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Biomedical subjects

P M Desmarchelier

Publications and source records attributed to P M Desmarchelier.

At least 19 recordsLinked to original sources
PubMed2004

In vitro studies on the colonization of bovine colonic mucosa by Shiga-toxigenic Escherichia coli (STEC).

This study investigated host-related factors that influence intestinal colonization by Shiga-toxigenic E. coli (STEC). A quantitative colonization assay was developed to comparatively measure attachment of STEC to bovine colonic tissues maintained in vitro. No differences were determined in colonization susceptibility between tissues derived from weaning calves and adult cattle, or for tissues from cattle fed grain and forage-based rations. Substrate conditions designed to represent various intra-enteric environments were tested for their effect on STEC/mucosal interaction. Under conditions corresponding to a well-fed ruminant (high volatile fatty acid and lactate concentrations, low pH), significantly less STEC colonized the mucosal surface of colonic biopsies. These results may help explain why fasted, poorly or intermittently fed cattle and pre-ruminant calves excrete STEC to a greater degree. Studies on the ecology of STEC within the ruminant gut help identify mechanisms to reduce their threat to public health.

Age Factors↗
PubMed1999

Use of pulse field gel electrophoresis for the epidemiological characterisation of coagulase positive Staphylococcus isolated from meat workers and beef carcasses.

Coagulase positive staphylococci isolated at an Australian abattoir, from beef carcasses, hands, and environmental samples, were typed by the DNA macrorestriction patterns obtained following PFGE of SmaI digests. The predominant PFGE pattern of isolates collected before evisceration was different from the dominant pattern of isolates collected after evisceration. PFGE patterns for isolates from workers' hands and from concordant carcasses were indistinguishable. PFGE types among isolates collected from non-evisceration abattoir personnel and clerical staff were distinct from patterns among isolates collected from the slaughter floor personnel. Twenty-nine (85%) of the 34 isolates collected from carcasses after evisceration exhibited PFGE patterns that were 93% homologous. During evisceration the carcasses were handled extensively and it is proposed that the hands of workers at this abattoir are the primary source of staphylococcal contamination of carcasses on the slaughter floor. This paper highlights the usefulness of PFGE as an epidemiological tool for determining the source of staphylococcal contamination in the food industry.

Abattoirs↗
PubMed1999

Incidence of coagulase positive Staphylococcus on beef carcasses in three Australian abattoirs.

The contamination of beef carcasses with coagulase-positive staphylococci (CPS) was studied at three beef abattoirs (A, B and C). The incidence and the number of CPS were determined on cattle hides immediately after slaughter and on three carcass sites (brisket, flank and round) at different points during processing along the slaughter line. The incidence of CPS on cattle hides ranged from 20 to 68.6%. At abattoir A, 6.5% of the carcasses sampled before evisceration were contaminated with CPS, compared to 40% of the carcasses after evisceration. The incidence on carcasses changed little during further processing; however, after chilling for 72 h, the incidence increased to 83%. After evisceration, the brisket and flank areas were more often contaminated than the round. A similar pattern of contamination was observed at abattoir B. At abattoir C, 26.7% of the samples collected before evisceration were contaminated and this fell to 16.7% after evisceration. After chilling for 72 h, the incidence of carcass contamination with CPS increased to 46.7%. The average number of CPS on contaminated carcasses prior to and after overnight chilling was less than 50 colony-forming units (cfu)/cm2 and, after weekend chilling, increased to 64 and 112 cfu/cm2 in abattoirs A and B, respectively. Of the isolates tested, 71.4% produced staphylococcal enterotoxin and 21% could not be classified phenotypically. The hands of workers and environmental sites associated with the evisceration process were examined for CPS at abattoir A. Hands were heavily contaminated and were the likely source of CPS contamination at this abattoir.

Abattoirs↗
PubMed1997

Isolation and identification of Burkholderia pseudomallei from soil using selective culture techniques and the polymerase chain reaction.

An environmental soil survey to detect Burkholderia pseudomallei was performed during the dry and wet seasons in Darwin, Northern Territory, Australia. Soil was sampled at regular intervals during a 15-month period at different depths from areas which were representative of the local, soil environment. Selective culture techniques using Ashdown's and Galimand and Dodin's methods and the polymerase chain reaction (PCR) using specific 16S rRNA primers were used to detect and identify the organism and determine its distribution within the soil stratum over the change in seasons. Results showed that Ashdown's method gave higher isolation rates in the dry season, and Galimand and Dodin's method gave higher isolation rates during the wet season. PCR of the soil enrichment proved to be a more sensitive method than culture and was also a useful confirmatory test in determining the identification of isolates where biochemical tests gave inconsistent results. The PCR primers were specific and able to detect 10(1) cfu g-1 soil and 10(4) cfu g-1 of soil using Ashdown's enrichment broth and Galimand and Dodin's broth, respectively. Overall the isolation of B. pseudomallei was greatest during the dry season and at the higher and lower soil depths, which is contradictory to epidemiological evidence that melioidosis occurs primarily during the wet season among patients exposed to contaminated surface soil and water.

Burkholderia pseudomallei↗
PubMed1996

WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L. monocytogenes chromosome.

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorph sm (RFLP) analysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 22 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used Ncil to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 4b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%. as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.

Bacterial Typing Techniques↗
PubMed1995

Detection of heat-stable enterotoxin genes among Australian Vibrio cholerae O1 strains.

DNA probes derived from the heat-stable enterotoxin gene of Vibrio cholerae non-O1 (stn), and the cholera toxin gene (ctx), were used to screen 199 strains of V. cholerae O1, which were isolated within Australia from 1977-1986. 13 environmental strains isolated from the riverine environment in Southeast Queensland in 1980 and 1981, hybridized with the stn and ctx DNA probes. The concentrated supernatant of 6 of these strains elicited fluid accumulation in the infant mouse assay both before and after heating at 100 degrees C for 5 min. Genetic relationships among the 13 stn+ strains were studied by a comparison of the rRNA-RFLPs (ribotyping) and by Southern blot analysis with a stn gene probe. The results indicate that there is a clonal relationship among the Australian stn+ strains and that there is an environmental reservoir of stn genes among Australian V. cholerae O1 isolates.

Animals↗
PubMed1995

An epidemiological study of Vibrio cholerae O1 in the Australian environment based on rRNA gene polymorphisms.

Since 1977, Vibrio cholerae O1 has been isolated from the Australian aquatic environment and periodically cholera cases have occurred following exposure to these environments. To study the relationships between clinical isolates and environmental isolates from rivers and aquatic life, widely distributed throughout the country, a wide range of molecular typing methods were employed. In this paper we report the analysis of the 180 Australian isolates (10 clinical and 170 environmental) using ribotyping. Seven ribotype patterns were observed among the Australian inaba isolates, 2 of which included all clinical inaba isolates and 84% environmental inaba isolates collected from 9 rivers and creeks in eastern Australia during an 8-year period. Isolates from epidemiologically related clinical cases, asymptomatic household contacts and sewage were indistinguishable. The ogawa isolates were more diverse, with 9 ribotypes observed among 24 isolates from 8 rivers during the same period. Ribotype patterns were not shared between the serotypes with the exception of one ogawa isolate which could be distinguished using PFGE. Ribotyping has been useful in confirming an association between epidemiologically related clinical isolates and the aquatic environment and the persistence of several clones of the O1 serovar in the Australian environment during an 8-year period.

Animals↗
PubMed1994

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces.

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.

Animals↗
PubMed1994

Detection of Pseudomonas pseudomallei by PCR and hybridization.

A molecular method for the detection of Pseudomonas pseudomallei was developed on the basis of the differences in the 23S rRNA sequences of related species of the genus Pseudomonas. An 18-base oligonucleotide probe, designed following partial sequencing of 23s ribosomal DNA (rDNA), was used for the identification and detection of P. pseudomallei either by hybridization or by direct PCR. Optimal detection was obtained by hybridization of the probe with PCR-amplified rDNA rather than with total genomic DNA or colony blots. One nanogram of template DNA amplified in a PCR mixture containing 14% glycerol could be detected in slot blots hybridized with the digoxigenin-labelled probe and the lumigen PPD detection system. Amplified rDNA sequences from 41 P. pseudomallei strains of various origins hybridized with the probe. The probe also hybridized with three Pseudomonas mallei reference strains under conditions of high stringency but failed to hybridize with amplified rDNA sequences from other closely related Pseudomonas spp. PCR with a conserved primer and the 18-base oligonucleotide probe (direct PCR) specifically amplified P. pseudomallei and P. mallei. By using these methods, approximately 10(4) P. pseudomallei cells per ml could be detected in artificially inoculated blood samples and in blood dried on filter paper following Chelex extraction. The detection limit in blood was increased to 10(2) cells per ml by concentration of bacteria from 0.5 ml of blood or by a 24-h blood culture enrichment prior to PCR. Approximately 10(3) cells per ml were detected in seeded sputum samples. The detection times by direct PCR and indirect PCR and then probe hybridization were approximately 5 h and 24 h, respectively. These results indicate that amplification of conserved rDNA sequences by PCR directly or by hybridization with a probe to PCR fragments offers promise for the detection of P. pseudomallei and P. mallei.

Animals↗
PubMed1994

Application of PCR to a clinical and environmental investigation of a case of equine botulism.

PCR for the detection of botulinum neurotoxin gene types A to E was used in the investigation of a case of equine botulism. Samples from a foal diagnosed with toxicoinfectious botulism in 1985 were reanalyzed by PCR and the mouse bioassay in conjunction with an environmental survey. Neurotoxin B was detected by mouse bioassay in culture enrichments of serum, spleen, feces, and intestinal contents. PCR results compared well with mouse bioassay results, detecting type B neurotoxin genes in these samples and also in a liver sample. Other neurotoxin types were not detected by either test. Clostridium botulinum type B was shown to be prevalent in soils collected from the area in which the foal was raised. Four methods were used to test for the presence of botulinum neurotoxin-producing organisms in 66 soil samples taken within a 5-km radius: PCR and agarose gel electrophoresis (types A to E), PCR and an enzyme-linked assay (type B), hybridization of crude alkaline cell lysates with a type B-specific probe, and the mouse bioassay (all types). Fewer soil samples were positive for C. botulinum type B by the mouse bioassay (15%) than by any of the DNA-based detection systems. Hybridization of a type B-specific probe to DNA dot blots (26% of the samples were positive) and PCR-enzyme-linked assay (77% of the samples were positive) were used for the rapid analysis of large numbers of samples, with sensitivity limits of 3 x 10(6) and 3,000 cells, respectively. Conventional detection of PCR products by gel electrophoresis was the most sensitive method (300-cell limit), and in the present environmental survey, neurotoxin B genes only were detected in 94% of the samples.

Animals↗
PubMed1994

Evaluation of the safety of domestic food preparation in Malaysia.

Food-handling practices were studied in 119 and 158 households, respectively, in an urban and a rural community in Peninsular Malaysia. Hazard analyses, including microbiological analysis of foods, were carried out in two households in each community and in a house that prepared food for distribution in the urban area. Kitchen hygiene was generally acceptable, although rated "poor" in some instances in the rural area. Food prepared for lunch was usually sufficient for dinner also, the leftover items being stored at ambient temperature until required. In the house that prepared food for distribution, breakfast was prepared during the evening, stored at ambient temperature overnight, and reheated before sale the next morning. There was a local preference for cooking food at temperatures close to boiling point; this reduced the numbers of vegetative cells but not those of spores. In some stored foods the populations of Staphylococcus aureus, Bacillus cereus and mesophilic aerobic bacteria increased, the last-mentioned reaching spoilage levels. Reheating reduced the populations of proliferating bacteria in most foods to acceptable levels but would not have destroyed heat-resistant enterotoxins. Because of their importance in combating acute bacterial foodborne disease, the control of the temperature and time factors during the cooking and storage of food should receive special attention in education on health and food safety.

Cooking↗
PubMed1993

Detection of the genes encoding botulinum neurotoxin types A to E by the polymerase chain reaction.

The polymerase chain reaction (PCR) was used as the basis for the development of highly sensitive and specific diagnostic tests for organisms harboring botulinum neurotoxin type A through E genes. Synthetic DNA primers were selected from nucleic acid sequence data for Clostridium botulinum neurotoxins. Individual components of the PCR for each serotype (serotypes A through E) were adjusted for optimal amplification of the target fragment. Each PCR assay was tested with organisms expressing each of the botulinum neurotoxin types (types A through G), Clostridium tetani, genetically related nontoxigenic organisms, and unrelated strains. Each assay was specific for the intended target. The PCR reliably identified multiple strains having the same neurotoxin type. The sensitivity of the test was determined with different concentrations of genomic DNA from strains producing each toxin type. As little as 10 fg of DNA (approximately three clostridial cells) was detected. C. botulinum neurotoxin types A, B, and E, which are most commonly associated with human botulism, could be amplified from crude DNA extracts, from vegetative cells, and from spore preparations. This suggests that there is great potential for the PCR in the identification and detection of botulinum neurotoxin-producing strains.

Base Sequence↗
PubMed1993

Molecular typing of Pseudomonas pseudomallei: restriction fragment length polymorphisms of rRNA genes.

The aim of this study was to develop a typing scheme for Pseudomonas pseudomallei by comparison of patterns of restriction fragment length polymorphisms in rRNA genes (ribotyping). BamHI restriction digests of 100 isolates from various animal (34), human (58), and environmental (6) sources, including six reference strains, were hybridized to Escherichia coli 16S and 23S rRNAs. A chemiluminescent labelling and detection system was used to visualize bands. On the basis of patterns, the strains were classified into 22 different groups, with the largest containing 29 isolates. While most of the ribotypes were not exclusive to a particular source, some ribotypes were restricted to a particular geographic area or to either a human or a particular animal species. Application of the typing scheme to isolates of four independent outbreaks among animals showed that certain ribotypes predominated. The study demonstrated ribotyping to be a useful tool in epidemiological investigations of melioidosis.

Animals↗
PubMed1993

Relationships among Pseudomonas pseudomallei isolates from patients with recurrent melioidosis.

Patients with melioidosis may present with recurrent infections after clinical resolution of their primary illness. Because there has been no satisfactory typing scheme for Pseudomonas pseudomallei, recrudescence could not be distinguished from reinfection. We determined the strain identity of primary and relapse isolates of P. pseudomallei from 25 patients with culture-proven melioidosis to answer whether secondary infections were due to the initial infecting strain or to the acquisition of a new strain. Fifty-four isolates were compared by the patterns of BamHI restriction digests produced after hybridization with a cDNA copy of Escherichia coli rRNA. Twenty-three patients had primary and relapse isolates with identical or highly similar ribotype patterns. The patterns of isolates from two patients were different; the primary and relapse isolates differed by a single fragment for one, and the other had identical primary and first-relapse isolates while the second-relapse isolate was markedly different. The results indicated that recurrent infection probably resulted from endogenous relapse in most of the melioidosis patients studied, although reinfection from an exogenous source was also possible in two cases.

Bacterial Typing Techniques↗
PubMed1992

Specific detection of Clostridium botulinum type B by using the polymerase chain reaction.

The polymerase chain reaction (PCR) and a radiolabeled oligonucleotide probe were used to specifically detect proteolytic and nonproteolytic Clostridium botulinum type B. Two synthetic primers deduced from the amino acid sequence data of type B neurotoxin were used to amplify a 1.5-kbp fragment corresponding to the light chain of the toxin. Although, nonspecific priming was observed when the PCR protocol was tested with other clostridial species, only the PCR product from C. botulinum type B isolates reacted with the radiolabeled internal probe. As little as 100 fg of DNA (approximately 35 clostridial cells) could be detected after only 25 amplification cycles.

Base Sequence↗
PubMed1990

A comparative study of clinical and food isolates of Listeria monocytogenes and related species.

Ninety-six isolates of presumptive or confirmed Listeria monocytogenes were obtained from local clinical (30 isolates) or food laboratories (66 isolates). Minimal biochemical analysis identified only 80% of these isolates as L. monocytogenes the remaining included L. seeligeri, 1%, or the non-haemolytic L. innocua, 19%. The 27 clinical and 50 food isolates, mainly from meat products, frozen confectionaries, and cheeses, confirmed as L. monocytogenes were compared biochemically and serologically. Twenty-one isolates, including some strains of L. innocua and L. seeligeri, were examined for pathogenicity in immunocompromized mice and 44 typed using bacterial restriction endonuclease DNA analysis (BRENDA). Only isolates of L. monocytogenes were found to be pathogenic. Biovar-typing of the isolates was unreliable and provided poor discrimination. Serogroups 1/2 and 4 predominated among clinical and food isolates and BRENDA provided better discrimination among isolates. Ten stable and reproducible restriction patterns were observed among the Listeria sp. isolates studied. Overall, a combination of techniques gave the best discrimination and indicated their potential for use as epidemiological tools.

Animals↗
PubMed1989

A molecular epidemiological study of Vibrio cholerae in Australia.

Two hundred and eight strains of Vibrio cholerae serovar O1 and 454 non-O1 strains, which were isolated within or imported into Australia, were tested for the production of cholera enterotoxin and for hybridization with gene probes for cholera enterotoxin. Genetic relationships among representative isolates were studied by a comparison of the restriction-endonuclease digest patterns of the chromosomal DNA and by Southern-blot analysis of toxigenic strains with the cholera-toxin gene probe. V. cholerae O1 isolates from patients and from the environment were predominantly toxigenic in contrast with the non-O1 isolates. The local O1 isolates were found to be diverse genetically and were unrelated to the imported O1 isolates, to those from other endemic areas and to non-O1 isolates.

Animals↗