The potential use of chilling to control the growth of Enterobacteriaceae on porcine carcasses and the incidence of E. coli O157:H7 in pigs

Aims:  To (i) monitor the presence of Enterobacteriaceae as indicators of faecal contamination on pig carcasses, (ii) examine the potential use of chilling as a critical control point (CCP) and establish its influence on pig carcass categorization by Decision 471/EC and (iii) determine the incidence of E. coli O157:H7 in pigs.
Methods and Results:  Porcine faecal samples and carcass swabs were collected before and after chilling at four Irish pig abattoirs and examined for Enterobacteriaceae and E. coli O157:H7. Chilling generally reduced Enterobacteriaceae counts on carcasses, but increases were also observed, particularly in one abattoir. E. coli O157:H7 was absent from carcasses before chilling, present on 0·21% after chilling and was recovered from 0·63% of faecal samples. All of the isolates were found to contain virulence genes associated with clinical illness in humans.
Conclusions:  The data show that overall chilling had the capacity to reduce the numbers of carcasses positive for the presence of Enterobacteriaceae .
Significance and Impact of Study:  The influence of chilling on the categorization of pig carcasses suggests that it has the potential to improve the numbers of acceptable carcasses and the process could be used as a CCP within a HACCP plan.     

Relationships between microbial population dynamics and putrescine and cadaverine accumulation during dry fermented sausage ripening

Aims:  To evaluate the concomitant effects of three technological variables (fermentation temperature, NaCl and glucose added to the meat batter) on diamines (cadaverine, putrescine and histamine) accumulation and microbial changes during ripening of dry fermented sausages.
Methods and Results:  The variables were modulated according to an experimental design and predictive mathematical models were obtained. The models indicated that the sausages were characterized by low histamine amount independently on the applied conditions. In contrast, putrescine and cadaverine accumulation was considerable and significantly affected by the three variables. The microbial population dynamics suggest that lactic acid bacteria (LAB) and microstaphylococci are favoured by increasing glucose concentration until 0·7 g kg⁻¹, while Enterobacteriaceae are negatively influenced by NaCl concentration and, to a lesser extent, by fermentation temperature.
Conclusions:  Data obtained showed a relationship between Enterobacteriaceae growth and cadaverine and putrescine accumulation in sausages during ripening. The conditions more favourable for LAB and microstaphylococci induced a reduced growth of Enterobacteriaceae with a consequent reduced accumulation of putrescine and cadaverine.
Significance and Impact of the Study:  The use of systematic experimental designs allows to individuate the technological conditions suitable to keep the aminogenic microflora under control, thus reducing the risk of diamines production during traditional fermented food manufacture.     

A phylogenetic approach to assessing the targets of microbial warfare

Bacteriocins are the most abundant and diverse defense systems in bacteria. As a result of the specific mechanisms of bacteriocin recognition and translocation into the target cell it is assumed that these toxins mediate intra-specific or population-level interactions. However, no published studies specifically address this question. We present here a survey of bacteriocin production in a collection of enteric bacteria isolated from wild mammals in Australia. A subset of the bacteriocin-producing strains was assayed for the ability to kill a broad range of enteric bacteria from the same bacterial collection. A novel method of estimating killing breadth was developed and used to compare the surveyed bacteriocins in terms of the phylogenetic range over which they kill. The most striking result is that although bacteriocin-producers kill members of their own species most frequently, some kill phylogenetically distant taxa more frequently than they kill closer relatives. This study calls into question the role these toxins play in natural populations. A significant number of bacteriocins are highly effective in killing inter-specific strains and thus bacteriocins may serve to mediate bacterial community interactions.     

The role of visible faecal material as a vehicle for generic Escherichia coli,coliform, and other enterobacteria contaminating poultry carcasses during slaughtering

AIMS: A comparison of Enterobacteriaceae, coliform and Escherichia coli counts in chicken carcasses with and without visible faecal contamination was conducted to evaluate the role of contamination as a vehicle for generic E. coli, coliform and other enterobacteria contaminating broiler chicken carcasses when processed under routine commercial operations. METHODS AND RESULTS: Samples were removed from the processing line immediately after evisceration, inside-outside shower and chilling for microbiological analysis. After evisceration, mean counts were significantly different only for E. coli (P < or = 0.05) in chicken carcasses with and without visible faecal contamination. While the spray wash practice was not efficient enough for complete removal of the visible contamination from carcasses, leading to microbiological reduction percentages lower than expected, 25 ppm chlorinated water chilling did reduce the contamination level considerably in all samples. CONCLUSIONS: Carcasses with and without visible faecal contamination harboured E. coli and other potentially hazardous enterobacteria. E. coli was the predominant strain isolated in all samples, Enterobacter cloacae being next most frequent. SIGNIFICANCE AND IMPACT OF THE STUDY: The zero tolerance of visible faecal contamination requirement alone is not sufficient to assure safety and to improve the microbial quality of carcasses.     

Phenotypic variability of beta-glucoside utilization and its correlation to pathogenesis process in a few enteric bacteria

Utilization of beta-glucosides is markedly variable in the members of the family Enterobacteriaceae. The results presented here provide molecular clues for evolutionary events that resulted in the phenotypic variability seen amongst the members of these species. The genomic hybridization of selected Enterobacteriaceae members with the Escherichia coli bgl and cel genes resulted in detection of a complete homolog of the bgl and cel operons in Shigella sonnei, a member that is evolutionarily closest to E. coli. However, the Salmonella group of organisms have been shown to carry only a homolog of bglR and bglG regions and the deletions of the bglF and bglB genes. Similarly, Proteus mirabilis, Enterobacter aerogenes and a non-enteric Gram-negative bacterium Pseudomonas aeruginosa have been shown to carry a homolog of the bglR and bglG regions and deletions of the bglF and bglB genes. The homolog of the cel operon could be identified in S. sonnei and Salmonella groups of organisms. Possible implications of these observations, in connection with the phenotypic variability seen in beta-glucoside utilization amongst these members, are discussed.     

The genes and enzymes for the catabolism of galactitol,D-tagatose,and related carbohydrates in<Emphasis Type="Italic"> Klebsiella oxytoca</Emphasis> M5a1 and other enteric bacteria display convergent evolution

Enteric bacteria (Enteriobacteriaceae) carry on their single chromosome about 4000 genes that all strains have in common (referred to here as obligatory genes), and up to 1300 facultative genes that vary from strain to strain and from species to species. In closely related species, obligatory and facultative genes are orthologous genes that are found at similar loci. We have analyzed a set of facultative genes involved in the degradation of the carbohydrates galactitol, D-tagatose, D-galactosamine and N-acetyl-galactosamine in various pathogenic and non-pathogenic strains of these bacteria. The four carbohydrates are transported into the cell by phosphotransferase (PTS) uptake systems, and are metabolized by closely related or even identical catabolic enzymes via pathways that share several intermediates. In about 60% of Escherichia coli strains the genes for galactitol degradation map to a gat operon at 46.8 min. In strains of Salmonella enterica, Klebsiella pneumoniae and K. oxytoca, the corresponding gat genes, although orthologous to their E. coli counterparts, are found at 70.7 min, clustered in a regulon together with three tag genes for the degradation of D-tagatose, an isomer of D-fructose. In contrast, in all the E. coli strains tested, this chromosomal site was found to be occupied by an aga/kba gene cluster for the degradation of D-galactosamine and N-acetyl-galactosamine. The aga/kba and the tag genes were paralogous either to the gat cluster or to the fru genes for degradation of D-fructose. Finally, in more then 90% of strains of both Klebsiella species, and in about 5% of the E. coli strains, two operons were found at 46.8 min that comprise paralogous genes for catabolism of the isomers D-arabinitol (genes atl or dal) and ribitol (genes rtl or rbt). In these strains gat genes were invariably absent from this location, and they were totally absent in S. enterica. These results strongly indicate that these various gene clusters and metabolic pathways have been subject to convergent evolution among the Enterobacteriaceae. This apparently involved recent horizontal gene transfer and recombination events, as indicated by major chromosomal rearrangements found in their immediate vicinity.Communicated by A. Kondorosi     

Antibodies against human muscle enolase recognize a 45-kDa bacterial cell wall outer membrane enolase-like protein

Enolase, is a glycolytic enzyme ubiquitous in higher organisms, where it forms tissue specific dimers of isoforms, also found in the cytoplasm of fermentative bacteria. The aim of this work was to identify enolase-like proteins in the cell wall of some Gram-negative bacteria using antibodies against human beta-enolase, an isoenzyme specific to skeletal and heart muscles. Cell wall outer membrane protein (OMP) preparations were obtained from 9 strains of Enterobacteriaceae and one of Pseudomonas aeruginosa. Specific enzymatic enolase activity was detected in the supernatant fractions of cytosolic and inner membrane material, but not in purified OMP preparations. Rabbit polyclonal antibodies specific against human beta-enolase were prepared and purified using immobilized human beta-enolase in affinity chromatography. In SDS-polyacrylamide gel electrophoresis and immunoblotting assay of purified OMP preparations, rabbit anti-enolase antibody interacted specifically with a few OMPs, of which a 45-kDa band also interacted with human sera of patients presenting Buerger disease and atherosclerosis. The most distinct interaction of human sera was observed with a 45-kDa OMP of Klebsiella pneumoniae. This protein was further isolated from K. pneumoniae cell mass in two ways, namely preparative SDS-polyacrylamide gel electrophoresis and specific affinity chromatography using immobilized affinity-purified rabbit antibody raised against human beta-enolase. The data obtained from tandem mass spectrometry tryptic peptide analysis and sequence comparison of human and bacterial enolases using protein databases, could reveal the similarity in the epitopes between membrane enolase-like protein from Klebsiella and human beta-enolase. The results show that the protein present in all studied strains has a common epitope on human beta-enolase. These data raise the question whether such a bacterial protein might be a marker for detecting and monitoring damage to skeletal and heart muscles.     

Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism (PCR-RFLP)

Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) has been developed to extend the identification of SHV beta-lactamases previously characterised by PCR-single strand conformational polymorphism (PCR-SSCP) analysis alone. Eight bacteria, each producing a different SHV beta-lactamase, were used in this study. These bacteria harbour bla(SHV-1), bla(SHV-2a), bla(SHV-3), bla(SHV-4), bla(SHV-5) (two strains), bla(SHV-11) and bla(SHV-12). All isolates were characterised by PCR-SSCP and PCR-RFLP with DdeI and NheI digestion. By a combination of these techniques, the genes encoding these beta-lactamases could be differentiated from each other. In addition, the PCR-RFLP technique theoretically can be applied to distinguish the genes encoding SHV-7, SHV-9, SHV-10, SHV-15, SHV-17 and SHV-24 from those encoding other SHV variants. We report a simple PCR-RFLP technique that can be used in epidemiological studies to enable the rapid characterisation of known SHV beta-lactamases in a combination with the previously published PCR-SSCP analysis.     

MASTDISCS combi Carba plus,a simple method for discriminating carbapenemase‐producing Enterobacteriaceae,including OXA‐48‐type producers

Accurate and rapid detection of carbapenemases and identification of their types in Enterobacteriaceae are both still major challenges for clinical laboratories in attempting to prevent the intrusion and transmission of carbapenemase‐producing Enterobacteriaceae. This study aimed to evaluate the performance of the MASTDISCS combi Carba plus disc system in identification of different carbapenemase types, including OXA‐48‐type carbapenemase, for which no specific enzyme inhibitors have so far been available. The simple disc system discriminates carbapenemases, including OXA‐48‐types exhibiting low carbapenem minimum inhibitory concentrations, by targeting Enterobacteriaceae isolates with a EUCAST meropenem screening cut‐off of ≥0.25 mg/L.     

Sources of microorganisms in pozol,a traditional Mexican fermented maize dough

Freshly prepared pozol, a traditional Mexican fermented maize dough, contained (c.f.u./g wet wt): lactic acid bacteria, 10⁴ to 10⁶; aerobic mesophiles, 10⁴ to 10⁵; Enterobacteriaceae, 10² to 10³; yeasts, 10² to 10⁴; and mould propagules, <10³. After 30 h at 28°C the numbers were, respectively: 10⁹, 7×10⁶, 5×10⁵, 10⁶ and 10⁴. Soaking alkali-treated grains overnight allowed lactic acid bacteria, aerobic mesophiles and Enterobacteriaceae to grow and these then constituted the primary microbial flora of the pozol dough. Grinding in a commercial mill inoculated the dough with lactic acid bacteria, aerobic mesophiles, Enterobacteriaceae and yeasts. Other processing stages, including the nature of the surface upon which the balls were made, handling of the dough, and air, contributed only minor numbers of microbes compared with the two major sources, soaking and grinding. The pH of pozol fell from an initial value of 7.3 to 4.6 after 30 h incubation at 28°C. The numbers of Enterobacteriaceae and other aerobic mesophilic bacteria remained constant between 11 and 30 h incubation and there was no evidence of the acidic conditions having any lethal effects on these organisms.