Peter M. Bennett

ISCR Elements: Novel Gene-Capturing Systems of the 21st Century?

SUMMARY “Common regions” (CRs), such as Orf513, are being increasingly linked to mega-antibiotic-resistant regions. While their overall nucleotide sequences show little identity to other mobile elements, amino acid alignments indicate that they possess the key motifs of IS91-like elements, which have been linked to the mobility ent plasmids in pathogenic Escherichia coli. Further inspection reveals that they possess an IS91-like origin of replication and termination sites (terIS), and therefore CRs probably transpose via a rolling-circle replication mechanism. Accordingly, in this review we have renamed CRs as ISCRs to give a more accurate reflection of their functional properties. The genetic context surrounding ISCRs indicates that they can procure 5′ sequences via misreading of the cognate terIS, i.e., “unchecked transposition.” Clinically, the most worrying aspect of ISCRs is that they are increasingly being linked with more potent examples of resistance, i.e., metallo-β-lactamases in Pseudomonas aeruginosa and co-trimoxazole resistance in Stenotrophomonas maltophilia. Furthermore, if ISCR elements do move via “unchecked RC transposition,” as has been speculated for ISCR1, then this mechanism provides antibiotic resistance genes with a highly mobile genetic vehicle that could greatly exceed the effects of previously reported mobile genetic mechanisms. It has been hypothesized that bacteria will surprise us by extending their “genetic construction kit” to procure and evince additional DNA and, therefore, antibiotic resistance genes. It appears that ISCR elements have now firmly established themselves within that regimen.

The occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs, human faeces, sewage and soil from urban sources

Eight hundred and twenty-two shop-bought food specimens, 136 soil and 692 faecal specimens were cultured for Listeria spp. in a regular, year round survey. 19.7% (162/822) of the foods, 93.9% (108/115) of the sewage, 14.7% (20/136) soils and 1% (7/692) of faeces yielded Listeria spp. with 10.5% foods, 60.0% sewage, 0.7% soils and 0.6% faeces containing L. monocytogenes. No seasonal variation was noted in isolates from either sewage or foods, with L. monocytogenes and L. innocua being the commonest species in both L. ivanovii when isolated from foods was strongly associated with mutton. Poultry was most likely to contain L. monocytogenes (65.6%, 21/32) and in the greatest numbers. A high percentage of beef (34.6%, 9/26), lamb (40%, 8/20), pork (28.1%, 9/32) and sausages (34.7%, 8/23) also contained L. monocytogenes. L. monocytogenes was rarely isolated from paté (1/40) or soft cheeses (1/251), both of which have been involved with foodborne listeriosis outbreak in the UK. Listeria spp. were commonest in faeces and soils in July to September but the predominant species isolated were different with L. monocytogenes and L. innocua the commonest from faeces and L. ivanovii and L. seeligeri the most common from soil.

Global Emergence of Trimethoprim/Sulfamethoxazole Resistance in Stenotrophomonas maltophilia Mediated by Acquisition of sul Genes

The first sul2 genes have been found in S. maltophilia from several different countries.

Sequence analysis of the L1 metallo-β-lactamase from Xanthomonas maltophilia

The amino acid sequence deduced from the L1 β-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-β-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 β-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-β-lactamases.

Plasmid Location and Molecular Heterogeneity of the L1 and L2 β-Lactamase Genes of Stenotrophomonas maltophilia

ABSTRACT An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 β-lactamase genes. The location of L1 andL2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes,L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1(L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lowerkcat/Km ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, andL2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.

Transposition of TnA Does Not Generate Deletions

SummaryWe have examined the incidence of loss of the TnA unit, Tn801, from RP1 under conditions where transposition of Tn801 to another replicon, R388, was readily detected. We found that the frequency of transposition of Tn801 from RP1 to R388 exceeded, by at least a factor of one hundred, the frequency at which it was deleted from RP1. We conclude that, in general, transposition of Tn801 does not generate derivatives of the donor plasmid which specifically lack Tn801. The relevance of these findings to the mechanism of transposition is discussed.

Characterisation of Tn501, a transposon determining resistance to mercuric ions

SummaryThe transposon encoding resistance to mercuric ions, Tn501, is 5.2 (±0.1)×106 daltons and is bounded by small inverted repeats. The restriction sites for the restriction endonucleases EcoRI, HindIII and SalGI have been mapped on the element.

Regional preference of insertion of Tn501 and Tn802 into RP1 and its derivatives

SummaryThe sites of insertion of Tn501 into RP1 and into derivatives of this plasmid that either lack the Tn801 (TnA) element or contain it in a different location have been determined. Similarly, the sites of insertion of Tn802 into a derivative of RP1 that lacks the Tn801 element and into recombinants of this plasmid with Tn501 were determined. ‘Hot spots’ for insertion were observed with both transposons; but it is clear that a particular DNA sequence is not sufficient to define a ‘hot spot’, since a particular region does contain many insertions when present in one plasmid but does not do so when part of another.

Fitness of Escherichia coli strains carrying expressed and partially silent IncN and IncP1 plasmids

BackgroundUnderstanding the survival of resistance plasmids in the absence of selective pressure for the antibiotic resistance genes they carry is important for assessing the value of interventions to combat resistant bacteria. Here, several poorly explored questions regarding the fitness impact of IncP1 and IncN broad host range plasmids on their bacterial hosts are examined; namely, whether related plasmids have similar fitness impacts, whether this varies according to host genetic background, and what effect antimicrobial resistance gene silencing has on fitness.ResultsFor the IncP1 group pairwise in vitro growth competition demonstrated that the fitness cost of plasmid RP1 depends on the host strain. For the IncN group, plasmids R46 and N3 whose sequence is presented for the first time conferred remarkably different fitness costs despite sharing closely related backbone structures, implicating the accessory genes in fitness. Silencing of antimicrobial resistance genes was found to be beneficial for host fitness with RP1 but not for IncN plasmid pVE46.ConclusionsThese findings suggest that the fitness impact of a given plasmid on its host cannot be inferred from results obtained with other host-plasmid combinations, even if these are closely related.

Evidence of Antibiotic Resistance Gene Silencing in Escherichia coli

ABSTRACT The possibility that unexpressed antibiotic resistance genes are carried by bacterial genomes is seldom investigated. Potential silencing of the resistance genes blaOXA-2, aadA1, sul1, and tetA carried on the plasmid pVE46 in a recent porcine isolate of Escherichia coli was investigated following oral inoculation of the strain into organic piglets. A small proportion of isolates recovered from feces did not express one or more resistance genes, despite retaining the pVE46 plasmid. Different combinations of unexpressed resistance genes were observed, and 12 representative isolates were selected for further study. Surprisingly, in most cases the resistance genes and their promoters, although not expressed, were intact, with fully wild-type sequences. Apart from four isolates exhibiting intermediate-level tetracycline resistance, no mRNA for the unexpressed genes was detected. Silencing of resistance genes was reversible at low frequencies between 10−6 and 10−10. Introduction of the plasmid from silenced isolates to another strain restored expression, indicating that gene silencing was a property of the host chromosome rather than the plasmid itself. When the same recent porcine E. coli strain carrying the unrelated plasmid RP1 was inoculated into piglets, three isolates (of 9,492) that no longer expressed RP1-encoded resistance genes were recovered. As with pVE46, in most cases the coding sequences and promoter regions of these genes were found to be intact, but they were not transcribed. Such gene silencing indicates a previously unrecognized form of transcriptional control that overrides standard expression signals to shut down gene expression. These findings suggest that unexpressed resistance genes may occur in the wild and hence may have clinical implications.