Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages

Escherichia coli, the most common cause of bacteraemia in humans in the UK, can also cause serious diseases in animals. However the population structure, virulence and antimicrobial resistance genes of those from extraintestinal organs of livestock animals are poorly characterised. The aims of this study were to investigate the diversity of these isolates from livestock animals and to understand if there was any correlation between the virulence and antimicrobial resistance genes and the genetic backbone of the bacteria and if these isolates were similar to those isolated from humans. Here 39 E. coli isolates from liver (n=31), spleen (n=5) and blood (n=3) of cattle (n=34), sheep (n=3), chicken (n=1) and pig (n=1) were assigned to 19 serogroups with O8 being the most common (n=7), followed by O101, O20 (both n=3) and O153 (n=2). They belong to 29 multi-locus sequence types, 20 clonal complexes with ST23 (n=7), ST10 (n=6), ST117 and ST155 (both n=3) being most common and were distributed among phylogenetic group A (n=16), B1 (n=12), B2 (n=2) and D (n=9). The pattern of a subset of putative virulence genes was different in almost all isolates. No correlation between serogroups, animal hosts, MLST types, virulence and antimicrobial resistance genes was identified. The distributions of clonal complexes and virulence genes were similar to other extraintestinal or commensal E. coli from humans and other animals, suggesting a zoonotic potential. The diverse and various combinations of virulence genes implied that the infections were caused by different mechanisms and infection control will be challenging

Comparative Analysis of ESBL-Positive Escherichia coli isolates from Animals and Humans from the UK, the Netherlands and Germany

The putative virulence and antimicrobial resistance gene contents of extended spectrum β-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring blaCTX-M-group-1 dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both blaCTX-M-group-1 and blaOXA-1-like genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6’)-Ib, catB3, blaOXA-1-like and blaCTX-M-group-1. forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans

Pathotyping Escherichia coli by Using Miniaturized DNA Microarrays▿ †

The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use

Genetic Diversity among Escherichia coli O157:H7 Isolates and Identification of Genes Linked to Human Infections▿ †

An Escherichia coli oligonucleotide microarray based on three sequenced genomes was validated for comparative genomic microarray hybridization and used to study the diversity of E. coli O157 isolates from human infections and food and animal sources. Among 26 test strains, 24 (including both Shiga toxin [Stx]-positive and -negative strains) were found to be related to the two sequenced E. coli O157:H7 strains, EDL933 and Sakai. However, these strains showed much greater genetic diversity than those reported previously, and most of them could not be categorized as either lineage I or II. Some genes were found more often in isolates from human than from nonhuman sources; e.g., ECs1202 and ECs2976, associated with stx2AB and stx1AB, were in all isolates from human sources but in only 40% of those from nonhuman sources. Some (but not all) lineage I-specific or -dominant genes were also more frequently associated with isolates from human. The results suggested that it might be more effective to concentrate our efforts on finding markers that are directly related to infection rather than those specific to certain lineages. In addition, two Stx-negative O157 cattle isolates (one confirmed to be H7) were significantly different from other Stx-positive and -negative E. coli O157:H7 strains and were more similar to MG1655 in their gene content. This work demonstrates that not all E. coli O157:H7 strains belong to the same clonal group, and those that were similar to E. coli K-12 might be less virulent

Cefotaxime resistant Escherichia coli collected from a healthy volunteer; characterisation and the effect of plasmid loss.

In this study 6 CTX-M positive E. coli isolates collected during a clinical study examining the effect of antibiotic use in a human trial were analysed. The aim of the study was to analyse these isolates and assess the effect of full or partial loss of plasmid genes on bacterial fitness and pathogenicity. A DNA array was utilised to assess resistance and virulence gene carriage. Plasmids were characterised by PCR-based replicon typing and addiction system multiplex PCR. A phenotypic array and insect virulence model were utilised to assess the effect of plasmid-loss in E. coli of a large multi-resistance plasmid. All six E. coli carrying bla CTX-M-14 were detected from a single participant and were identical by pulse field gel electrophoresis and MLST. Plasmid profiling and arrays indicated absence of a large multi-drug resistance (MDR) F-replicon plasmid carrying blaTEM, aadA4, strA, strB, dfrA17/19, sul1, and tetB from one isolate. Although this isolate partially retained the plasmid it showed altered fitness characteristics e.g. inability to respire in presence of antiseptics, similar to a plasmid-cured strain. However, unlike the plasmid-cured or plasmid harbouring strains, the survival rate for Galleria mellonella infected by the former strain was approximately 5-times lower, indicating other possible changes accompanying partial plasmid loss. In conclusion, our results demonstrated that an apparently healthy individual can harbour bla CTX-M-14 E. coli strains. In one such strain, isolated from the same individual, partial absence of a large MDR plasmid resulted in altered fitness and virulence characteristics, which may have implications in the ability of this strain to infect and any subsequent treatment

Gel picture showing the electrophoretic separation of plasmids.

<p>The plasmids carried by ARD1257 (B), ARD1258 (C), and ARD1258C (D) migrating in 0.8% agarose, for 270 min at 150 v, 4°C, are shown. Lane A shows the reference plasmid bands from strain 39R861. The reference plasmid bands are from top to bottom; 148 kb, 63 kb, 36 kb, genomic DNA band, 6.8 kb. Lane A–C were run on a separate occasion to lane D but under identical run conditions with a reference plasmid.</p

Difference in melanisation of <i>Galleria mellonella</i> following infection with <i>E. coli</i> isolates.

<p>Images of <i>Galleria mellonella</i> infected with ARD1257 and ARD1258, demonstrating the melanisation that occurred following infection with ARD1257.</p

Survival of <i>Galleria mellonella</i> with <i>E. coli</i> isolates.

<p>The mean percentage survival rate of <i>Galleria mellonella</i> infected with different <i>E. coli</i> strains after 24 h. Error bars indicate the Standard deviation from replicate experiments.</p

Growth curves in LB media.

<p>The growth curves for ARD1258, ARD1257, ARD1258C and MG1655 determined over 24°C in LB media are shown. The absorbance in corrected for the blank and represents the mean of triplicate wells from three individual experiments; error bars indicate a 95% confidence interval.</p