ABSTRACT A multiplex PCR was developed for the rapid detection of genes encoding Shiga toxins 1 and 2 ( stx 1 and stx 2 ), intimin ( eaeA ), and enterohemolysin A ( hlyA ) in 444 fecal samples derived from healthy and clinically affected cattle, sheep, pigs, and goats. The method involved non-solvent-based extraction of nucleic acid from an aliquot of an overnight culture of feces in EC (modified) broth. The detection limit of the assay for both fecal samples and pure cultures was between 18 and 37 genome equivalents. stx 1 and hlyA were the most commonly encountered virulence factors.
Knowledge of mobile genetic elements that capture and disseminate antimicrobial resistance genes between diverse environments, particularly across human-animal boundaries, is key to understanding the role anthropogenic activities have in the evolution of antimicrobial resistance. Plasmids that circulate within the Enterobacteriaceae and the Proteobacteria more broadly are well placed to acquire resistance genes sourced from separate niche environments and provide a platform for smaller mobile elements such as IS26 to assemble these genes into large, complex genomic structures. Here, we characterised two atypical Z/I1 hybrid plasmids, pSTM32-108 and pSTM37-118, hosting antimicrobial resistance and virulence associated genes within endemic pathogen Salmonella enterica serovar Typhimurium 1,4,[5],12:i:-, sourced from Australian swine production facilities during 2013. We showed that the plasmids found in S. Typhimurium 1,4,[5],12:i:- are close relatives of two plasmids identified from Escherichia coli of human and bovine origin in Australia circa 1998. The older plasmids, pO26-CRL125 and pO111-CRL115, encoded a putative serine protease autotransporter and were host to a complex resistance region composed of a hybrid Tn21-Tn1721 mercury resistance transposon and composite IS26 transposon Tn6026. This gave a broad antimicrobial resistance profile keyed towards first generation antimicrobials used in Australian agriculture but also included a class 1 integron hosting the trimethoprim resistance gene dfrA5. Genes encoding resistance to ampicillin, trimethoprim, sulphonamides, streptomycin, aminoglycosides, tetracyclines and mercury were a feature of these plasmids. Phylogenetic analyses showed very little genetic drift in the sequences of these plasmids over the past 15 years; however, some alterations within the complex resistance regions present on each plasmid have led to the loss of various resistance genes, presumably as a result of the activity of IS26. These alterations may reflect the specific selective pressures placed on the host strains over time. Our studies suggest that these plasmids and variants of them are endemic in Australian food production systems.
Abstract Genomics is a cornerstone of modern pathogen epidemiology yet demonstrating transmission in a One Health context is challenging, as strains circulate and evolve within and between diverse hosts and environments. To identify phylogenetic linkages and better define relevant measures of genomic relatedness in a One Health context, we collated 5471 Escherichia coli genome sequences from Australia originating from humans ( n = 2996), wild animals ( n = 870), livestock ( n = 649), companion animals ( n = 375), environmental sources ( n = 292) and food ( n = 289) spanning over 36 years. Of the 827 multi-locus sequence types (STs) identified, 10 STs were commonly associated with cross-source genomic clusters, including the highly clonal ST131, pandemic zoonotic lineages such as ST95, and emerging human ExPEC ST1193. Here, we show that assessing genomic relationships at ≤ 100 SNP threshold enabled detection of cross-source linkage otherwise obscured when applying typical outbreak-oriented relatedness thresholds ( ≤ 20 SNPs) and should be considered in interrogation of One Health genomic datasets.
Salmonella genomic island 1 (SGI1) is an integrative genetic island first described in Salmonella enterica serovars Typhimurium DT104 and Agona in 2000. Variants of it have since been described in multiple serovars of S. enterica, as well as in Proteus mirabilis, Acinetobacter baumannii, Morganella morganii, and several other genera. The island typically confers resistance to older, first-generation antimicrobials; however, some variants carry blaNDM-1, blaVEB-6, and blaCTX-M15 genes that encode resistance to frontline, clinically important antibiotics, including third-generation cephalosporins. Genome sequencing studies of avian pathogenic Escherichia coli (APEC) identified a sequence type 117 (ST117) isolate (AVC96) with genetic features found in SGI1. The complete genome sequence of AVC96 was assembled from a combination of Illumina and single-molecule real-time (SMRT) sequence data. Analysis of the AVC96 chromosome identified a variant of SGI1-B located 18 bp from the 3' end of trmE, also known as the attB site, a known hot spot for the integration of genomic islands. This is the first report of SGI1 in wild-type E. coli The variant, here named SGI1-B-Ec1, was otherwise unremarkable, apart from the identification of ISEc43 in open reading frame (ORF) S023.IMPORTANCE SGI1 and variants of it carry a variety of antimicrobial resistance genes, including those conferring resistance to extended-spectrum β-lactams and carbapenems, and have been found in diverse S. enterica serovars, Acinetobacter baumannii, and other members of the Enterobacteriaceae SGI1 integrates into Gram-negative pathogenic bacteria by targeting a conserved site 18 bp from the 3' end of trmE For the first time, we describe a novel variant of SGI1 in an avian pathogenic Escherichia coli isolate. The presence of SGI1 in E. coli is significant because it represents yet another lateral gene transfer mechanism to enhancing the capacity of E. coli to acquire and propagate antimicrobial resistance and putative virulence genes. This finding underscores the importance of whole-genome sequencing (WGS) to microbial genomic epidemiology, particularly within a One Health context. Further studies are needed to determine how widespread SGI1 and variants of it may be in Australia.
Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) cells were isolated from 191 fecal samples from cattle with gastrointestinal infections (diagnostic samples) collected in New South Wales, Australia. By using a multiplex PCR, E. coli cells possessing combinations of stx1, stx2, eae, and ehxA were detected by a combination of direct culture and enrichment in E. coli (EC) (modified) broth followed by plating on vancomycin-cefixime-cefsulodin blood (BVCC) agar for the presence of enterohemolytic colonies and on sorbitol MacConkey agar for the presence of non-sorbitol-fermenting colonies. The high prevalence of the intimin gene eae was a feature of the STEC (35 [29.2%] of 120 isolates) and contrasted with the low prevalence (9 [0.5%] of 1,692 fecal samples possessed STEC with eae) of this gene among STEC recovered during extensive sampling of feces from healthy slaughter-age cattle in Australia (M. Hornitzky, B. A. Vanselow, K. Walker, K. A. Bettelheim, B. Corney, P. Gill, G. Bailey, and S. P. Djordjevic, Appl. Environ. Microbiol. 68:6439-6445, 2002). Forty-seven STEC serotypes were identified, including O5:H-, O8:H19, O26:H-, O26:H11, O113:H21, O157:H7, O157:H- and Ont:H- which are known to cause severe disease in humans and 23 previously unreported STEC serotypes. Serotypes Ont:H- and O113:H21 represented the two most frequently isolated STEC isolates and were cultured from nine (4.7%) and seven (3.7%) animals, respectively. Fifteen eae-positive E. coli serotypes, considered to represent atypical EPEC, were identified, with O111:H- representing the most prevalent. Using both techniques, STEC cells were cultured from 69 (36.1%) samples and EPEC cells were cultured from 30 (15.7%) samples, including 9 (4.7%) samples which yielded both STEC and EPEC. Culture on BVCC agar following enrichment in EC (modified) broth was the most successful method for the isolation of STEC (24.1% of samples), and direct culture on BVCC agar was the most successful method for the isolation of EPEC (14.1% samples). These studies show that diarrheagenic calves and cattle represent important reservoirs of eae-positive E. coli.
The role of agriculture in the transfer of drug resistant pathogens to humans is widely debated and poorly understood. Escherichia coli is a valuable indicator organism for contamination and carriage of antimicrobial resistance in foods. Whilst whole genome sequences for E. coli from animals and associated meats are common, sequences from produce are scarce. Produce may acquire drug resistant E. coli from animal manure fertilizers, contaminated irrigation water and wildlife, particularly birds. Whole genome sequencing was used to characterize 120 tetracycline (TET) resistant E. coli from store-bought, ready-to-eat cilantro, arugula and mixed salad from two German cities. E. coli were recovered on the day of purchase and after 7 days of refrigeration. Cilantro was far more frequently contaminated with TET resistant E. coli providing 102 (85%) sequenced strains. Phylogroup B1 dominated the collection (n=84, 70%) with multi-locus sequence types B1-ST6186 (n=37, 31%), C-ST165 (n=17, 14%), B1-ST58 (n=14, 12%), B1-ST641 (n=8, 7%) and C-ST88 (n=5, 4%) frequently identified. Notably, seven strains of diverse ST carried genetic indicators of ColV virulence plasmid carriage. A number of previously identified and novel integrons associated with insertion elements including IS26 were also identified. Storage may affect the lineages of E. coli isolated however further studies are needed. Our study indicates produce predominantly carry E. coli with a commensal phylogroup and a variety of antimicrobial resistance and virulence-associated traits. Genomic surveillance of bacteria that contaminate produce should be a matter of public health importance in order to develop a holistic understanding of the environmental dimensions of antimicrobial resistance.
Many bacterial pathogens require adhesion to the mucosal epithelium to establish colonisation and employ numerous strategies to then avoid clearance by the host immune system. One such strategy involves expressing plasminogen receptors on the cell surface. Recently we showed that Mycoplasma hyopneumoniae is adept at capturing porcine plasminogen onto cell surface adhesins. This interaction promotes the conversion of bound plasminogen to plasmin where it plays an important role in regulating lung inflammation. Cell surface plasmin triggers a proteolytic cascade that is thought to promote dissemination of the pathogen from the initial site of colonisation. M. hyopneumoniae is a genome-reduced pathogen that has lost the genes required to synthesise amino acids and is thus reliant on the host for amino acids for growth. We have shown M. hyopneumoniae expresses a glutamyl-aminopeptidase (MHJ_0125) and a leucyl-aminopeptidase (MHJ_0461) on the extracellular surface of the cell membrane and both are perceived as playing a key role in the generation of a pool of free amino acids for growth during pathogenesis. MHJ_0461 displays a catalytic preference for leucine, phenylalanine, and methionine, whilst MHJ_0125 demonstrates a preference for glutamic acid and alanine. In addition to their catalytic functions as aminopeptidases, both enzymes bind porcine plasminogen, promoting its conversion to plasmin by tPA, and display an affinity for highly sulphated glycosaminoglycans. MHJ_0461 was also shown to bind extracellular DNA. These studies highlight the multifunctional properties of surface proteins in M. hyopneumoniae and the increasing pool of evidence that moonlighting proteins play important roles during microbial pathogenesis.
Summary Mycoplasma hyopneumoniae , the causative agent of porcine enzootic pneumonia, colonizes the respiratory cilia of affected swine causing significant economic losses to swine production worldwide. Heparin is known to inhibit adherence of M. hyopneumoniae to porcine respiratory epithelial cilia. M. hyopneumoniae cells bind heparin but the identity of the heparin‐binding proteins is limited. Proteomic analysis of M. hyopneumoniae lysates identified 27 kDa (P27), 110 kDa (P110) and 52 kDa (P52) proteins representing different regions of a 159 kDa (P159) protein derived from mhp494. These cleavage fragments were surface located and present at all growth stages. Following purification of four recombinant proteins spanning P159 (F1 P159 , F2 P159 , F3 P159 and F4 P159 ), only F3 P159 and F4 P159 bound heparin in a dose‐dependent manner ( K d values 142.37 ± 22.01 nM; 75.37 ± 7.34 nM respectively). Scanning electron microscopic studies showed M. hyopneumoniae bound intimately to porcine kidney epithelial‐like cells (PK15 cells) but these processes were inhibited by excess heparin and F4 P159 . Similarly, latex beads coated with F2 P159 and F4 P159 adhered to and entered PK15 cells, but heparin, F2 P159 and F4 P159 was inhibitory. These findings indicate that P159 is a post‐translationally cleaved, glycosaminoglycan‐binding adhesin of M. hyopneumoniae .
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