Identification and evolution of ICE-PmuST394: a novel integrative conjugative element in Pasteurella multocida ST394.

BACKGROUND: The emergence of macrolide and tetracycline resistance within Pasteurella multocida isolated from feedlot cattle and the dominance of ST394 in Australia was reported recently. OBJECTIVES: To establish the genetic context of the resistance genes in P. multocida 17BRD-035, the ST394 reference genome, and conduct a molecular risk assessment of their ability to disperse laterally. METHODS: A bioinformatic analysis of the P. multocida 17BRD-035 genome was conducted to determine if integrative conjugative elements (ICEs) carrying resistance genes, which hamper antibiotic treatment options locally, are in circulation in Australian feedlots. RESULTS: A novel element, ICE-PmuST394, was characterized in P. multocida 17BRD-035. It was also identified in three other isolates (two ST394s and a ST125) in Australia and is likely present in a genome representing P. multocida ST79 from the USA. ICE-PmuST394 houses a resistance module carrying two variants of the blaROB gene, blaROB-1 and blaROB-13, and the macrolide esterase gene, estT. The resistance gene combination on ICE-PmuST394 confers resistance to ampicillin and tilmicosin, but not to tulathromycin and tildipirosin. Our analysis suggests that ICE-PmuST394 is circulating both by clonal expansion and horizontal transfer but is currently restricted to a single feedlot in Australia. CONCLUSIONS: ICE-PmuST394 carries a limited number of unusual antimicrobial resistance genes but has hotspots that facilitate genomic recombination. The element is therefore amenable to hosting more resistance genes, and therefore its presence (or dispersal) should be regularly monitored. The element has a unique molecular marker, which could be exploited for genomic surveillance purposes locally and globally.

Identification of a novel lineage of plasmids within phylogenetically diverse subclades of IncHI2-ST1 plasmids.

IncHI2-ST1 plasmids play an important role in co-mobilizing genes conferring resistance to critically important antibiotics and heavy metals. Here we present the identification and analysis of IncHI2-ST1 plasmid pSPRC-Echo1, isolated from an Enterobacter hormaechei strain from a Sydney hospital, which predates other multi-drug resistant IncHI2-ST1 plasmids reported from Australia. Our time-resolved phylogeny analysis indicates pSPRC-Echo1 represents a new lineage of IncHI2-ST1 plasmids and show how their diversification relates to the era of antibiotics.

Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease.

BACKGROUND: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. RESULTS: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. CONCLUSIONS: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc.

Comparative genomic analysis of a multiple antimicrobial resistant enterotoxigenic E. coli O157 lineage from Australian pigs.

BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) are a major economic threat to pig production globally, with serogroups O8, O9, O45, O101, O138, O139, O141, O149 and O157 implicated as the leading diarrhoeal pathogens affecting pigs below four weeks of age. A multiple antimicrobial resistant ETEC O157 (O157 SvETEC) representative of O157 isolates from a pig farm in New South Wales, Australia that experienced repeated bouts of pre- and post-weaning diarrhoea resulting in multiple fatalities was characterized here. Enterohaemorrhagic E. coli (EHEC) O157:H7 cause both sporadic and widespread outbreaks of foodborne disease, predominantly have a ruminant origin and belong to the ST11 clonal complex. Here, for the first time, we conducted comparative genomic analyses of two epidemiologically-unrelated porcine, disease-causing ETEC O157; E. coli O157 SvETEC and E. coli O157:K88 734/3, and examined their phylogenetic relationship with EHEC O157:H7. RESULTS: O157 SvETEC and O157:K88 734/3 belong to a novel sequence type (ST4245) that comprises part of the ST23 complex and are genetically distinct from EHEC O157. Comparative phylogenetic analysis using PhyloSift shows that E. coli O157 SvETEC and E. coli O157:K88 734/3 group into a single clade and are most similar to the extraintestinal avian pathogenic Escherichia coli (APEC) isolate O78 that clusters within the ST23 complex. Genome content was highly similar between E. coli O157 SvETEC, O157:K88 734/3 and APEC O78, with variability predominantly limited to laterally acquired elements, including prophages, plasmids and antimicrobial resistance gene loci. Putative ETEC virulence factors, including the toxins STb and LT and the K88 (F4) adhesin, were conserved between O157 SvETEC and O157:K88 734/3. The O157 SvETEC isolate also encoded the heat stable enterotoxin STa and a second allele of STb, whilst a prophage within O157:K88 734/3 encoded the serum survival gene bor. Both isolates harbor a large repertoire of antibiotic resistance genes but their association with mobile elements remains undetermined. CONCLUSIONS: We present an analysis of the first draft genome sequences of two epidemiologically-unrelated, pathogenic ETEC O157. E. coli O157 SvETEC and E. coli O157:K88 734/3 belong to the ST23 complex and are phylogenetically distinct to EHEC O157 lineages that reside within the ST11 complex.

Tn6026 and Tn6029 are found in complex resistance regions mobilised by diverse plasmids and chromosomal islands in multiple antibiotic resistant Enterobacteriaceae.

Transposons flanked by direct copies of IS26 are important contributors to the evolution of multiple antibiotic resistance. Tn6029 and Tn6026 are examples of composite transposons that have become widely disseminated on small and large plasmids with different incompatibility markers in pathogenic and commensal Escherichia coli and various serovars of Salmonella enterica. Some of the plasmids that harbour these transposons also carry combinations of virulence genes. Recently, Tn6029 and Tn6026 and derivatives thereof have been found on chromosomal islands in both established and recently emerged pathogens. While Tn6029 and Tn6026 carry genes encoding resistance to older generation antibiotics, they also provide a scaffold for the introduction of genes encoding resistance to a wide variety of clinically relevant antibiotics that are mobilised by IS26. As a consequence, Tn6029 and Tn6026 or variants are likely to increasingly feature in complex resistance regions in multiple antibiotic resistant Enterobacteriaceae that threaten the health of humans and food production animals.

Genomic profiling of Pasteurella multocida isolated from feedlot cases of bovine respiratory disease.

Pasteurella multocida causes a range of diseases in many host species throughout the world, including bovine respiratory disease (BRD) which is predominantly seen in feedlot cattle. This study assessed genetic diversity among 139 P. multocida isolates obtained from post-mortem lung swabs of BRD-affected feedlot cattle in four Australian states: New South Wales, Queensland, South Australia, and Victoria during 2014-2019. Whole-genome sequencing (WGS) was used to determine capsular serogroup, lipopolysaccharide genotypes, multi-locus sequence types and phylogenetic relationships. Two capsular types (A and D), with most isolates (132/139; 95%) belonging to type A; and three lipopolysaccharide (LPS) genotypes were identified (L1 [6/139; 4.3%], L3 [124/139; 89.2%] and L6 [9/139; 6.4%)]). Multi-locus sequence types (STs) ST9, ST13, ST17, ST20, ST36, ST50, ST58, ST79, ST124, ST125, ST132, ST167, ST185, ST327, ST394, and three novel STs [ST396, ST397, and ST398] were identified, with ST394 (59/139; 42.4%) and ST79 (44/139; 32%) the most prevalent in all four states. Isolates displaying phenotypic resistance to single, dual or multiple antibiotics (macrolide, tetracycline and aminopenicillins) were predominantly ST394 (23/139; 17%). Laterally mobile elements identified in the resistant ST394 isolates included small plasmids, encoding macrolide and/or tetracycline resistance, distributed in all states; and chromosomally located integrative conjugative elements (ICEs) (4 ST394 and 1 ST125) from the same Queensland feedlot. This study highlights the genomic diversity, epidemiological relationships and AMR associations in bovine P. multocida isolates from Australia and provides insight into the unique ST prevalence compared to other major beef-producing countries.

Phylogenomic analysis of a global collection of Escherichia coli ST38: evidence of interspecies and environmental transmission?

IMPORTANCE: Extraintestinal pathogenic Escherichia coli (ExPEC) sequence type (ST) 38 is one of the top 10 human pandemic lineages. Although a major cause of urinary tract and blood stream infections, ST38 has been poorly characterized from a global phylogenomic perspective. A comprehensive genome-scale analysis of 925 ST38 isolate genomes identified two broad ancestral clades and linkage of discrete ST38 clusters with specific bla CTX-M variants. In addition, the clades and clusters carry important virulence genes, with diverse but poorly characterized plasmids. Numerous putative interhost and environment transmission events were identified here by the presence of ST38 clones (defined as isolates with ≤35 SNPs) within humans, companion animals, food sources, urban birds, wildlife, and the environment. A small cluster of international ST38 clones from diverse sources, likely representing progenitors of a hospital outbreak that occurred in Brisbane, Australia, in 2017, was also identified. Our study emphasizes the importance of characterizing isolate genomes derived from nonhuman sources and geographical locations, without any selection bias.

Integrative omics identifies conserved and pathogen-specific responses of sepsis-causing bacteria.

Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.

Correction: Alhamami et al. First Emergence of Resistance to Macrolides and Tetracycline Identified in Mannheimia haemolytica and Pasteurella multocida Isolates from Beef Feedlots in Australia. Microorganisms 2021, 9, 1322.

The authors wish to make the following corrections to this paper [...].

Genome sequence of Vibrio rotiferianus strain DAT722.

Vibrio rotiferianus is a marine pathogen capable of causing disease in various aquatic organisms. We announce the genome sequence of V. rotiferianus DAT722, which has a large chromosomal integron containing 116 gene cassettes and is a model organism for studying the role of this system in vibrio evolution.

Dissemination of multiple drug resistance genes by class 1 integrons in Klebsiella pneumoniae isolates from four countries: a comparative study.

A comparative genetic analysis of 42 clinical Klebsiella pneumoniae isolates, resistant to two or more antibiotics belonging to the broad-spectrum ß-lactam group, sourced from Sydney, Australia, and three South American countries is presented. The study focuses on the genetic contexts of class 1 integrons, mobilizable genetic elements best known for their role in the rapid evolution of antibiotic resistance among Gram-negative pathogens. It was found that the class 1 integrons in this cohort were located in a number of different genetic contexts with clear regional differences. In Sydney, IS26-associated Tn21-like transposons on IncL/M plasmids contribute greatly to the dispersal of integron-associated multiple-drug-resistant (MDR) loci. In contrast, in the South American countries, Tn1696-like transposons on an IncA/C plasmid(s) appeared to be disseminating a characteristic MDR region. A range of mobile genetic elements is clearly being recruited by clinically important mobile class 1 integrons, and these elements appear to be becoming more common with time. This in turn is driving the evolution of complex and laterally mobile MDR units and may further complicate antibiotic therapy.

Molecular Analysis of an IncF ColV-Like Plasmid Lineage That Carries a Complex Resistance Locus with a Trackable Genetic Signature.

IncF ColV plasmids are important plasmid incompatibility group that are currently restricted to the Enterobacteriaceae. These plasmids carry an important repertoire of virulence-associated genes (VAGs) that contribute to the ability of avian pathogenic Escherichia coli to cause disease in poultry. VAGs found on ColV plasmids have also been linked to urosepsis and meningitis in humans but the mechanisms that elicit these disease conditions are not well understood. Recently we described the sequence of a ColV plasmid pSDJ2009-52F that carried the typical repertoire of VAGs and a complex resistance gene locus flanked by IS26, an insertion element that plays an important role in mobilizing antibiotic resistance genes on plasmids and genomic islands. We recovered complete ColV-like plasmid sequences from public databases that shared >80% sequence identity with pSDJ2009-52F in geographically diverse regions of the world over a 20-year timeframe. Previously we noted that pSDJ2009-52F carries a unique genetic signature in the class 1 integron within the complex resistance locus that was presumably created by the action of IS26. Here we show that most ColV-like plasmids that are closely related to pSDJ2009-52F also carry the same signature. Our studies provide insight into how these signature-bearing plasmids and the mobile genetic elements they carry traffic between E. coli sequence types over large geographic distances.

Genomic Characterisation of a Multiple Drug Resistant IncHI2 ST4 Plasmid in Escherichia coli ST744 in Australia.

Antibiotic resistance genes (ARGs) including those from the blaCTX-M family and mcr-1 that encode resistance to extended spectrum ß-lactams and colistin, respectively, have been linked with IncHI2 plasmids isolated from swine production facilities globally but not in IncHI2 plasmids from Australia. Here we describe the first complete sequence of a multiple drug resistance Australian IncHI2-ST4 plasmid, pTZ41_1P, from a commensal E. coli from a healthy piglet. pTZ41_1P carries genes conferring resistance to heavy-metals (copper, silver, tellurium and arsenic), ß-lactams, aminoglycosides and sulphonamides. The ARGs reside within a complex resistance locus (CRL) that shows considerable sequence identity to a CRL in pSDE_SvHI2, an IncHI2:ST3 plasmid from an enterotoxigenic E. coli with serotype O157:H19 of porcine origin that caused substantial losses to swine production operations in Australia in 2007. pTZ41_1P is closely related to IncHI2 plasmids found in E. coli and Salmonella enterica from porcine, avian and human sources in Europe and China but it does not carry genes encoding resistance to clinically-important antibiotics. We identified regions of IncHI2 plasmids that contribute to the genetic plasticity of this group of plasmids and highlight how they may readily acquire new resistance gene cargo. Genomic surveillance should be improved to monitor IncHI2 plasmids.

Genomic profiling of Escherichia coli isolates from bacteraemia patients: a 3-year cohort study of isolates collected at a Sydney teaching hospital.

This study sought to assess the genetic variability of Escherichia coli isolated from bloodstream infections (BSIs) presenting at Concord Hospital, Sydney during 2013-2016. Whole-genome sequencing was used to characterize 81 E. coli isolates sourced from community-onset (CO) and hospital-onset (HO) BSIs. The cohort comprised 64 CO and 17 HO isolates, including 35 multidrug-resistant (MDR) isolates exhibiting phenotypic resistance to three or more antibiotic classes. Phylogenetic analysis identified two major ancestral clades. One was genetically diverse with 25 isolates distributed in 16 different sequence types (STs) representing phylogroups A, B1, B2, C and F, while the other comprised phylogroup B2 isolates in subclades representing the ST131, ST73 and ST95 lineages. Forty-seven isolates contained a class 1 integron, of which 14 carried blaCTX -M-gene. Isolates with a class 1 integron carried more antibiotic resistance genes than isolates without an integron and, in most instances, resistance genes were localized within complex resistance loci (CRL). Resistance to fluoroquinolones could be attributed to point mutations in chromosomal parC and gyrB genes and, in addition, two isolates carried a plasmid-associated qnrB4 gene. Co-resistance to fluoroquinolone and broad-spectrum beta-lactam antibiotics was associated with ST131 (HO and CO), ST38 (HO), ST393 (CO), ST2003 (CO) and ST8196 (CO and HO), a novel ST identified in this study. Notably, 10/81 (12.3 %) isolates with ST95 (5 isolates), ST131 (2 isolates), ST88 (2 isolates) and a ST540 likely carry IncFII-IncFIB plasmid replicons with a full spectrum of virulence genes consistent with the carriage of ColV-like plasmids. Our data indicate that IncF plasmids play an important role in shaping virulence and resistance gene carriage in BSI E. coli in Australia.

Escherichia coli ST8196 is a novel, locally evolved, and extensively drug resistant pathogenic lineage within the ST131 clonal complex.

The H30Rx subclade of Escherichia coli ST131 is a clinically important, globally dispersed pathogenic lineage that typically displays resistance to fluoroquinolones and extended spectrum ß-lactams. Isolates EC233 and EC234, variants of ST131-H30Rx with a novel sequence type (ST) 8196, isolated from unrelated patients presenting with bacteraemia at a Sydney Hospital in 2014 are characterised here. EC233 and EC234 are phylogroup B2, serotype O25:H4A, and resistant to ampicillin, amoxicillin, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, norfloxacin and gentamicin and are likely clonal. Both harbour an IncFII_2 plasmid (pSPRC_Ec234-FII) that carries most of the resistance genes on an IS26 associated translocatable unit, two small plasmids and a novel IncI1 plasmid (pSPRC_Ec234-I). SNP-based phylogenetic analysis of the core genome of representatives within the ST131 clonal complex places both isolates in a subclade with three clinical Australian ST131-H30Rx clade-C isolates. A MrBayes phylogeny analysis of EC233 and EC234 indicates ST8196 share a most recent common ancestor with ST131-H30Rx strain EC70 isolated from the same hospital in 2013. Our study identified genomic hallmarks that define the ST131-H30Rx subclade in the ST8196 isolates and highlights a need for unbiased genomic surveillance approaches to identify novel high-risk MDR E. coli pathogens that impact healthcare facilities.

Osteoarticular Infection in Three Young Thoroughbred Horses Caused by a Novel Gram Negative Cocco-Bacillus.

We describe three cases of osteoarticular infection (OAI) in young thoroughbred horses in which the causative organism was identified by MALDI-TOF as Kingella species. The pattern of OAI resembled that reported with Kingella infection in humans. Analysis by 16S rRNA PCR enabled construction of a phylogenetic tree that placed the isolates closer to Simonsiella and Alysiella species, rather than Kingella species. Average nucleotide identity (ANI) comparison between the new isolate and Kingella kingae and Alysiella crassa however revealed low probability that the new isolate belonged to either of these species. This preliminary analysis suggests the organism isolated is a previously unrecognised species.

Tn6060, a transposon from a genomic island in a Pseudomonas aeruginosa clinical isolate that includes two class 1 integrons.

A 25,441-bp transposon was recovered from a Pseudomonas aeruginosa clinical isolate. While the transposition module was >99% identical to sequence of Tn1403, the element had been subject to rearrangements, with two In70.2-like class 1 integrons inserted into it in an unusual "tail-to-tail" configuration. One cassette array was the same as that in In70.2; however, the second was different, generating a transposon that collectively includes six resistance cassettes.

High contiguity genome sequence of a multidrug-resistant hospital isolate of Enterobacter hormaechei.

BACKGROUND: Enterobacter hormaechei is an important emerging pathogen and a key member of the highly diverse Enterobacter cloacae complex. E. hormaechei strains can persist and spread in nosocomial environments, and often exhibit resistance to multiple clinically important antibiotics. However, the genomic regions that harbour resistance determinants are typically highly repetitive and impossible to resolve with standard short-read sequencing technologies. RESULTS: Here we used both short- and long-read methods to sequence the genome of a multidrug-resistant hospital isolate (C15117), which we identified as E. hormaechei. Hybrid assembly generated a complete circular chromosome of 4,739,272 bp and a fully resolved plasmid of 339,920 bp containing several antibiotic resistance genes. The strain also harboured a 34,857 bp repeat encoding copper resistance, which was present in both the chromosome and plasmid. Long reads that unambiguously spanned this repeat were required to resolve the chromosome and plasmid into separate replicons. CONCLUSION: This study provides important insights into the evolution and potential spread of antimicrobial resistance in a nosocomial E. hormaechei strain. More broadly, it further exemplifies the power of long-read sequencing technologies, particularly the Oxford Nanopore platform, for the characterisation of bacteria with complex resistance loci and large repeat elements.