Culture and molecular method for detection of Mycobacterium tuberculosis complex and Mycobacterium avium subsp. paratuberculosis in milk and dairy products.

A combined molecular and cultural method for the detection of the Mycobacterium tuberculosis complex (MTBC) and Mycobacterium avium subsp. paratuberculosis was developed and tested with artificially contaminated milk and dairy products. Results indicate that the method can be used for a reliable detection as a basis for first risk assessments.

Ribotyping of Clostridium perfringens from industrially produced ground meat.

AIMS: Clostridium (Cl.) perfringens is a common cause of food poisoning outbreaks. Ribosomal DNA analysis (ribotyping), a method which analyses restriction fragment length polymorphisms in the chromosomal genes that encode rRNA, has been shown to be useful for microbial species identification and subtyping. METHODS AND RESULTS: The current study has used ribotyping to examine 111 Cl. perfringens isolates from industrially produced ground meat in order to collect a basis for a contamination survey. Among the 111 isolates 107 distinctly different ribopatterns were detected. In only four cases two Cl. perfringens isolates showed an identical ribopattern. The isolates gave identical ribotype patterns in three different runs, carried out 3-4 months apart from each other. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The discriminatory index for EcoRI ribotyping of the Cl. perfringens isolates was 0 x 99. Results showed that ribotyping is suitable for subtyping Cl. perfringens isolates from raw meat. Ribotyping appeared to be a useful tool for profound epidemiologic studies of Cl. perfringens-contamination in food production and processing.

Comparison of protocols for pulsed-field gel electrophoresis of Clostridia.

Genotyping of bacterial strains via pulsed-field gel electrophoresis has to be considered an important tool for epidemiological investigations in food hygiene as well as in other areas. Yet, a major disadvantage of this method is its long duration. Therefore, rapid procedures for DNA isolation and restriction are being sought. One such protocol was modified and further shortened to two days. This short protocol was used for macrorestriction analysis of 34 strains of 25 different Clostridium species. Parallel analyses were performed using a conventional 5-day protocol in order to compare the long and the short method by running the DNA samples obtained via both protocols on the same gel. In the case of nine strains, none of the two methods yielded satisfactory results, whereas for three strains the long protocol proved to be preferable to the short one. Comparable results were obtained using both methods in the case of 22 strains belonging to 17 different Clostridium species.

Impedance microbiology: applications in food hygiene.

Impedance microbiology is a rapid method that enables qualitative and quantitative tracing of microorganisms by measuring the change in the electrical conductivity. With direct impedance technology, the change in the conductivity of a liquid culture medium serves as a measuring parameter, whereas with indirect impediometry, the change in the electrical conductivity of a reaction solution, which occurs through the absorption of gases from the inoculated bacterial culture, is measured. Most investigations concerning the applicability of impediometry in food microbiology deal with the impedimetric detection or enumeration of Enterobacteriaceae, especially the detection of Salmonella. However, impediometry has been applied to other bacterial groups or species as well. Furthermore, a great number of published findings concern the impedimetric determination of the total bacterial count. The successful application of this fast method on further areas of food hygiene, such as tracing antibiotics and testing additives for their antimicrobiological effect, has also been described. In general the use of impediometry for the application areas stated has been judged positively. However, the time and expense required by the user to optimize the method, the deficits when testing slightly contaminated sample material or determining the bacterial count in those cases in which the microorganisms are sublethally damaged, and the necessity of performing individual calibration for each food category limit the applicability of impediometry.

Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene.

Clostridium perfringens continues to be a common cause of food-borne disease. It produces an enterotoxin (CPE) which is released upon lysis of the vegetative cell during sporulation in the intestinal tract. Catering premises with insufficient cooling and reheating devices often seem to be the cause of outbreaks of C. perfringens food poisoning. Typing of C. perfringens is of great importance for investigating sources of food poisoning cases and for studying the epidemiology of this microorganism. This report describes the examination of 155 C. perfringens isolates by molecular methods. Isolates were taken from 10 food poisoning outbreaks and cases (n = 34, food and fecal isolates) and from meat and fish pastes (n = 121). Isolates were characterized by plasmid profiling, ribotyping, and/or macrorestriction analysis by pulsed-field gel electrophoresis (PFGE). Results show that all three methods are suitable for classifying C. perfringens isolates below the species level. Ribopatterns and PFGE patterns can be interpreted more easily than plasmid profiling results and can be recommended for contamination studies and epidemiologic investigation of food poisonings associated with C. perfringens.

Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species.

While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming. In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species. The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species. For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species. In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days. In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel. Briefly, the procedure was as follows. After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase. This was followed by a 1-h proteinase K treatment. Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE. After a 2-h restriction with SmaI, electrophoresis was carried out overnight.

Prevalence of the enterotoxin gene and clonality of Clostridium perfringens strains associated with food-poisoning outbreaks.

The prevalence of the enterotoxin gene in a well-characterized collection of 71 Clostridium perfringens strains from 36 separate food-poisoning cases or outbreaks was analyzed with the polymerase chain reaction (PCR). The clonality of 39 strains originating from 14 outbreaks where at least two isolates were available was studied with pulsed-field gel electrophoresis (PFGE) using SmaI and ApaI restriction endonucleases. The cpe gene PCR assay was found to correlate well with Clostridium perfringens enterotoxin (CPE) production in vitro with reverse passive latex agglutination. Of the C. perfringens food and clinical food-poisoning isolates 24 (86%) and 38 (88%) were cpe-positive, respectively. Different PFGE patterns indicated that multiple cpe-positive clones are frequently present within one outbreak. The existence of cpe-positive and negative isolates with identical or nearly identical PFGE patterns in a single outbreak suggests that the cpe gene may be in a movable genetic element.

Strain differentiation of Clostridium perfringens by bacteriocin typing, plasmid profiling and ribotyping.

Bacteriocin typing, plasmid profiling and ribotyping were used to type 34 food and patient Clostridium perfringens isolates from 10 food poisoning cases, respectively, outbreaks. In nine cases/outbreaks bacteriocin patterns showed identical main groups. Subgroups differed within all cases/outbreaks. Plasmid profiles were identical for all isolates within each of three outbreaks. In eight food poisoning cases and outbreaks, all the ribotypes of each food and stool isolate were found to be identical. All three typing methods give valuable results for the characterization of C. perfringens beyond the species level. Bacteriocin typing represents a suitable addition to plasmid typing, particularly since the results do not show any correlation between losses of plasmids and changes in bacteriocin sensitivity patterns. Ribotyping was found to be a suitable tool to determine the genetic relationship of C. perfringens isolates in the context of food-borne poisoning.

Ribotyping for strain characterization of Clostridium perfringens isolates from food poisoning cases and outbreaks.

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases.