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Effective implementation of antibiotic stewardship, especially in critical care, is limited by a lack of direct comparative investigations on how different antibiotics impact the microbiota and antibiotic resistance rates. We investigated the impact of two commonly used antibiotics, third-generation cephalosporins (3GC) and piperacillin/tazobactam (TZP) on the endotracheal, perineal and faecal microbiota of intensive care patients in Australia. Patients exposed to either 3GC, TZP, or no β-lactams (control group) were sampled over time and 16S rRNA amplicon sequencing was performed to examine microbiota diversity and composition. While neither treatment significantly affected diversity, numerous changes to microbiota composition were associated with each treatment. The shifts in microbiota composition associated with 3GC exposure differed from those observed with TZP, consistent with previous reports in animal models. This included a significant increase in Enterobacteriaceae and Enterococcaceae abundance in endotracheal and perineal microbiota for those administered 3GC compared to the control group. Culture-based analyses did not identify any significant changes in the prevalence of specific pathogenic or antibiotic-resistant bacteria. Exposure to clinical antibiotics has previously been linked to reduced microbiota diversity and increased antimicrobial resistance, but our results indicate that these effects may not be immediately apparent after short-term real-world exposures.

Bacteriophage (phage) therapy is re-emerging a century after it began. Activity against antibiotic-resistant pathogens and a lack of serious side effects make phage therapy an attractive treatment option in refractory bacterial infections. Phages are highly specific for their bacterial targets, but the relationship between in vitro activity and in vivo efficacy remains to be rigorously evaluated. Pharmacokinetic and pharmacodynamic principles of phage therapy are generally based on the classic predator-prey relationship, but numerous other factors contribute to phage clearance and optimal dosing strategies remain unclear. Combinations of fully characterised, exclusively lytic phages prepared under good manufacturing practice are limited in their availability. Safety has been demonstrated but randomised controlled trials are needed to evaluate efficacy.

The spread of multidrug resistance via mobile genetic elements is a major clinical and veterinary concern. Pathogenic Escherichia coli harbour antibiotic resistance and virulence genes mainly on plasmids, but also bacteriophages and hybrid phage-like plasmids. In this study, the genomes of three E. coli phage-like plasmids, pJIE250-3 from a human E. coli clinical isolate, pSvP1 from a porcine ETEC O157 isolate, and pTZ20_1P from a porcine commensal E. coli, were sequenced (PacBio RSII), annotated and compared. All three elements are coliphage P1 variants, each with unique adaptations. pJIE250-3 is a P1-derivative that has lost lytic functions and contains no accessory genes. In pTZ20_1P and pSvP1, a core P1-like genome is associated with insertion sequence-mediated acquisition of plasmid modules encoding multidrug resistance and virulence, respectively. The transfer ability of pTZ20_1P, carrying antibiotic resistance markers, was also tested and, although this element was not able to transfer by conjugation, it was able to lysogenize a commensal E. coli strain with consequent transfer of resistance. The incidence of P1-like plasmids (~7%) in our E. coli collections correlated well with that in public databases. This study highlights the need to investigate the contribution of phage-like plasmids to the successful spread of antibiotic resistant pathotypes.

The bacterial L-form is induced by exposure to cell wall targeting antibiotics or innate immune effectors such as lysozyme and is likely to be important in many human infections. Here, we demonstrate that the osmotically fragile L-form is a distinct physiological state in Escherichia coli that is highly tolerant of oxidative stress and resistant to powerful antibiotics and common therapeutic bacteriophages. L-forms quickly revert (

Effective antimicrobial stewardship requires a better understanding of the impact of different antibiotics on the gut microflora. Studies with humans are confounded by large interindividual variability and difficulty in identifying control cohorts. However, controlled murine models can provide valuable information. In this study, we examined the impact of a penicillin-like antibiotic (piperacillin-tazobactam [TZP]) or a third-generation cephalosporin (ceftriaxone [CRO]) on the murine gut microbiota by analysis of changes in fecal microbiome composition by 16S rRNA amplicon sequencing and standard microbiology. Resistance to colonization by multidrug-resistant Escherichia coli sequence type 131 (ST131) and Klebsiella pneumoniae ST258 was also tested. Changes in microbiome composition and a significant (P

Effective antimicrobial stewardship requires a better understanding of the impact of different antibiotics on the gut microflora. Studies in humans are confounded by large inter-individual variability and difficulty in identifying control cohorts. However, controlled murine models can provide valuable information. We examined the impact of a penicillin-like antibiotic (piperacillin/tazobactam, TZP) or a third-generation cephalosporin (ceftriaxone, CRO) on the murine gut microbiota. We analyzed gut microbiome composition by 16S-rRNA amplicon sequencing and effects on the Enterobacteriaceae by qPCR and standard microbiology. Colonization resistance to multidrug resistant Escherichia coli ST131 and Klebsiella pneumoniae ST258 was also tested. Changes in microbiome composition and a significant (p

© 2020 The Authors. Sequence type (ST)73 has emerged as one of the most frequently isolated extraintestinal pathogenic Escherichia coli. To examine the localized diversity of ST73 clonal groups, including their mobile genetic element profile, we sequenced the genomes of 16 multiple-drug resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia, between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccurred, over a 2 year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 clone D i2 reference genome shows high bootstrap support and clusters into four major groups that correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes, but the presence of iss, pic and several iron-acquisition operons was notable.

Multidrug resistant (MDR) carbapenemase-producing (CP) Klebsiella pneumoniae, belonging to clonal group CG258, is capable of causing severe disease in humans and is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as therapeutic alternatives to antibiotics. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the genomes of 18 Kumoniae target strains and tested them against novel lytic bacteriophages (n = 65). The genomes of K pneumoniae carrying blaNDM and blaKPC were sequenced and CG258 isolates selected for bacteriophage susceptibility testing. The local K pneumoniae CG258 population was dominated by sequence type ST258 clade 1 (86%) with variations in capsular locus (cps) and prophage content. CG258-specific bacteriophages primarily targeted the capsule, but successful infection is also likely blocked in some by immunity conferred by existing prophages. Five tailed bacteriophages against K pneumoniae ST258 clade 1 were selected for further characterization. Our findings show that effective control of K pneumoniae CG258 with bacteriophage will require mixes of diverse lytic viruses targeting relevant cps variants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic bacteriophage candidates against this serious pathogen.

Plasmids are important in carrying antibiotic resistance and other genes between bacterial cells, and a number of methods can be employed to characterize plasmids from clinical isolates. Single colonies typically obtained as part of hospital workflow can undergo S1 nuclease treatment to linearize plasmids followed by pulsed-field gel electrophoresis to enable determination of the number and sizes of plasmids present. Hybridization of S1/PFGE gels can be used to associate replicon types and passenger genes, such as those conferring antibiotic resistance, with a particular plasmid band. Individual plasmids, obtained by conjugation or transformation, can be compared by gel electrophoresis following restriction digestion of plasmid DNA prepared by alkaline lysis methods, including using specialized kits.

Multidrug resistant carbapenemase-producingKlebsiella pneumoniaecapable of causing severe disease in humans is classified as an urgent threat by health agencies worldwide. Bacteriophages are being actively explored as potential therapeutics against these multidrug resistant pathogens. We are currently developing bacteriophage therapy against carbepenem-resistantK. pneumoniaebelonging to the genetically diverse, globally disseminated clonal group CG258. In an effort to define a robust experimental approach for effective selection of lytic viruses for therapy, we have fully characterized the bacterial genomes of 18 target strains, tested them against novel lytic bacteriophages, and generated phage-susceptibility profiles. The genomes ofK. pneumoniaeisolates carryingblaNDMandblaKPCwere sequenced and isolates belonging to CG258 were selected for susceptibility testing using a panel of lytic bacteriophages (n=65). The localK. pneumoniaeCG258 population was dominated by isolates belonging to sequence type ST258 clade 1 (86%). The primary differences between ST258 genomes were variations in the capsular locus (cps) and in prophage content. We showed that CG258-specific lytic phages primarily target the capsule, and that successful infection is blocked in many, post-adsorption, by immunity conferred by existing prophages. Five bacteriophages specifically active againstK. pneumoniaeST258 clade 1 (n=5) belonging to the Caudovirales order were selected for further characterization. Our findings show that effective control ofK. pneumoniaeCG258 with phage will require mixes of diverse lytic viruses targeting all relevantcpsvariants and allowing for variable prophage content. These insights will facilitate identification and selection of therapeutic phage candidates against this serious pathogen.ImportanceBacteriophages are natural agents that exclusively and selectively kill bacteria and have the potential to be useful in the treatment of multidrug resistant infections.K. pneumoniaeCG258 is a main agent of life-threatening sepsis that is often resistant to last-line antibiotics. Our work highlights some key requirements for developing bacteriophage preparations targeting this pathogen. By defining the genomic profile of our clinicalK. pneumoniaeCG258 population and matching it with bacteriophage susceptibility patterns, we found that bacteriophage ability to lyse each strain correlates well withK. pneumoniaeCG258 structural subtypes (capsule variants). This indicates that preparation of bacteriophage therapeutics targeting this pathogen should aim at including phages against each bacterial capsular subtype. This necessitates a detailed understanding of the diversity of circulating isolates in different geographical areas in order to make rational therapeutic choices.

ST73 has emerged as one of the most frequently isolated extraintestinal pathogenic E. coli (ExPEC). To examine the localised diversity of ST73 clonal groups including their mobile genetic elements profile, we sequenced the genomes of 16 multiple drug-resistant ST73 isolates from patients with urinary tract infection from a single hospital in Sydney, Australia between 2009 and 2011. Genome sequences were used to generate a SNP-based phylogenetic tree to determine the relationship of these isolates in a global context with ST73 sequences (n=210) from public databases. There was no evidence of a dominant outbreak strain of ST73 in patients from this hospital, rather we identified at least eight separate groups, several of which reoccur, over a two-year period. The inferred phylogeny of all ST73 strains (n=226) including the ST73 Clone D i2 reference genome shows high bootstrap support and clusters into four major groups which correlate with serotype. The Sydney ST73 strains carry a wide variety of virulence-associated genes but the presence of iss, pic and several iron acquisition operons was notable.ImpactST73 is a major clonal lineage of ExPEC that causes urinary tract infections often with uroseptic sequelae but has not garnered substantial scientific interest as the globally disseminated ST131. Isolation of multiple antimicrobial resistant variants of ExPEC ST73 have increased in frequency, but little is known about the carriage of class 1 integrons in this sequence type and the plasmids that are likely to mobilise them. This pilot study examines the ST73 isolates within a single hospital in Sydney Australia and provides the first large-scale core-genome phylogenetic analysis of ST73 utilizing public sequence read datasets. We used this analysis to identify at least 8 sub-groups of ST73 within this single hospital. Mobile genetic elements associated with antibiotic resistance were less diverse and only three class 1 integron structures were identified, all sharing the same basic structure suggesting that the acquisition of drug resistance is a recent event. Genomic epidemiological studies are needed to further characterise established and emerging clonal populations of multiple drug resistant ExPEC to identify sources and aid outbreak investigations.

Postpartum women are at increased risk of developing puerperal sepsis caused by group A Streptococcus (GAS). Specific GAS serotypes, including M1 and M28, are more commonly associated with puerperal sepsis. However, the mechanisms of GAS genital tract infection are not well understood. We utilized a murine genital tract carriage model to demonstrate that M1 and M28 GAS colonization triggers TNF-α, IL-1β, and IL-17A production in the female genital tract. GAS-induced IL-17A significantly influences streptococcal carriage and alters local inflammatory responses in two genetically distinct inbred strains of mice. An absence of IL-17A or the IL-1 receptor was associated with reduced neutrophil recruitment to the site of infection; and clearance of GAS was significantly attenuated in IL-17A−/− mice and Rag1−/− mice (that lack mature lymphocytes) but not in mice deficient for the IL-1 receptor. Together, these findings support a role for IL-17A in contributing to the control of streptococcal mucosal colonization and provide new insight into the inflammatory mediators regulating host-pathogen interactions in the female genital tract.

The past 50 years has witnessed the emergence of new viral and bacterial pathogens with global effect on human health. The hyperinvasive group A Streptococcus (GAS) M1T1 clone, first detected in the mid-1980s in the United States, has since disseminated worldwide and remains a major cause of severe invasive human infections. Although much is understood regarding the capacity of this pathogen to cause disease, much less is known of the precise evolutionary events selecting for its emergence. We used high-throughput technologies to sequence a World Health Organization strain collection of serotype M1 GAS and reconstructed its phylogeny based on the analysis of core genome single-nucleotide polymorphisms. We demonstrate that acquisition of a 36-kb genome segment from serotype M12 GAS and the bacteriophage-encoded DNase Sda1 led to increased virulence of the M1T1 precursor and occurred relatively early in the molecular evolutionary history of this strain. The more recent acquisition of the phage-encoded superantigen SpeA is likely to have provided selection advantage for the global dissemination of the M1T1 clone. This study provides an exemplar for the evolution and emergence of virulent clones from microbial populations existing commensally or causing only superficial infection.—Maamary, P. G., Ben Zakour, N. L., Cole, J. N., Hollands, A., Aziz, R. K., Barnett, T. C., Cork, A. J., Henningham, A., Sanderson-Smith, M., McArthur, J. D., Venturini, C., Gillen, C. M., Kirk, J. K., Johnson, D. R., Taylor, W. L., Kaplan, E. L., Kotb, M., Nizet, V., Beatson, S. A., Walker, M. J. Tracing the evolutionary history of the pandemic group A streptococcal M1T1 clone.

Streptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies for disruption of streptokinase mediated plasminogen activation which could be employed to treat severe invasive S. pyogenes infections.

The rise of multiple antibiotic resistance in clinically relevant bacteria has created a global crisis with increasing burden on healthcare systems. The need to optimise alternative therapies to antibiotics, particularly in high risk nosocomial settings, is therefore immediate. Bacteriophages are specialised lethal viruses of bacteria, and an underused clinical resource for the treatment of severe infections refractory to antibiotics. Both the gaps in knowledge of bacteriophage biology, particularly the details of host-pathogen dynamic interactions, and legislative hurdles related to the regulation of natural microorganisms for therapy have delayed progress in bacteriophage clinical applications. At the Westmead Institute for Medical Research (WIMR), in collaboration with Westmead Hospital (Western Sydney Local Health District, WSLHD) and the University of Sydney (USyd), we have been investigating rational design protocols for routine bacteriophage application in clinical practice and testing bacteriophage therapeutics on patients suffering from multidrug resistant (MDR) severe infections.

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome1,2,3. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone4,5, with bacteriophage-encoded determinants DNase Sda16 and superantigen SpeA27 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS8,9,10, led to our investigation of the next most common cause of scarlet fever, emm1 GAS8,9. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones10,11 in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.

A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1-6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage ΦHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, ΦHKU.vir and a new prophage, ΦHKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever-associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.

The resurgence of invasive disease caused by Streptococcus pyogenes (group A Streptococcus [GAS]) in the past 30 years has paralleled the emergence and global dissemination of the highly virulent M1T1 clone. The GAS M1T1 clone has diverged from the ancestral M1 serotype by horizontal acquisition of two unique bacteriophages, encoding the potent DNase Sda1/SdaD2 and the superantigen SpeA, respectively. The phage-encoded DNase promotes escape from neutrophil extracellular traps and is linked to enhanced virulence of the M1T1 clone. In this study, we successfully used in vitro lysogenic conversion to transfer the Sda1-encoding phage from the M1T1 clonal strain 5448 to the nonclonal M1 isolate SF370 and determined the impact of this horizontal gene transfer event on virulence. Although Sda1 was expressed in SF370 lysogens, no capacity of the phage-converted strain to survive human neutrophil killing, switch to a hyperinvasive covRS mutant form, or cause invasive lethal infection in a humanized plasminogen mouse model was observed. This work suggests that the hypervirulence of the M1T1 clone is due to the unique synergic effect of the M1T1 clone bacteriophage-specific virulence factor Sda1 acting in concert with the M1T1 clone-specific genetic scaffold.

Enterohemorrhagic Escherichia coli (EHEC) and atypical enteropathogenic E. coli (aEPEC) are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL 125 (125 kb) from a human O26:H- EHEC, and pO111-CRL115 (115kb) from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3′-conserved segment. Complete sequence analysis showed that pO26-CRL125 and pO111-CRL115 are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL125 but absent in pO111-CRL115, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL125 imperfect repeats was identified in pO111-CRL115 precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3′-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex antibiotic resistance gene locus. Comparative sequence analysis of these closely related plasmids reveals aspects of plasmid evolution in pathogenic E. coli from different hosts. © 2013 Venturini et al.

Between June and November 2010, a concerning rise in the number of cases of puerperal sepsis, a postpartum pelvic bacterial infection contracted by women after childbirth, was observed in the New South Wales, Australia, hospital system. Group A streptococcus (GAS; Streptococcus pyogenes ) isolates PS001 to PS011 were recovered from nine patients. Pulsed-field gel electrophoresis and emm sequence typing revealed that GAS of emm1.40 , emm75.0 , emm77.0 , emm89.0 , and emm89.9 were each recovered from a single patient, ruling out a single source of infection. However, emm28.8 GAS were recovered from four different patients. To investigate the relatedness of these emm28 isolates, whole-genome sequencing was undertaken and the genome sequences were compared to the genome sequence of the emm28.4 reference strain, MGAS6180. A total of 186 single nucleotide polymorphisms were identified, for which the phylogenetic reconstruction indicated an outbreak of a polyclonal nature. While two isolates collected from different hospitals were not closely related, isolates from two puerperal sepsis patients from the same hospital were indistinguishable, suggesting patient-to-patient transmission or infection from a common source. The results of this study indicate that traditional typing protocols, such as pulsed-field gel electrophoresis, may not be sensitive enough to allow fine epidemiological discrimination of closely related bacterial isolates. Whole-genome sequencing presents a valid alternative that allows accurate fine-scale epidemiological investigation of bacterial infectious disease.

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes. © 2012 Dawes et al.

Enterohemorrhagic Escherichia coli (EHEC) strains are zoonotic pathogens responsible for a range of severe human disease. The repertoire of virulence determinants promoting EHEC disease is encoded on both the main chromosome and virulence plasmid. We examined a multiply antibiotic-resistant O26 EHEC strain for carriage of resistance genes on the virulence plasmid. The EHEC virulence plasmid containing a complex antibiotic-resistance gene locus, designated as pO26-CRL, was purified from EHEC O26:H(-) (patient with hemorrhagic colitis) and subjected to shotgun-sequencing and bioinformatic analysis. Determination of the 111,481-bp sequence of pO26-CRL revealed genes encoding a functional enterohemolysin operon (ehxCABD), STEC-specific extracellular serine protease (espP), putative EHEC adhesin (toxB), catalase/peroxidase (katP), and myristoyl transferase (msbB) involved in lipid A synthesis. A 22,609-bp Tn21 derivative is inserted within the conjugal transfer gene traC and encodes resistance to trimethoprim, streptomycin, sulfathiozole, kanamycin, neomycin, beta-lactams, and mercuric chloride. Plasmid pO26-CRL is nonconjugative but is mobilizable. This is the first report of an EHEC virulence plasmid containing a complex antibiotic resistance locus, and raises the concern that antibiotic use will coselect for virulence determinants, leading to increased disease potential in both commensal and pathogenic E. coli populations.-Venturini, C., Beatson, S. A., Djordjevic, S. P., Walker, M. J. Multiple antibiotic resistance gene recruitment onto the enterohemorrhagic Escherichia coli virulence plasmid.