray application onto eggs in the hatchery. These cultures consist of many non-culturable and obligately anaerobic bacteria. Due to their undefined nature it is difficult to obtain approval from regulatory agencies to use these preparations as direct fed microbials for poultry. In this study, 10 batches of prototype NTCs were produced using an identical protocol over a period of 2 years. Traditional microbiological techniques and a molecular culture-independent methodology, polymerase chain reaction combined with denaturing gradient gel electrophoresis (PCR-DGGE), were applied to characterise these cultures and also to examine if the constituents of the NTCs were identical. Culture-dependent analysis of these cultures included plating on a variety of selective and semi-selective agars, examination of colony morphology, Gram-staining and a series of biochemical tests (API, BioMerieux, France). Two sets of PCR-DGGE studies were performed. These involved the amplification of universal and subsequently lactic acid bacteria (LAB)-specific hypervariable regions of a 16S rRNA gene by PCR. Resultant amplicons were subjected to DGGE. Sequence analysis was performed on subsequent bands present in resultant DGGE profiles using the Basic Local Alignment Search Tool (BLAST). Microbiological culturing techniques tended to isolate common probiotic bacterial species from the genera Lactobacillus, Lactococcus, Bifidobacterium, Enterococcus, Clostridium, Escherichia, Pediococcus and Enterobacterium as well as members of the genera, Actinomyces, Bacteroides, Propionibacterium, Capnocytophaga, Proteus, and Klebsiella. Bacteroides, Enterococcus, Escherichia, Brevibacterium, Klebsiella, Lactobacillus, Clostridium, Bacillus, Eubacterium, Serratia, Citrobacter, Enterobacter, Pectobacterium and Pantoea spp. in addition to unculturable bacteria were identified as constituents of the NTCs using universal PCR-DGGE analysis. A number of the sequences detected by LAB-specific PCR-DGGE were homologous to those of a number of Lactobacillus spp., including L. fermentum, L. pontis, L. crispatus, L. salivarius, L. casei, L. suntoryeus, L. vaginalis, L. gasseri, L. aviaries, L. johnsonii, L. acidophilus, and L. mucosae in addition to a range of unculturable lactobacilli. While NTCs are successful due to their complexity, the presence of members of Lactobacillus spp. amongst other probiotic genera, in these samples possibly lends to the success of the NTC cultures as probiotics or competitive exclusion products in poultry over the decades. PCR-DGGE proved to be an effective tool in detecting non-culturable organisms present in these complex undefined cultures. In conclusion, while the culture-dependent identification methods or PCR-DGGE alone cannot comprehensively elucidate the bacterial species present in such complex cultures, their complementarity provides useful information on the identity of the constituents of NTCs and will aid in future development of defined probiotics. Moreover, for the purpose of analysing prototype NTCs during their development, PCR-DGGE overcomes the limitations associated with conventional culturing methods including their low sensitivities, inability to detect unculturable bacteria and unknown species, very slow turnabout time and poor reproducibility. This study demonstrated that PCR-DGGE is indeed more valuable in detecting predominant microbial populations between various NTCs than as an identification methodology, being more applicable as a quality control method used to analyse for batch-to-batch variation during NTC production.
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AIMS: To determine the sensitivity and specificity of two automated enzyme immunoassays (EIA), EiaFoss and Minividas, and a conventional microbiological culture technique for detecting thermophilic Campylobacter spp. in turkey samples. METHODS AND RESULTS: A total of 286 samples (faecal, meat, neckskin and environmental samples) were collected over a period of 4 months at a turkey slaughterhouse and meat-cutting plant in Denmark. Faecal and environmental samples were tested by the conventional culture method and by the two EIAs, whereas meat and neckskin samples were tested by the two EIAs only. Two enrichment broths were used, Campylobacter Enrichment Broth (CEB) and Preston Broth (PB). Verification of positive test results was carried out by conventional culture on selective solid media. The specificities of all methods were high. The sensitivities of the EIAs were higher than that of the conventional culture technique but varied depending on the type of sample and enrichment broth. For neckskin samples, the Minividas had a significantly higher sensitivity than the EiaFoss and using PB instead of CEB as the enrichment broth significantly improved the sensitivity for both EIAs. CONCLUSIONS: Both EIAs provided more accurate results than the conventional culture technique. Furthermore, neckskin samples enriched in PB resulted in more positive test results and Campylobacter growth than samples enriched in CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: The Eiafoss and Minividas proved to be reliable methods for detecting Campylobacter spp. in various samples. However, the results emphasize the need for the development of specific enrichment protocols for specific samples.
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Infection control teams rely on microbiology laboratories for accurate, reproducible data to trace the spread of microorganisms in the hospital. Since few problems are recognized immediately, laboratories should save important nosocomial pathogens so that outbreak strains will be available for analysis by more sophisticated techniques, particularly since automation has reduced the amount of epidemiologically useful data generated by routine procedures. Traditional supplemental reference procedures include phage typing, biotyping, serotyping and bacteriocin typing. Recently, analysis of antibiotic resistance determinants has provided the hospital epidemiologist with additional tools. Resistant strains have been characterized by their production of specific antibiotic inactivating enzymes. Agarose gel electrophoresis and restriction endonuclease analysis of plasmid DNA have permitted precise characterization of nosocomial strains, and the spread of antibiotic resistance genes can now be followed by sensitive and specific DNA probes. DNA probes also show promise for distinguishing nosocomial strains bearing known virulence factors from strains with less pathogenic potential. Molecular genetics techniques have found an additional role in elucidating the epidemiology of nosocomial viruses, especially the herpesviruses.
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The Compu-pet 100 diluter/dispenser is a versatile and accurate instrument which can be employed for a wide variety of microbiological tests and techniques. Time in performing tests is often more than halved when the diluter is used. Large numbers of tests can be performed with minimal fatigue.
In a field study the hygiene level of trucks for the transport of pigs was measured. In total, on 56 times the hygiene level of a truck has been measured with two different methods, one microbiological method and one bioluminescence technique. During the field study 1.913 hygiene samples were taken. The average hygiene-level after cleaning and disinfection improved. The improvement was based on education and advisory about hygiene which immediately could be used by the truck-drivers. The fact that the results of hygiene measuring was shown directly was also a stimulation to the drivers; they improved their cleaning and disinfection job. The use of detergents by cleaning trucks is not required, although it contributes to a better hygiene-level. The introduction of plastic materials in stead of wood also improved the overall-hygiene in the transportation of animals.