Complete Nucleotide Sequence of Two Multidrug-Resistant IncR Plasmids from Klebsiella pneumoniae

ABSTRACTWe report here the complete nucleotide sequence of two IncR replicons encoding multidrug resistance determinants, including β-lactam (blaDHA-1,blaSHV-12), aminoglycoside (aphA1,strA,strB), and fluoroquinolone (qnrB4,aac6′-1b-cr) resistance genes. The plasmids have backbones that are similar to each other, including the replication and stability systems, and contain a wide variety of transposable elements carrying known antibiotic resistance genes. This study confirms the increasing clinical importance of IncR replicons as resistance gene carriers.

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Functional and genetic characterization of an incF-type multidrug resistance plasmid isolated from fresh spinach

10.1101/358887 ◽

2018 ◽

Author(s):

Adeyinka O. Ajayi ◽

Benjamin J. Perry ◽

Christopher K. Yost

Keyword(s):

Antibiotic Resistance ◽

Multidrug Resistance ◽

Food Safety ◽

Sequence Analysis ◽

Dna Sequence ◽

Resistance Genes ◽

Plasmid Dna ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Leafy Greens

AbstractThe presence of antibiotic-resistant bacteria and clinically-relevant antibiotic resistance genes within raw foods is an on-going food safety concern. It is particularly important to be aware of the microbial quality of fresh produce because foods such as leafy greens including lettuce and spinach are minimally processed and often consumed raw therefore they often lack a microbial inactivation step. This study characterizes the genetic and functional aspects of a mobile, multidrug resistance plasmid, pLGP4, isolated from fresh spinach bought from a farmers’ market. pLGP4 was isolated using a bacterial conjugation approach. The functional characteristics of the plasmid were determined using multidrug resistance profiling and plasmid stability assays. pLGP4 was resistant to six of the eight antibiotics tested and included ciprofloxacin and meropenem. The plasmid was stably maintained within host strains in the absence of an antibiotic selection. The plasmid DNA was sequenced using an Illumina MiSeq high throughput sequencing approach and assembled into contigs using SPAdes. PCR mapping and Sanger DNA sequencing of PCR amplicons was used to complete the plasmid DNA sequence. Comparative sequence analysis determined that the plasmid was similar to plasmids that have been frequently associated with multidrug resistant clinical isolates of Klebsiella spp. DNA sequence analysis showed pLGP4 harboured qnrB1 and several other antibiotic resistance genes including three β-lactamases: blaTEM-1, blaCTX-M-15 and blaOXA-1. The detection of a multidrug-resistant, clinically-relevant plasmid on fresh spinach emphasizes the importance for vegetable producers to implement evidence-based food safety approaches into their production practises to ensure the food safety of leafy greens.

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Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

10.1101/678573 ◽

2019 ◽

Cited By ~ 1

Author(s):

Marinelle Rodrigues ◽

Sara W. McBride ◽

Karthik Hullahalli ◽

Kelli L. Palmer ◽

Breck A. Duerkop

Keyword(s):

Antibiotic Resistance ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Bacterial Infections ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Conjugative Plasmid ◽

Antibiotic Resistant ◽

Resistance Determinants

AbstractThe innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiontEnterococcus faecalisis associated with HAIs and some strains are MDR. Therefore, novel strategies to controlE. faecalispopulations are needed. We previously characterized anE. faecalisType II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers toE. faecalisfor the selective removal of antibiotic resistance genes. Usingin vitrocompetition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistantE. faecalisby several orders of magnitude. Finally, we show thatE. faecalisdonor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinantsin vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.ImportanceCRISPR-Cas nucleic acid targeting systems hold promise for the amelioration of multidrug-resistant enterococci, yet the utility of such tools in the context of the intestinal environment where enterococci reside is understudied. We describe the development of a CRISPR-Cas antimicrobial, deployed on a conjugative plasmid, for the targeted removal of antibiotic resistance genes from intestinalEnterococcus faecalis. We demonstrate that CRISPR-Cas targeting reduces antibiotic resistance ofE. faecalisby several orders of magnitude in the intestine. Although barriers exist that influence the penetrance of the conjugative CRISPR-Cas antimicrobial among target recipientE. faecaliscells, the removal of antibiotic resistance genes inE. faecalisupon uptake of the CRISPR-Cas antimicrobial is absolute. In addition, cells that obtain the CRISPR-Cas antimicrobial are immunized against the acquisition of new antibiotic resistance genes. This study suggests a potential path toward plasmid based CRISPR-Cas therapies in the intestine.

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Complete Sequence of the FII Plasmid p42-2, CarryingblaCTX-M-55,oqxAB,fosA3, andfloRfrom Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00475-16 ◽

2016 ◽

Vol 60 (7) ◽

pp. 4336-4338 ◽

Cited By ~ 11

Author(s):

Qiu E. Yang ◽

Timothy Rutland Walsh ◽

Bao Tao Liu ◽

Meng Ting Zou ◽

Hui Deng ◽

...

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Multidrug Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Complete Sequence ◽

Content Type ◽

Resistance Determinants

ABSTRACTWe sequenced a novel conjugative multidrug resistance IncF plasmid, p42-2, isolated fromEscherichia colistrain 42-2, previously identified in China. p42-2 is 106,886 bp long, composed of a typical IncFII-type backbone (∼54 kb) and one distinct acquired DNA region spanning ∼53 kb, harboring 12 antibiotic resistance genes [blaCTX-M-55,oqxA,oqxB,fosA3,floR,tetA(A),tetA(R),strA,strB,sul2,aph(3′)-II, and ΔblaTEM-1]. The spread of these multidrug resistance determinants on the same plasmid is of great concern and, because of coresistance to antibiotics from different classes, is therapeutically challenging.

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Environmental Spread of New Delhi Metallo-β-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh

Applied and Environmental Microbiology ◽

10.1128/aem.00793-17 ◽

2017 ◽

Vol 83 (15) ◽

Cited By ~ 32

Author(s):

Mohammad Aminul Islam ◽

Moydul Islam ◽

Rashedul Hasan ◽

M. Iqbal Hossain ◽

Ashikun Nabi ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Tap Water ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

New Delhi ◽

Multidrug Resistant Bacteria ◽

Content Type ◽

Wastewater Samples

ABSTRACT Resistance to carbapenem antibiotics through the production of New Delhi metallo-β-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the bla NDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among bla NDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including bla CTX-M-1 (80%), bla CTX-M-15 (63%), bla TEM (76%), bla SHV (33%), bla CMY-2 (16%), bla OXA-48-like (2%), bla OXA-1 (53%), and bla OXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying bla NDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community. IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the bla NDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

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CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

mSphere ◽

10.1128/msphere.00064-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 43

Author(s):

Valerie J. Price ◽

Wenwen Huo ◽

Ardalan Sharifi ◽

Kelli L. Palmer

Keyword(s):

Antibiotic Resistance ◽

High Risk ◽

Enterococcus Faecalis ◽

Resistance Genes ◽

Treatment Options ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Content Type ◽

New Genes ◽

Restriction Modification

ABSTRACT Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics. Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis. IMPORTANCE Enterococcus faecalis is a bacterium that normally inhabits the gastrointestinal tracts of humans and other animals. Although these bacteria are members of our native gut flora, they can cause life-threatening infections in hospitalized patients. Antibiotic resistance genes appear to be readily shared among high-risk E. faecalis strains, and multidrug resistance in these bacteria limits treatment options for infections. Here, we find that CRISPR-Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, significantly impact the spread of antibiotic resistance genes in E. faecalis populations. The loss of these systems in high-risk E. faecalis suggests that they are immunocompromised, a tradeoff that allows them to readily acquire new genes and adapt to new antibiotics.

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Antimicrobial Resistance and CRISPR Typing Among Salmonella Isolates From Poultry Farms in China

Frontiers in Microbiology ◽

10.3389/fmicb.2021.730046 ◽

2021 ◽

Vol 12 ◽

Author(s):

Cui Li ◽

Yulong Wang ◽

Yufeng Gao ◽

Chao Li ◽

Boheng Ma ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Antibiotic Resistance Gene ◽

Antibiotic Resistance Genes ◽

Direct Repeat ◽

Epidemiological Data ◽

Multidrug Resistant ◽

Typing Method ◽

Crispr Locus ◽

Research Areas

Although knowledge of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been applied in many research areas, comprehensive studies of this system in Salmonella, particularly in analysis of antibiotic resistance, have not been reported. In this work, 75 Salmonella isolates obtained from broilers or broilers products were characterized to determine their antimicrobial susceptibilities, antibiotic resistance gene profiles, and CRISPR array diversities, and genotyping was explored. In total, 80.00% (60/75) of the strains were multidrug resistant, and the main pattern observed in the isolates was CN-AZM-AMP-AMC-CAZ-CIP-ATM-TE-SXT-FOS-C. The resistance genes of streptomycin (aadA), phenicol (floR-like and catB3-like), β-lactams (blaTEM, blaOXA, and blaCTX), tetracycline [tet(A)-like], and sulfonamides (sul1 and sul2) appeared at higher frequencies among the corresponding resistant isolates. Subsequently, we analyzed the CRISPR arrays and found 517 unique spacer sequences and 31 unique direct repeat sequences. Based on the CRISPR spacer sequences, we developed a novel typing method, CRISPR locus three spacer sequences typing (CLTSST), to help identify sources of Salmonella outbreaks especially correlated with epidemiological data. Compared with multi-locus sequence typing (MLST), conventional CRISPR typing (CCT), and CRISPR locus spacer pair typing (CLSPT), discrimination using CLTSST was weaker than that using CCT but stronger than that using MLST and CLSPT. In addition, we also found that there were no close correlations between CRISPR loci and antibiotics but had close correlations between CRISPR loci and antibiotic resistance genes in Salmonella isolates.

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Accumulation and expression of multiple antibiotic resistance genes in Arcobacter cryaerophilus that thrives in sewage

PeerJ ◽

10.7717/peerj.3269 ◽

2017 ◽

Vol 5 ◽

pp. e3269 ◽

Cited By ~ 15

Author(s):

Jess A. Millar ◽

Rahul Raghavan

Keyword(s):

Antibiotic Resistance ◽

Wastewater Treatment ◽

Resistance Genes ◽

Treatment Plant ◽

Antibiotic Resistance Genes ◽

Environmental Water ◽

Multidrug Resistant ◽

Municipal Sewage ◽

Animal Origin ◽

Resistant Bacteria

We explored the bacterial diversity of untreated sewage influent samples of a wastewater treatment plant in Tucson, AZ and discovered that Arcobacter cryaerophilus, an emerging human pathogen of animal origin, was the most dominant bacterium. The other highly prevalent bacteria were members of the phyla Bacteroidetes and Firmicutes, which are major constituents of human gut microbiome, indicating that bacteria of human and animal origin intermingle in sewage. By assembling a near-complete genome of A. cryaerophilus, we show that the bacterium has accumulated a large number of antibiotic resistance genes (ARGs) probably enabling it to thrive in the wastewater. We also determined that a majority of ARGs was being expressed in sewage, suggestive of trace levels of antibiotics or other stresses that could act as a selective force that amplifies multidrug resistant bacteria in municipal sewage. Because all bacteria are not eliminated even after several rounds of wastewater treatment, ARGs in sewage could affect public health due to their potential to contaminate environmental water.

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Molecular detection of antibiotic resistance genes in multidrug-resistant Staphylococcus aureus isolates from dog dental plaque

BULGARIAN JOURNAL OF VETERINARY MEDICINE ◽

10.15547/bjvm.2099 ◽

2019 ◽

Vol 22 (4) ◽

pp. 419-427

Author(s):

S. Nouri Gharajalar ◽

M. Onsori

Keyword(s):

Staphylococcus Aureus ◽

Antibiotic Resistance ◽

Multiplex Pcr ◽

Resistance Genes ◽

Dental Plaque ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Diffusion Method ◽

Pcr Assay ◽

Multiplex Pcr Assay

Multidrug resistant Staphylococcus aureus strains are a major health care problem both in humans and animals. In this work we described three multiplex PCR assays for detection of clinically relevant antibiotic resistance genes in S. aureus isolated from dog dental plaques. Thirty dental plaque samples were collected; then cultural, biochemical and molecular tests performed for isolation and identification of S. aureus from samples. The antibiotic susceptibility of the isolates were checked by Kirby Bauer disc diffusion method and the prevalence of antibiotic resistance genes determined using multiplex PCR assay. As a result S. aureus was isolated from 18 dog plaque samples. Fifteen of these isolates were resistant to penicillin. The mecA gene was more prevalent than blaZ among penicillin-resistant bacteria. Ten of the isolates were resistant to tetracycline. The percentage of tetM was higher than tetK among them. Also, 10 of the isolates were resistant to cefazolin among them bla TEM detected in higher rate than blaSHV and blaOXA-1. Hence multiplex PCR assay is a suitable method for detection of antibiotic resistance patterns of S. aureus isolates.

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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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Characterization of antibiotic resistance genes in the species of the rumen microbiota

Nature Communications ◽

10.1038/s41467-019-13118-0 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 9

Author(s):

Yasmin Neves Vieira Sabino ◽

Mateus Ferreira Santana ◽

Linda Boniface Oyama ◽

Fernanda Godoy Santos ◽

Ana Júlia Silva Moreira ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Animal Health ◽

Antibiotic Resistance Genes ◽

Tetracycline Resistance ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Ruminal Bacteria ◽

Genes Encoding

AbstractInfections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

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