Molecular characterization of plasmids encoding blaCTX-M from faecal Escherichia coli in travellers returning to the UK from South Asia.

BACKGROUND: The global prevalence of extended-spectrum beta-lactamase-producing Escherichia coli is rising and is dominated by blaCTX-M spread by plasmids. Travellers to South Asia from Western Europe have high rates of acquisition of faecal CTX-M-producing E. coli (CTX-M-EC). AIMS: To determine the conjugative ability of CTX-M-EC acquired by healthy volunteers after travel to South Asia, the proportion of travel-acquired CTX-M-EC where blaCTX-M is encoded on a plasmid vs on the bacterial chromosome, and the relatedness of travel-acquired CTX-M-EC plasmids to previously sequenced plasmids. METHODS: Faecal samples were collected pre- and post-travel from 23 volunteers who visited South Asia, and CTX-M-EC were cultured. After short- and long-read sequencing, 10 plasmid sequences were identified and compared with previously sequenced plasmids in GenBank. Conjugation to E. coli K-12 was undertaken using filter mating. FINDINGS: Thirty-five percent of CTX-M-EC isolates tested transferred the blaCTX-M plasmid by conjugation. Travel-acquired CTX-M-EC carried blaCTX-M on a plasmid in 62% of isolates, whereas 38% of isolates had blaCTX-M on the chromosome. CTX-M-EC plasmids acquired after travel to South Asia had close homology to previously described epidemic plasmids which are widely disseminated in humans, animals and the natural environment. CONCLUSION: Globally successful epidemic plasmids are involved in the spread of CTX-M-EC. Targeted strategies may be used to displace such plasmids from the host strain as part of efforts in infection prevention and control in healthcare settings. Bacteria with blaCTX-M plasmids were readily acquired by healthy volunteers, and were carried on return to the UK, providing opportunities for onward dissemination.

Genetic & virulence profiling of ESBL-positive E. coli from nosocomial & veterinary sources.

CTX-M genes are the most prevalent ESBL globally, infiltrating nosocomial, community and environmental settings. Wild and domesticated animals may act as effective vectors for the dissemination of CTX-producing Enterobacteriaceae. This study aimed to contextualise blaCTX-M-14-positive, cephalosporin-resistant Enterobacteriaceae human infections and compared resistance and pathogenicity markers with veterinary isolates. Epidemiologically related human (n=18) and veterinary (n=4) blaCTX-M-14-positive E. coli were fully characterised. All were typed by XbaI pulsed field gel electrophoresis and ST. Chromosomal/plasmidic locations of blaCTX-M-14 were deduced by S1-nuclease digestion, and association with ISEcp1 was investigated by sequencing. Conjugation experiments assessed transmissibility of plasmids carrying blaCTX-M-14. Presence of virulence determinants was screened by PCR assay and pathogenicity potential was determined by in vitro Galleria mellonella infection models. 84% of clinical E. coli originated from community patients. blaCTX-M-14 was found ubiquitously downstream of ISEcp1 upon conjugative plasmids (25-150 kb). blaCTX-M-14 was also found upon the chromosome of eight E. coli isolates. CTX-M-14-producing E. coli were found at multiple hospital sites. Clonal commonality between patient, hospitals and livestock microbial populations was found. In vivo model survival rates from clinical isolates (30%) and veterinary isolates (0%) were significantly different (p<0.05). Co-transfer of blaCTX-M-14 and virulence determinants was demonstrated. There is evidence of clonal spread of blaCTX-M-14-positive E. coli involving community patients and farm livestock. blaCTX-M-14 positive human clinical isolates carry a lower intrinsic pathogenic potential than veterinary E. coli highlighting the need for greater veterinary practices in preventing dissemination of MDR E. coli among livestock.

blaNDM-1 is a chimera likely constructed in Acinetobacter baumannii.

Alignment of DNA sequences found upstream of aphA6 and all bla(NDM-1) genes displays 100% identity. This identity continues 19 bp into the bla(NDM-1) gene such that the first 6 amino acids of aphA6 and bla(NDM-1) are the same. Furthermore, the percent GC content (GC%) of aphA6 is considerably lower than that of bla(NDM-1) and the GC% within the bla(NDM-1) structural gene changes dramatically after the first 19 bp. This is unequivocal evidence that bla(NDM-1) is a chimera.

Molecular characterization of VIM-producing Klebsiella pneumoniae from Scandinavia reveals genetic relatedness with international clonal complexes encoding transferable multidrug resistance.

VIM-producing Klebsiella pneumoniae (VPKP) has been identified as a source of hospital outbreaks and is prevalent particularly in the Mediterranean region. In this study we have characterized eight VPKP isolates identified in Scandinavia during 2005-2008. With the exception of one isolate, all were from patients with recent history of hospitalization abroad (Greece, n = 6; Turkey, n = 1). Multilocus sequence typing (MLST) resulted in five sequence types (STs), ST36 (n = 1), ST147 (n = 4), ST272 (n = 1), ST273 (n = 1) and ST383 (n = 1), which except for ST272 were part of putative international clonal complexes. All were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum ß-lactamases (CTX-M-3, SHV-5 and SHV-12), 16S rRNA methylases (ArmA) and plasmid-mediated quinolone resistance determinants (QnrS). One isolate harboured a novel VIM-variant (VIM-26) while VIM-1 and VIM-19 were detected in six and one isolate, respectively. Two different genetic structures surrounding the bla(VIM) gene were identified in four isolates. In two isolates bla(VIM-1) and bla(VIM-26) were located in an integron similar to In-e541 (intI1;bla(VIM-1/-26);aacA7; dhfrI;aadA1;3'CS) while in the other two isolates bla(VIM-1) was located in an integron lacking 3'CS but with an IS26 element in the 3'end (intI1;bla(VIM-1);aac(6')-Ib;IS26), as identified in the IncN plasmid pKOX105. The bla(VIM) -genes were located on transferable plasmids ranging from ∼40 to ∼240 kb and associated with Tn21 in four isolates. PCR-based replicon typing indicated association of bla(VIM) with IncN (n = 3) and A/C (n = 1) broad-host-range plasmids but also with unknown replicons (n = 4). In conclusion, Scandinavian VPKP is associated with importation and genetically related to international clones encoding transferable plasmid-mediated multidrug resistance.

Characterization of a carbapenemase-producing clinical isolate of Bacteroides fragilis in Scandinavia: genetic analysis of a unique insertion sequence.

In 2003 a Bacteroides fragilis blood culture isolate (K2-28) was recovered from a 61-y-old male with severe general atherosclerosis during treatment with meropenem. K2-28 was shown to possess a functional metallo-beta-lactamase with a reduction in imipenem MIC from 256 to 3 mg/l in the presence of EDTA using the MBL E-test strip. PCR results were for positive for the cfiA gene. Analysis of the cfiA from K2-28 revealed it was 100% identical to previously described cfiA-1 genes. Analysis of the upstream region of cfiA revealed a novel insertion sequence (IS) element, being most similar (94% identity) to IS612 recently described from Japan designating the element within the IS4 family. The element possessed a perfect terminal inverted repeat sequence at the distal ends of the IS element and provided a putative promoter for transcription of the cfiA gene. The distance between the hybrid promoter and the cfiA start codon was 158 base pairs and inserted into a different DNA sequence upstream of cfiA to that previously reported. The -10 promoter region was most similar to that of IS613 (100%) and the -35 promoter region to IS612 (100%), demonstrating the plasticity of these genetic regions.

The variable P5 proteins of typeable and non-typeable Haemophilus influenzae target human CEACAM1.

Haemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non-typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non-typeable H. influenzae targets human CEACAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non-typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1-transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell-bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.

Prevalence of stephanofilariasis in young Bos indicus cattle in northern Australia.

Four consecutive annual calf crops of known genotype from a single property in northern Australia were examined for the presence of stephanofilarial lesions. The animals ranged in age from 60 to 348 days at the time of examination. Initial lesions of stephanofilariasis developed adjacent to the medial canthi of the eyes; animals as young as 69 days of age were found infected. In all years, prevalence of lesions increased with age for all genotypes, and was significantly lower in genotypes with higher Bos indicus content (P less than 0.01). Males had a higher prevalence of lesions than females in only one year, and in two of the years, dark-coloured animals had a higher prevalence of lesions than light-coloured ones.

The toxicity of jute (Corchorus olitorius) seed to pigs.

Finely ground Corchorus olitorius seed was administered as a daily drench to 1 pig at 100mg/kg body weight and as a single drench to 2 pigs at 500 mg/kg body weight. The latter 2 died within 48 hours, and the former was killed on the 7th day as it approached a moribund state. Clinical signs observed were anorexia, vomiting and dysentery. Autopsy revealed severe haemorrhagic enteritis. The seed was also fed to pigs at levels of 0.05%, 0.1% and 0.5% by weight of the ration for 4 weeks. Levels of 0.05% of greater caused reductions in voluntary feed intake and weight gain.

The effect of dehorning Brahman crossbred animals of four age groups on subsequent bodyweight gain.

The effect of dehorning Brahman crossbred cattle of four age groups on their subsequent bodyweight performance was monitored in 4 drafts of cattle between 1975 and 1980. During the 6 weeks following dehorning, bodyweight change of dehorned cattle was less (P less than 0.05) than that of horned cattle in half of the groups. There were no differences between treatments, in bodyweight gain to 12 months after dehorning, following any dehorning date or in final bodyweight, for each draft. The data suggest that age of dehorning is not critical.