A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line.

Pork contributes significantly to the public health disease burden caused by Salmonella infections. During the slaughter process pig carcasses can become contaminated with Salmonella. Contamination at the slaughter-line is initiated by pigs carrying Salmonella on their skin or in their faeces. Another contamination route could be resident flora present on the slaughter equipment. To unravel the contribution of these two potential sources of Salmonella a quantitative study was conducted. Process equipment (belly openers and carcass splitters), faeces and carcasses (skin and cutting surfaces) along the slaughter-line were sampled at 11 sampling days spanning a period of 4 months. Most samples taken directly after killing were positive for Salmonella. On 96.6% of the skin samples Salmonella was identified, whereas a lower number of animals tested positive in their rectum (62.5%). The prevalence of Salmonella clearly declined on the carcasses at the re-work station, either on the cut section or on the skin of the carcass or both (35.9%). Throughout the sampling period of the slaughter-line the total number of Salmonella per animal was almost 2log lower at the re-work station in comparison to directly after slaughter. Seven different serovars were identified during the study with S. Derby (41%) and S. Typhimurium (29%) as the most prominent types. A recurring S. Rissen contamination of one of the carcass splitters indicated the presence of an endemic 'house flora' in the slaughterhouse studied. On many instances several serotypes per individual sample were found. The enumeration of Salmonella and the genotyping data gave unique insight in the dynamics of transmission of this pathogen in a slaughter-line. The data of the presented study support the hypothesis that resident flora on slaughter equipment was a relevant source for contamination of pork

Molecular Methods for Detection of Antibiotic Resistance

The combination of molecular characterization of resistant strains with precise identification of the antibiotic resistance gene(s) or mutation(s) and the genetic elements involved in the dissemination of these genes is an effective approach in the control of the spread of antibiotic resistance. Molecular methods also help to determine the location of the gene and to differentiate between horizontal gene transfer and clonal spread. This chapter describes polymerase chain reaction (PCR) and microarray analysis in detail, and specifies applications of these methods in the investigation of antibiotic-resistant strains. Numerous PCR assays for the detection of antibiotic resistance genes have been developed and the development of microarrays for the simultaneous detection of a large number of these genes and the genetic elements involved in their dissemination is in progress. The implementation of molecular methods in routine analysis can be achieved only when it is supported by the proper validation of the methods and the availability of the necessary controls, reference strains, and educated personnel. The molecular characterization of antibiotic-resistant strains can help to identify atypical resistant strains, describe new outbreak strains at an early stage, elucidate the epidemiology of resistant strains at a genotypic level, and explain the processes leading to the selection of resistant and virulent strains. In addition, molecular methods can allow proper risk assessment with respect to the use of antimicrobial substances. If the transcriptional and translational expression of antibiotic resistance genes can be better understood, molecular methods may replace phenotypic measurements

Human γ-crystallin genes. A gene family on its way to extinction

During hominoid evolution the γ-crystallins of the lens have decreased in quantity as well as complexity, a change correlated with an increased water content of the lens. To trace the molecular basis for the decrease in γ-crystallin gene expression, we have characterized the structure and expression of the human γ-crystallin gene family. We show that the human γ-crystallin gene family consists of six complete genes (γA, γB, γC, γD, ψγE and ψγF) and one second exon fragment, the γG gene. Model experiments showed that, although the γG sequence is bordered by consensus splice sites, it is most likely transcriptionally inactive in the lens. In the human embryonic lens the γC and γD genes accounted for 81 % of the γ-crystallin transcripts, the γA gene contributed 14% and the γB gene only 5%. The composition of the γ-crystallin mRNA pool changed only after birth, with the γD transcript as the only detectable transcript at ten years of age. The relative activities of the γA, γC and γD promoters in a transient expression system were in agreement with the ratio of their in vivo RNA levels, suggesting that the difference in accumulation of these transcripts is due to differences in the rate of transcription. The γB promoter was much more active than expected and had lost its tissue-specificity. Model experiments showed that the low yield of the γB transcript is due to post-transcriptional processes, most likely RNA instability mediated by third exon sequences. Together with previous data, our results show that the decrease in expression of the γ-crystallin genes in the human lens is the consequence of gene loss (γG), inactivation of coding sequences (ψγE and ψγF), decrease in rate of transcription (γA), increase in rate of RNA turn-over (γB) and a delay in the onset of transcription during development

Acquired antibiotic resistance genes:an overview

In this review an overview is given on antibiotic resistance (AR) mechanisms with special attentions to the AR genes described so far preceded by a short introduction on the discovery and mode of action of the different classes of antibiotics. As this review is only dealing with acquired resistance, attention is also paid to mobile genetic elements such as plasmids, transposons, and integrons, which are associated with AR genes, and involved in the dispersal of antimicrobial determinants between different bacteria

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.Work on antibiotic resistance in LAB&B strains at the authors' laboratories was supported by an EU project within the sixth Framework Programme (ref. 506214). M.S.A. was awarded a postdoctoral fellowship from the Secretaria de Estado de Universidades e Investigación of the Spanish Ministry of Education and Science (ref. SB2004-0165).Peer reviewe

Molecular hazard identification of non-O157 Shiga toxin-producing Escherichia coli (STEC)

The complexity regarding Shiga toxin-producing Escherichia coli (STEC) in food safety enforcement as well as clinical care primarily relates to the current inability of an accurate risk assessment of individual strains due to the large variety in serotype and genetic content associated with (severe) disease. In order to classify the clinical and/or epidemic potential of a STEC isolate at an early stage it is crucial to identify virulence characteristics of putative pathogens from genomic information, which is referred to as ‘predictive hazard identification’. This study aimed at identifying associations between virulence factors, phylogenetic groups, isolation sources and seropathotypes. Most non-O157 STEC in the Netherlands belong to phylogroup B1 and are characterized by the presence of ehxA, iha and stx2, but absence of eae. The large variability in the number of virulence factors present among serogroups and seropathotypes demonstrated that this was merely indicative for the virulence potential. While all the virulence gene associations have been worked out, it appeared that there is no specific pattern that would unambiguously enable hazard identification for an STEC strain. However, the strong correlations between virulence factors indicate that these arrays are not a random collection but are rather specific sets. Especially the presence of eae was strongly correlated to the presence of many of the other virulence genes, including all non-LEE encoded effectors. Different stx-subtypes were associated with different virulence profiles. The factors ehxA and ureC were significantly associated with HUS-associated strains (HAS) and not correlated to the presence of eae. This indicates their candidacy as important pathogenicity markers next to eae and stx2a

A multiplex ligation detection assay for the characterization of Salmonella enterica strains

A proof of principle of a multi-target assay for genotyping Salmonella has been developed targeting 62 genomic marker sequences of Salmonella related to pathogenicity. The assay is based on multiplex ligation detection reaction (LDR) followed by customized ArrayTube (R) microarray detection. The feasibility of the developed assay was verified in a method comparison study with conventional PCR using 16 Salmonella 'test' strains comprising eight serovars. Subsequently, the feasibility of the LDR microarray assay was also tested by analyzing 41 strains belonging to 23 serovars. With the exception of four serovars each serovar was characterized by a unique virulence associated gene repertoire. The LDR microarray platform proved to be a convenient, rapid and easy to use tool with potential in tracing a Salmonella contamination in the food chain, for outbreak studies, and to provide data for risk assessors that support bio-traceability models. (C) 2010 Elsevier B.V. All rights reserve