Whole-Genome Sequencing of Gentamicin-Resistant Campylobacter coli Isolated from U.S. Retail Meats Reveals Novel Plasmid-Mediated Aminoglycoside Resistance Genes

ABSTRACTAminoglycoside resistance inCampylobacterhas been routinely monitored in the United States in clinical isolates since 1996 and in retail meats since 2002. Gentamicin resistance first appeared in a single human isolate ofCampylobacter coliin 2000 and in a single chicken meat isolate in 2007, after which it increased rapidly to account for 11.3% of human isolates and 12.5% of retail isolates in 2010. Pulsed-field gel electrophoresis analysis indicated that gentamicin-resistantC. coliisolates from retail meat were clonal. We sequenced the genomes of two strains of this clone using a next-generation sequencing technique in order to investigate the genetic basis for the resistance. The gaps of one strain were closed using optical mapping and Sanger sequencing, and this is the first completed genome ofC. coli. The two genomes are highly similar to each other. A self-transmissible plasmid carrying multiple antibiotic resistance genes was revealed within both genomes, carrying genes encoding resistance to gentamicin, kanamycin, streptomycin, streptothricin, and tetracycline. Bioinformatics analysis and experimental results showed that gentamicin resistance was due to a phosphotransferase gene,aph(2″)-Ig, not described previously. The phylogenetic relationship of this newly emerged clone to otherCampylobacterspp. was determined by whole-genome single nucleotide polymorphisms (SNPs), which showed that it clustered with the other poultry isolates and was separated from isolates from livestock.

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Comparative Genomics of Klebsiella pneumoniae Strains with Different Antibiotic Resistance Profiles

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00052-11 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4267-4276 ◽

Cited By ~ 48

Author(s):

Vinod Kumar ◽

Peng Sun ◽

Jessica Vamathevan ◽

Yong Li ◽

Karen Ingraham ◽

...

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Susceptible Strain ◽

Clinical Isolates ◽

Whole Genome ◽

Content Type ◽

Extended Spectrum

ABSTRACTThere is a global emergence of multidrug-resistant (MDR) strains ofKlebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology ofK. pneumoniaestrains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum β-lactamases (ESBLs), have been extensively studied, only four complete genomes ofK. pneumoniaeare available. To better understand the multidrug resistance factors inK. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum β-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other publishedK. pneumoniaegenomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to otherK. pneumoniaestrains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data onK. pneumoniaestrains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.

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Whole-Genome Sequencing for Detecting Antimicrobial Resistance in Nontyphoidal Salmonella

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01030-16 ◽

2016 ◽

Vol 60 (9) ◽

pp. 5515-5520 ◽

Cited By ~ 148

Author(s):

Patrick F. McDermott ◽

Gregory H. Tyson ◽

Claudine Kabera ◽

Yuansha Chen ◽

Cong Li ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

The United States ◽

Whole Genome ◽

Content Type ◽

Structural Resistance ◽

Retail Meat

ABSTRACTLaboratory-basedin vitroantimicrobial susceptibility testing is the foundation for guiding anti-infective therapy and monitoring antimicrobial resistance trends. We used whole-genome sequencing (WGS) technology to identify known antimicrobial resistance determinants among strains of nontyphoidalSalmonellaand correlated these with susceptibility phenotypes to evaluate the utility of WGS for antimicrobial resistance surveillance. Six hundred fortySalmonellaof 43 different serotypes were selected from among retail meat and human clinical isolates that were tested for susceptibility to 14 antimicrobials using broth microdilution. The MIC for each drug was used to categorize isolates as susceptible or resistant based on Clinical and Laboratory Standards Institute clinical breakpoints or National Antimicrobial Resistance Monitoring System (NARMS) consensus interpretive criteria. Each isolate was subjected to whole-genome shotgun sequencing, and resistance genes were identified from assembled sequences. A total of 65 unique resistance genes, plus mutations in two structural resistance loci, were identified. There were more unique resistance genes (n =59) in the 104 human isolates than in the 536 retail meat isolates (n =36). Overall, resistance genotypes and phenotypes correlated in 99.0% of cases. Correlations approached 100% for most classes of antibiotics but were lower for aminoglycosides and beta-lactams. We report the first finding of extended-spectrum β-lactamases (ESBLs) (blaCTX-M1andblaSHV2a) in retail meat isolates ofSalmonellain the United States. Whole-genome sequencing is an effective tool for predicting antibiotic resistance in nontyphoidalSalmonella, although the use of more appropriate surveillance breakpoints and increased knowledge of new resistance alleles will further improve correlations.

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High Prevalence and Predominance of the aph(2″)-If Gene Conferring Aminoglycoside Resistance in Campylobacter

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00112-17 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 19

Author(s):

Hong Yao ◽

Dejun Liu ◽

Yang Wang ◽

Qijing Zhang ◽

Zhangqi Shen

Keyword(s):

Horizontal Transmission ◽

Foodborne Pathogen ◽

Systemic Infection ◽

Campylobacter Coli ◽

Aminoglycoside Resistance ◽

Content Type ◽

High Prevalence ◽

Gentamicin Resistance ◽

Long Range Pcr ◽

High Level

ABSTRACT Campylobacter is a major foodborne pathogen, and previous studies revealed that Campylobacter isolates from food-producing animals are increasingly resistant to gentamicin in China. The molecular epidemiology and genetic mechanisms responsible for gentamicin resistance in China have not been well understood. In this study, 607 Campylobacter isolates of chicken and swine origins collected in 2014 were analyzed, revealing that 15.6% (25/160) of the Campylobacter jejuni isolates and 79.9% (357/447) of the Campylobacter coli isolates were resistant to gentamicin. PCR detection of the gentamicin resistance genes indicated that aph(2″)-If was more prevalent than the previously identified aacA/aphD gene and has become the dominant gentamicin resistance determinant in Campylobacter. Transformation and whole-genome sequencing as well as long-range PCR discovered that aph(2″)-If was located on a chromosomal segment inserted between two conserved genes, Cj0299 and panB. Cloning of aph(2″)-If into gentamicin-susceptible C. jejuni NCTC 11168 confirmed its function in conferring high-level resistance to gentamicin and kanamycin. Molecular typing by pulsed-field gel electrophoresis suggested that both regional expansion of a particular clone and horizontal transmission were involved in the dissemination of the aph(2″)-If gene in Campylobacter. To our knowledge, this is the first report describing the high prevalence of a chromosomally encoded aph(2″)-If gene in Campylobacter. The high prevalence and predominance of this gene might be driven by the use of aminoglycoside antibiotics in food animal production in China and potentially compromise the usefulness of gentamicin as a therapeutic agent for Campylobacter-associated systemic infection.

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Phylogeographical Analyses and Antibiotic Resistance Genes of Acinetobacter johnsonii Highlight Its Clinical Relevance

mSphere ◽

10.1128/msphere.00581-20 ◽

2020 ◽

Vol 5 (4) ◽

Author(s):

Santiago Castillo-Ramírez ◽

Valeria Mateo-Estrada ◽

Gerardo Gonzalez-Rocha ◽

Andrés Opazo-Capurro

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Clinical Importance ◽

Antibiotic Resistance Genes ◽

Genomic Diversity ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Content Type ◽

Acinetobacter Johnsonii ◽

Well Differentiated

ABSTRACT Acinetobacter johnsonii has been severely understudied and its population structure and the presence of antibiotic resistance genes (ARGs) are very much uncertain. Our phylogeographical analysis shows that intercontinental transmission has occurred frequently and that different lineages are circulating within single countries; notably, clinical and nonclinical strains are not well differentiated from one another. Importantly, in this species recombination is a significant source of single nucleotide polymorphisms. Furthermore, our results show this species could be an important reservoir of ARGs since it has a significant amount of ARGs, and many of them show signals of horizontal gene transfer. Thus, this study clearly points out the clinical importance of A. johnsonii and the urgent need to better appreciate its genomic diversity.

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Cotransfer of Antibiotic Resistance Genes and a hylEfm-Containing Virulence Plasmid in Enterococcus faecium

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00242-09 ◽

2009 ◽

Vol 53 (10) ◽

pp. 4240-4246 ◽

Cited By ~ 50

Author(s):

Cesar A. Arias ◽

Diana Panesso ◽

Kavindra V. Singh ◽

Louis B. Rice ◽

Barbara E. Murray

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Enterococcus Faecium ◽

Antibiotic Resistance Genes ◽

The United States ◽

Virulence Plasmid ◽

Gene Encoding ◽

Healthy Human ◽

Gentamicin Resistance ◽

High Level

ABSTRACT The hylEfm gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hylEfm -containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hylEfm gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hylEfm , whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hylEfm -positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hylEfm plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

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Whole-Genome Sequencing Analysis Accurately Predicts Antimicrobial Resistance Phenotypes in Campylobacter spp.

Applied and Environmental Microbiology ◽

10.1128/aem.02873-15 ◽

2015 ◽

Vol 82 (2) ◽

pp. 459-466 ◽

Cited By ~ 132

Author(s):

S. Zhao ◽

G. H. Tyson ◽

Y. Chen ◽

C. Li ◽

S. Mukherjee ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Resistance Genes ◽

Antimicrobial Agents ◽

The United States ◽

23S Rrna ◽

Whole Genome ◽

Sequencing Analysis ◽

Content Type

ABSTRACTThe objectives of this study were to identify antimicrobial resistance genotypes forCampylobacterand to evaluate the correlation between resistance phenotypes and genotypes usingin vitroantimicrobial susceptibility testing and whole-genome sequencing (WGS). A total of 114Campylobacterspecies isolates (82C. coliand 32C. jejuni) obtained from 2000 to 2013 from humans, retail meats, and cecal samples from food production animals in the United States as part of the National Antimicrobial Resistance Monitoring System were selected for study. Resistance phenotypes were determined using broth microdilution of nine antimicrobials. Genomic DNA was sequenced using the Illumina MiSeq platform, and resistance genotypes were identified using assembled WGS sequences through blastx analysis. Eighteen resistance genes, includingtet(O),blaOXA-61,catA,lnu(C),aph(2″)-Ib,aph(2″)-Ic,aph(2′)-If,aph(2″)-Ig,aph(2″)-Ih,aac(6′)-Ie-aph(2″)-Ia,aac(6′)-Ie-aph(2″)-If,aac(6′)-Im,aadE,sat4,ant(6′),aad9,aph(3′)-Ic, andaph(3′)-IIIa, and mutations in two housekeeping genes (gyrAand 23S rRNA) were identified. There was a high degree of correlation between phenotypic resistance to a given drug and the presence of one or more corresponding resistance genes. Phenotypic and genotypic correlation was 100% for tetracycline, ciprofloxacin/nalidixic acid, and erythromycin, and correlations ranged from 95.4% to 98.7% for gentamicin, azithromycin, clindamycin, and telithromycin. All isolates were susceptible to florfenicol, and no genes associated with florfenicol resistance were detected. There was a strong correlation (99.2%) between resistance genotypes and phenotypes, suggesting that WGS is a reliable indicator of resistance to the nine antimicrobial agents assayed in this study. WGS has the potential to be a powerful tool for antimicrobial resistance surveillance programs.

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Novel Aminoglycoside Resistance Transposons and Transposon-Derived Circular Forms Detected in Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02143-15 ◽

2016 ◽

Vol 60 (3) ◽

pp. 1801-1818 ◽

Cited By ~ 29

Author(s):

Nabil Karah ◽

Chinmay Kumar Dwibedi ◽

Karin Sjöström ◽

Petra Edquist ◽

Anders Johansson ◽

...

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Point Mutations ◽

Antibiotic Resistance Genes ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Aminoglycoside Resistance ◽

Content Type ◽

Carbapenem Resistant

Acinetobacter baumanniihas emerged as an important opportunistic pathogen equipped with a growing number of antibiotic resistance genes. Our study investigated the molecular epidemiology and antibiotic resistance features of 28 consecutive carbapenem-resistant clinical isolates ofA. baumanniicollected throughout Sweden in 2012 and 2013. The isolates mainly belonged to clonal complexes (CCs) with an extensive international distribution, such as CC2 (n= 16) and CC25 (n= 7). Resistance to carbapenems was related toblaOXA-23(20 isolates),blaOXA-24/40-like(6 isolates),blaOXA-467(1 isolate), and ISAba1-blaOXA-69(1 isolate). Ceftazidime resistance was associated withblaPER-7in the CC25 isolates. Two classical point mutations were responsible for resistance to quinolones in all the isolates. Isolates with high levels of resistance to aminoglycosides carried the 16S rRNA methylasearmAgene. The isolates also carried a variety of genes encoding aminoglycoside-modifying enzymes. Several novel structures involved in aminoglycoside resistance were identified, including Tn6279, ΔTn6279, Ab-ST3-aadB, and different assemblies of Tn6020and TnaphA6. Importantly, a number of circular forms related to the IS26or ISAba125composite transposons were detected. The frequent occurrence of these circular forms in the populations of several isolates indicates a potential role of these circular forms in the dissemination of antibiotic resistance genes.

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Whole-Genome Sequence of ablaOXA-48-Harboring Raoultella ornithinolytica Clinical Isolate from Lebanon

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02773-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2548-2550 ◽

Cited By ~ 10

Author(s):

Charbel Al-Bayssari ◽

Abiola Olumuyiwa Olaitan ◽

Thongpan Leangapichart ◽

Liliane Okdah ◽

Fouad Dabboussi ◽

...

Keyword(s):

Antibiotic Resistance ◽

Clinical Isolate ◽

Genome Sequence ◽

Resistance Genes ◽

Molecular Mechanisms ◽

Antibiotic Resistance Genes ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Genetic Organization

ABSTRACTWe analyzed the whole-genome sequence of ablaOXA-48-harboringRaoultella ornithinolyticaclinical isolate from a patient in Lebanon. The size of theRaoultella ornithinolyticaCMUL058 genome was 5,622,862 bp, with a G+C content of 55.7%. We deciphered all the molecular mechanisms of antibiotic resistance, and we compared our genome to other availableR. ornithinolyticagenomes in GenBank. The resistome consisted of 9 antibiotic resistance genes, including a plasmidicblaOXA-48gene whose genetic organization is also described.

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Genomic Analysis of the Multidrug-Resistant Acinetobacter baumannii Strain MDR-ZJ06 Widely Spread in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01134-10 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4506-4512 ◽

Cited By ~ 77

Author(s):

Hua Zhou ◽

Tongwu Zhang ◽

Dongliang Yu ◽

Borui Pi ◽

Qing Yang ◽

...

Keyword(s):

Antibiotic Resistance ◽

Acinetobacter Baumannii ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Genomic Analysis ◽

Multidrug Resistant ◽

Whole Genome Sequence ◽

Whole Genome ◽

Content Type ◽

Resistance Island

ABSTRACTWe previously reported that the multidrug-resistant (MDR)Acinetobacter baumanniistrain MDR-ZJ06, belonging to European clone II, was widely spread in China. In this study, we report the whole-genome sequence of this clinically important strain. A 38.6-kb AbaR-type genomic resistance island (AbaR22) was identified in MDR-ZJ06. AbaR22 has a structure similar to those of the resistance islands found inA. baumanniistrains AYE and AB0057, but it contained only a few antibiotic resistance genes. The region of resistant gene accumulation as previously described was not found in AbaR22. In the chromosome of the strain MDR-ZJ06, we identified the geneblaoxa-23in a composite transposon (Tn2009). Tn2009shared the backbone with otherA. baumanniitransponsons that harborblaoxa-23, but it was bracketed by two ISAba1elements which were transcribed in the same orientation. MDR-ZJ06 also expressed thearmAgene on its plasmid pZJ06, and this gene has the same genetic environment as thearmAgene of theEnterobacteriaceae. These results suggest variability of resistance acquisition even in closely relatedA. baumanniistrains.

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Comparative Genomics Reveals a Well-Conserved Intrinsic Resistome in the Emerging Multidrug-Resistant Pathogen Cupriavidus gilardii

mSphere ◽

10.1128/msphere.00631-19 ◽

2019 ◽

Vol 4 (5) ◽

Cited By ~ 2

Author(s):

Cristian Ruiz ◽

Ashley McCarley ◽

Manuel Luis Espejo ◽

Kerry K. Cooper ◽

Dana E. Harmon

Keyword(s):

Antibiotic Resistance ◽

Comparative Genomics ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistance Mechanisms ◽

Antibiotics Resistance ◽

Whole Genome ◽

Intrinsic Resistance ◽

Content Type

ABSTRACT The Gram-negative bacterium Cupriavidus gilardii is an emerging multidrug-resistant pathogen found in many environments. However, little is known about this species or its antibiotic resistance mechanisms. We used biochemical tests, antibiotic susceptibility experiments, and whole-genome sequencing to characterize an environmental C. gilardii isolate. Like clinical isolates, this isolate was resistant to meropenem, gentamicin, and other antibiotics. Resistance to these antibiotics appeared to be related to the large number of intrinsic antibiotic resistance genes found in this isolate. As determined by comparative genomics, this resistome was also well conserved in the only two other C. gilardii strains sequenced to date. The intrinsic resistome of C. gilardii did not include the colistin resistance gene mcr-5, which was in a transposon present only in one strain. The intrinsic resistome of C. gilardii was comprised of (i) many multidrug efflux pumps, such as a homolog of the Pseudomonas aeruginosa MexAB-OprM pump that may be involved in resistance to meropenem, other β-lactams, and aminoglycosides; (ii) a novel β-lactamase (OXA-837) that decreases susceptibility to ampicillin but not to other β-lactams tested; (iii) a new aminoglycoside 3-N-acetyltransferase [AAC(3)-IVb, AacC10] that decreases susceptibility to gentamicin and tobramycin; and (iv) a novel partially conserved aminoglycoside 3ʺ-adenylyltransferase [ANT(3ʺ)-Ib, AadA32] that decreases susceptibility to spectinomycin and streptomycin. These findings provide the first mechanistic insight into the intrinsic resistance of C. gilardii to multiple antibiotics and its ability to become resistant to an increasing number of drugs during therapy. IMPORTANCE Cupriavidus gilardii is a bacterium that is gaining increasing attention both as an infectious agent and because of its potential use in the detoxification of toxic compounds and other biotechnological applications. In recent years, however, there has been an increasing number of reported infections, some of them fatal, caused by C. gilardii. These infections are hard to treat because this bacterium is naturally resistant to many antibiotics, including last-resort antibiotics, such as carbapenems. Moreover, this bacterium often becomes resistant to additional antibiotics during therapy. However, little is known about C. gilardii and its antibiotic resistance mechanisms. The significance of our research is in providing, for the first time, whole-genome information about the natural antibiotic resistance genes found in this bacterium and their conservation among different C. gilardii strains. This information may provide new insights into the appropriate use of antibiotics in combating infections caused by this emerging pathogen.

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