Zuowei Wu

Comprehensive resistome analysis reveals the prevalence of NDM and MCR-1 in Chinese poultry production

By 2030, the global population will be 8.5 billion, placing pressure on international poultry production, of which China is a key producer1. From April 2017, China will implement the withdrawal of colistin as a growth promoter, removing over 8,000 tonnes per year from the Chinese farming sector2. To understand the impact of banning colistin and the epidemiology of multi-drug-resistant (MDR) Escherichia coli (using blaNDM and mcr-1 as marker genes), we sampled poultry, dogs, sewage, wild birds and flies. Here, we show that mcr-1, but not blaNDM, is prevalent in hatcheries, but blaNDM quickly contaminates flocks through dogs, flies and wild birds. We also screened samples directly for resistance genes to understand the true breadth and depth of the environmental and animal resistome. Direct sample testing for blaNDM and mcr-1 in hatcheries, commercial farms, a slaughterhouse and supermarkets revealed considerably higher levels of positive samples than the blaNDM- and mcr-1-positive E. coli, indicating a substantial segment of unseen resistome—a phenomenon we have termed the ‘phantom resistome’. Whole-genome sequencing identified common blaNDM-positive E. coli shared among farms, flies, dogs and farmers, providing direct evidence of carbapenem-resistant E. coli transmission and environmental contamination.

Multi-omics Approaches to Deciphering a Hypervirulent Strain of Campylobacter jejuni

Campylobacter jejuni clone SA recently emerged as the predominant cause of sheep abortion in the United States and is also associated with foodborne gastroenteritis in humans. A distinct phenotype of this clone is its ability to induce bacteremia and abortion. To facilitate understanding the pathogenesis of this hypervirulent clone, we analyzed a clinical isolate (IA3902) of clone SA using multi-omics approaches. The genome of IA3902 contains a circular chromosome of 1,635,045 bp and a circular plasmid of 37,174 bp. Comparative genomic analysis revealed that IA3902 is most closely related to C. jejuni NCTC11168, which is a reference strain and was previously shown to be non-abortifacient in pregnant animals. Despite the high genomic synteny and sequence homology, there are 12 variable regions (VRs) and 8,696 single-nucleotide polymorphisms and indels between the two genomes. Notably, the variable genes in the capsular polysaccharides biosynthesis and O-linked glycosylation loci of IA3902 are highly homogenous to their counterparts in C. jejuni subsp. doylei and C. jejuni G1, which are known to be frequently associated with bacteremia. Transcriptomic and proteomic profiles were conducted to compare IA3902 with NCTC11168, which revealed that the pathways of energy generation, motility, and serine utilization were significantly up-regulated in IA3902, whereas the pathways of iron uptake and proline, glutamate, aspartate, and lactate utilization were significantly down-regulated. These results suggest that C. jejuni clone SA has evolved distinct genomic content and gene expression patterns that modulate surface polysacharide structures, motilitiy, and metabolic pathways. These changes may have contributed to its hyper-virulence in abortion induction.

Adaptive mechanisms of Campylobacter jejuni to erythromycin treatment

BackgroundMacrolide is the drug of choice to treat human campylobacteriosis, but Campylobacter resistance to this antibiotic is rising. The mechanisms employed by Campylobacter jejuni to adapt to erythromycin treatment remain unknown and are examined in this study. The transcriptomic response of C. jejuni NCTC 11168 to erythromycin (Ery) treatment was determined by competitive microarray hybridizations. Representative genes identified to be differentially expressed were further characterized by constructing mutants and assessing their involvement in antimicrobial susceptibility, oxidative stress tolerance, and chicken colonization.ResultsFollowing the treatment with an inhibitory dose of Ery, 139 genes were up-regulated and 119 were down-regulated. Many genes associated with flagellar biosynthesis and motility was up-regulated, while many genes involved in tricarboxylic acid cycle, electron transport, and ribonucleotide biosynthesis were down-regulated. Exposure to a sub-inhibitory dose of Ery resulted in differential expression of much fewer genes. Interestingly, two putative drug efflux operons (cj0309c-cj0310c and cj1173-cj1174) were up-regulated. Although mutation of the two operons did not alter the susceptibility of C. jejuni to antimicrobials, it reduced Campylobacter growth under high-level oxygen. Another notable finding is the consistent up-regulation of cj1169c-cj1170c, of which cj1170c encodes a known phosphokinase, an important regulatory protein in C. jejuni. Mutation of the cj1169c-cj1170c rendered C. jejuni less tolerant to atmospheric oxygen and reduced Campylobacter colonization and transmission in chickens.ConclusionsThese findings indicate that Ery treatment elicits a range of changes in C. jejuni transcriptome and affects the expression of genes important for in vitro and in vivo adaptation. Up-regulation of motility and down-regulation of energy metabolism likely facilitate Campylobacter to survive during Ery treatment. These findings provide new insight into Campylobacter adaptive response to antibiotic treatment and may help to understand the mechanisms underlying antibiotic resistance development.

Retrospective analysis of genome sequences revealed the wide dissemination of optrA in Gram-positive bacteria.

Sir, Oxazolidinones, including linezolid and tedizolid, are highly effective against infections of enterococci, staphylococci and streptococci, which are clinically important Gram-positive pathogens. cfr is the first gene that is transferable and causes resistance to oxazolidinones; cfr also mediates resistance to phenicols, lincosamides, pleuromutilins and streptogramin A. Recently, Wang et al. first reported a novel transferable resistance gene, optrA, which confers resistance to oxazolidinones and phenicols in enterococcal isolates in China. Shortly after, the optrA gene was detected in clinical enterococcal isolates in Italy and in Staphylococcus sciuri in China. WGS is now routine and thousands of bacterial genomes are available in public databases. To investigate the prevalence of optrA in the sequenced genomes, we retrieved the optrA sequence and traced this gene in the GenBank database (Table S1, available as Supplementary data at JAC Online). We found that optrA presented in two enterococcal isolates from human blood in the USA, two environmental samples from swine manure metagenome in China and six Streptococcus suis isolates from healthy pigs in China. These results are the first reported presence of optrA in clinical enterococci in the USA, and in environmental microorganisms and streptococci in China. optrA genes in those isolates showed 99% nucleotide identity with the original optrA from Enterococcus faecalis pE349. Comparison of the OptrA amino acid sequences revealed seven variants with alterations at nine positions (Table S1). Further analysis showed that the genetic context of optrA in two US isolates, especially that from E. faecalis 599, was similar to that in the chromosome of E. faecalis E016 though seemingly lacking Tn558 and the Derm(A)-like segment, and distinct from that in the plasmid of E. faecalis pE349 and S. sciuri pWo28-3 (Figure 1). The genetic contexts of optrA in two swine manure samples, IN-7 and IN-8 (Figure 1), were similar to those in plasmid pFX13, i.e. flanked by IS1216E, with the Derm(A)-like gene located upstream instead of downstream of optrA. We also found that optrA coexisted with tet(O/W/32/O) and tet(L) in sample IN-7 and with lsa(A) and tet(O/W/32/O) in sample IN-8 (Figure 1). The genetic composition of the mosaic segments is unique and no similar structure was found in known mobile genetic elements. We found that six S. suis isolates from healthy pigs in 2011 also harbour optrA (Table S1). This is surprising as oxazolidinones, often last-resort drugs in clinical treatment, have not been proved to be used in livestock and optrA has not been reported in streptococci. All optrA-carrying segments except strain YS39 were flanked downstream by IS1216E (Figure 1). As the genetic contexts of YS35 and YS39 were not intact due to the over-truncated contigs, we only discuss the other four isolates. In isolate YS57, the 8.1 kb optrA contig was comprised of optrA, an upstream araC and downstream an Derm(A)-like gene, an S-adenosylmethionine (SAM)-dependent methyltransferase (met) gene and two hypothetical genes. The optrA gene block was flanked by IS1216E at both ends in the same orientation, which was found only in plasmids before. The optrA-carrying IS1216E fragment was inserted between SNF2 and agg, two conserved genes of the 89K pathogenicity island (PAI), which presented in a strain of human S. suis outbreaks in China in 2005. However, the other conserved genes of the 89K PAI were split into other contigs due to incomplete genome assembly. Significantly, the 89K PAI belongs to the ICESa2603 family of integrative conjugative elements, which is widely distributed in and demonstrates horizontal transfer between streptococci and enterococci. Figure S1 shows the schematic genetic diagram of the optrA-carrying contig of YS57 with Streptococcus agalactiae ICESa2603 and S. suis 89K. On contigs of YS21, YS49 and YS50, the 7.4 kb optrA fragment contains optrA, an upstream truncated araC, a nickase gene and three hypothetical genes. Upstream of optrA, the fragment was flanked by a truncated IS1272-like element not fully sequenced (Figure 1). Downstream of optrA, the fragment was flanked by an IS3L-like element and IS1216E. The optrA fragment was integrated into a larger prophage by replacing the Mega-like element of Streptococcus pyogenes Um46.1 and the cadA/C-tet(W) fragment of S. suis USsUD.1 (Figure S1). The Um46.1 prophage family, which was originally found integrated in the 30-terminal part of rum loci in S. pyogenes and thereafter in S. agalactiae and S. suis, is transferable to other streptococci. These results suggest the important role of IS1216E in chromosomal

Rising fluoroquinolone resistance in Campylobacter isolated from feedlot cattle in the United States

Antibiotic resistance, particularly to fluoroquinolones and macrolides, in the major foodborne pathogen Campylobacter is considered a serious threat to public health. Although ruminant animals serve as a significant reservoir for Campylobacter, limited information is available on antibiotic-resistant Campylobacter of bovine origin. Here, we analyzed the antimicrobial susceptibilities of 320 C. jejuni and 115 C. coli isolates obtained from feedlot cattle farms in multiple states in the U.S. The results indicate that fluoroquinolone resistance reached to 35.4% in C. jejuni and 74.4% in C. coli, which are significantly higher than those previously reported in the U.S. While all fluoroquinolone resistant (FQR) C. coli isolates examined in this study harbored the single Thr-86-Ile mutation in GyrA, FQRC. jejuni isolates had other mutations in GyrA in addition to the Thr-86-Ile change. Notably, most of the analyzed FQRC. coli isolates had similar PFGE (pulsed field gel electrophoresis) patterns and the same MLST (multilocus sequence typing) sequence type (ST-1068) regardless of their geographic sources and time of isolation, while the analyzed C. jejuni isolates were genetically diverse, suggesting that clonal expansion is involved in dissemination of FQRC. coli but not C. jejuni. These findings reveal the rising prevalence of FQRCampylobacter in the U.S. and provide novel information on the epidemiology of antibiotic-resistant Campylobacter in the ruminant reservoir.

Emergence of a plasmid-borne multidrug resistance gene cfr(C) in foodborne pathogen Campylobacter

Objectives To identify and characterize a novel cfr variant that recently emerged and confers multidrug resistance in Campylobacter , a major foodborne pathogen. Methods WGS was initially used to identify the cfr (C) gene in Campylobacter isolates and its function was further verified by cloning into an antibiotic-susceptible Campylobacter jejuni strain. Distribution of cfr (C) in various Campylobacter isolates was determined by PCR analysis. Genotyping of cfr (C)-positive strains was done by PFGE and MLST. Results The cfr (C) gene is predicted to encode a protein that shares 55.1% and 54.9% identity with Cfr and Cfr(B), respectively. cfr (C) was located on a conjugative plasmid of ∼48 kb. Cloning of cfr (C) into C. jejuni NCTC 11168 and conjugative transfer of the cfr (C)-containing plasmid confirmed its role in conferring resistance to phenicols, lincosamides, pleuromutilins and oxazolidinones, and resulted in an 8-256-fold increase in their MICs in both C. jejuni and Campylobacter coli . The cfr (C) gene was detected in multiple C. coli (34 of 344; 10%) isolates derived from different cattle farms in different states, and molecular typing of the cfr (C)-positive C. coli isolates revealed its spread mainly via clonal expansion. Conclusions These results identify cfr (C) as a new multidrug resistance mechanism in Campylobacter and suggest the potential transmission of this mechanism via the foodborne route, warranting enhanced efforts to monitor its spread in Campylobacter and other foodborne pathogens.

Identification and functional analysis of two toxin-antitoxin systems in Campylobacter jejuni.

Toxin–antitoxin (TA) systems are widely distributed in bacteria and play an important role in maintaining plasmid stability. The leading foodborne pathogen, Campylobacter jejuni, can carry multiple plasmids associated with antibiotic resistance or virulence. Previously a virulence plasmid named pVir was identified in C. jejuni 81‐176 and IA3902, but determining the role of pVir in pathogenesis has been hampered because the plasmid cannot be cured. In this study, we report the identification of two TA systems that are located on the pVir plasmid in 81‐176 and IA3902, respectively. The virA (proteic antitoxin)/virT (proteic toxin) pair in IA3902 belongs to a Type II TA system, while the cjrA (RNA antitoxin)/cjpT (proteic toxin) pair in 81‐176 belongs to a Type I TA system. Notably, cjrA (antitoxin) represents the first noncoding small RNA demonstrated to play a functional role in Campylobacter physiology to date. By inactivating the TA systems, pVir was readily cured from Campylobacter, indicating their functionality in Campylobacter. Using pVir‐cured IA3902, we demonstrated that pVir is not required for abortion induction in the guinea pig model. These findings establish the key role of the TA systems in maintaining plasmid stability and provide a means to evaluate the function of pVir in Campylobacter pathobiology.

A single nucleotide change in mutY increases the emergence of antibiotic-resistant Campylobacter jejuni mutants

OBJECTIVES Mutator strains play an important role in the emergence of antibiotic-resistant bacteria. Campylobacter jejuni is a leading cause of foodborne illnesses worldwide and is increasingly resistant to clinically important antibiotics. The objective of this study was to identify the genetic basis that contributes to a mutator phenotype in Campylobacter and determine the role of this phenotype in the development of antibiotic resistance. METHODS A C. jejuni isolate (named CMT) showing a mutator phenotype was subjected to WGS analysis. Comparative genomics, site-specific reversion and mutation, and gene knockout were conducted to prove the mutator effect was caused by a single nucleotide change in the mutY gene of C. jejuni. RESULTS The C. jejuni CMT isolate showed ∼ 100-fold higher mutation frequency to ciprofloxacin than the WT strain. Under selection by ciprofloxacin, fluoroquinolone-resistant mutants emerged readily from the CMT isolate. WGS identified a single nucleotide change (G595 → T) in the mutY gene of the CMT isolate. Further experiments using defined mutant constructs proved its specific role in elevating mutation frequencies. The mutY point mutation also led to an ∼ 700-fold increase in the emergence of ampicillin-resistant mutants, indicating its broader impact on antibiotic resistance. Structural modelling suggested the G595 → T mutation probably affects the catalytic domain of MutY and consequently abolishes the anti-mutator function of this DNA repair protein. CONCLUSIONS The G595 → T mutation in mutY abolishes its anti-mutator function and confers a mutator phenotype in Campylobacter, promoting the emergence of antibiotic-resistant Campylobacter.

Wide but Variable Distribution of a Hypervirulent Campylobacter jejuni Clone in Beef and Dairy Cattle in the United States

ABSTRACT Campylobacter jejuni clone SA is the major cause of sheep abortion and contributes significantly to foodborne illnesses in the United States. Clone SA is hypervirulent because of its distinct ability to produce systemic infection and its predominant role in clinical sheep abortion. Despite the importance of clone SA, little is known about its distribution and epidemiological features in cattle. Here we describe a prospective study on C. jejuni clone SA prevalence in 35 feedlots in 5 different states in the United States and a retrospective analysis of clone SA in C. jejuni isolates collected by National Animal Health Monitoring System (NAHMS) dairy studies in 2002, 2007, and 2014. In feedlot cattle feces, the overall prevalence of Campylobacter organisms was 72.2%, 82.1% of which were C. jejuni. Clone SA accounted for 5.8% of the total C. jejuni isolates, but its prevalence varied by feedlot and state. Interestingly, starlings on the feedlots harbored C. jejuni in feces, including clone SA, suggesting that these birds may play a role in the transmission of Campylobacter. In dairy cattle, the overall prevalence of clone SA was 7.2%, but a significant decrease in the prevalence was observed from 2002 to 2014. Whole-genome sequence analysis of the dairy clone SA isolates revealed that it was genetically stable over the years and most of the isolates carried the tetracycline resistance gene tet(O) in the chromosome. These findings indicate that clone SA is widely distributed in both beef and dairy cattle and provide new insights into the molecular epidemiology of clone SA in ruminants. IMPORTANCE C. jejuni clone SA is a major cause of small-ruminant abortion and an emerging threat to food safety because of its association with foodborne outbreaks. Cattle appear to serve as a major reservoir for this pathogenic organism, but there is a major gap in our knowledge about the epidemiology of clone SA in beef and dairy cattle. By taking advantage of surveillance studies conducted on a national scale, we found a wide but variable distribution of clone SA in feedlot cattle and dairy cows in the United States. Additionally, the work revealed important genomic features of clone SA isolates from cattle. These findings provide critically needed information for the development of preharvest interventions to control the transmission of this zoonotic pathogen. Control of C. jejuni clone SA will benefit both animal health and public health, as it is a zoonotic pathogen causing disease in both ruminants and humans.

A zero-inflated Poisson model for insertion tolerance analysis of genes based on Tn-seq data

MOTIVATION Transposon insertion sequencing (Tn-seq) is an emerging technology that combines transposon mutagenesis with next-generation sequencing technologies for the identification of genes related to bacterial survival. The resulting data from Tn-seq experiments consist of sequence reads mapped to millions of potential transposon insertion sites and a large portion of insertion sites have zero mapped reads. Novel statistical method for Tn-seq data analysis is needed to infer functions of genes on bacterial growth. RESULTS In this article, we propose a zero-inflated Poisson model for analyzing the Tn-seq data that are high-dimensional and with an excess of zeros. Maximum likelihood estimates of model parameters are obtained using an expectation-maximization (EM) algorithm, and pseudogenes are utilized to construct appropriate statistical tests for the transposon insertion tolerance of normal genes of interest. We propose a multiple testing procedure that categorizes genes into each of the three states, hypo-tolerant, tolerant and hyper-tolerant, while controlling the false discovery rate. We evaluate the proposed method with simulation studies and apply the proposed method to a real Tn-seq data from an experiment that studied the bacterial pathogen, Campylobacter jejuniAvailability and implementation: We provide R code for implementing our proposed method at http://github.com/ffliu/TnSeq A users guide with example data analysis is also available there. CONTACT pliu@iastate.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.