Multidrug Resistance Region Research Articles

The highly diverse plasmid population found in Escherichia coli colonizing travellers to Laos and its role in antimicrobial resistance gene carriage.

Increased colonization by antimicrobial-resistant organisms is closely associated with international travel. This study investigated the diversity of mobile genetic elements involved with antimicrobial resistance (AMR) gene carriage in extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli that colonized travellers to Laos. Long-read sequencing was used to reconstruct complete plasmid sequences from 48 isolates obtained from the daily stool samples of 23 travellers over a 3 week period. This method revealed a collection of 105 distinct plasmids, 38.1 % (n=40) of which carried AMR genes. The plasmids in this population were diverse, mostly unreported and included 38 replicon types, with F-type plasmids (n=23) the most prevalent amongst those carrying AMR genes. Fine-scale analysis of all plasmids identified numerous AMR gene contexts and emphasized the importance of IS elements, specifically members of the IS6/IS26 family, in the evolution of complex multidrug resistance regions. We found a concerning convergence of ESBL and colistin resistance determinants, with three plasmids from two different F-type lineages carrying bla CTX-M and mcr genes. The extensive diversity seen here highlights the worrying probability that stable new vehicles for AMR will evolve in E. coli populations that can disseminate internationally through travel networks.

Comparative genomic analysis of Colistin resistant Escherichia coli isolated from pigs, a human and wastewater on colistin withdrawn pig farm

In this study, genomic and plasmid characteristics of Escherichia coli were determined with the aim of deducing how mcr genes may have spread on a colistin withdrawn pig farm. Whole genome hybrid sequencing was applied to six mcr-positive E. coli (MCRPE) strains isolated from pigs, a farmworker and wastewater collected between 2017 and 2019. Among these, mcr-1.1 genes were identified on IncI2 plasmids from a pig and wastewater, and on IncX4 from the human isolate, whereas mcr-3 genes were found on plasmids IncFII and IncHI2 in two porcine strains. The MCRPE isolates exhibited genotypic and phenotypic multidrug resistance (MDR) traits as well as heavy metal and antiseptic resistance genes. The mcr-1.1-IncI2 and IncX4 plasmids carried only colistin resistance genes. Whereas, the mcr-3.5-IncHI2 plasmid presented MDR region, with several mobile genetic elements. Despite the MCRPE strains belonged to different E. coli lineages, mcr-carrying plasmids with high similarities were found in isolates from pigs and wastewater recovered in different years. This study highlighted that several factors, including the resistomic profile of the host bacteria, co-selection via adjunct antibiotic resistance genes, antiseptics, and/or disinfectants, and plasmid-host fitness adaptation may encourage the maintenance of plasmids carrying mcr genes in E. coli.

Complete Genetic Analysis of Plasmids Carrying Multiple Resistance, Virulence, and Phage-Like Genes in Foodborne Escherichia coli Isolate.

Bacterial antimicrobial resistance, especially phenotypic resistance to multiple drugs (MDR), has posed a serious threat to public health worldwide. To clarify the mechanism of transmission of multidrug resistance encoding plasmids in Enterobacterales, all seven plasmids of an Escherichia coli (E. coli) strain 1108 obtained from a chicken meat sample were extracted and sequenced by Illumina Nextseq 500 and MinION platforms. Plasmids in strain 1108 possessed 16 known antimicrobial resistance genes, with p1108-NDM (~97K) being the most variable plasmid. The multidrug resistance region of p1108-NDM was punctuated by eight IS26 insertion sequences; thus, four MDR regions were found in the backbone of this plasmid. The plasmid p1108-MCR (~65K) was found to lack the ISApl1 element and harbor the blaCTX-M-64-ISEcp1 transposition unit. Moreover, the ISEcp1-blaCMY-2 transposition unit was found in plasmid p1108-CMY2 (~98K), whereas plasmid p1108-emrB (~102K) was associated with resistance to erythromycin (emrB) and streptomycin (aadA22). p1108-IncY (96K) was a phage P1-like plasmid, while p1108-IncFIB (~194K) was found to harbor a virulence region similar to ColV plasmids, and they were found to encode a conserved conjugative transfer protein but harbor no resistance genes. Finally, no mobile element and resistant genes were found in p1108-ColV (~2K). Carriage of mcr-1-encoding elements in carbapenemase-producing Escherichia coli will potentially render all antimicrobial treatment regimens ineffective. Enhanced surveillance and effective intervention strategies are urgently needed to control the transmission of such multidrug resistance plasmids. IMPORTANCE Antimicrobial resistance (AMR) has been increasingly prevalent in agricultural and clinical fields. Understanding the genetic environment involved in AMR genes is important for preventing transmission and developing mitigation strategies. In this study, we investigated the genetic features of an E. coli strain (1108) isolated from food product and harboring 16 AMR genes, including blaNDM-1 and mcr-1 genes encoding resistance to last line antibiotics, meropenem, and colistin. Moreover, this strain also carried virulence genes such as iroBCDEN, iucABCD, and iutA. Our findings confirmed that multiple conjugative plasmids that were formed through active recombination and translocation events were associated with transmission of AMR determinants. Our data warrant the continuous monitoring of emergence and further transmission of these important MDR pathogens.

A new class A beta-lactamase gene blaCAE-1 coexists with blaAFM-1 in a novel untypable plasmid in Comamonas aquatica

Antimicrobial resistance, especially carbapenem resistance, poses a serious threat to global public health. Here, a carbapenem-resistant Comamonasaquatica isolate SCLZS63 was recovered from hospital sewage. Whole-genome sequencing showed that SCLZS63 has a 4,048,791-bp circular chromosome and three plasmids. The carbapenemase gene blaAFM-1 is located on the 143,067-bp untypable plasmid p1_SCLZS63, which is a novel type of plasmid with two multidrug-resistant (MDR) regions. Notably, a novel class A serine β-lactamase gene, blaCAE-1, coexists with blaAFM-1 in the mosaic MDR2 region. Cloning assay showed that CAE-1 confers resistance to ampicillin, piperacillin, cefazolin, cefuroxime, and ceftriaxone, and elevates the MIC of ampicillin-sulbactam two-fold in Escherichia coli DH5α, suggesting that CAE-1 functions as a broad-spectrum β-lactamase. Amino acid sequences analysis suggested that blaCAE-1 may originate from Comamonadaceae. The blaAFM-1 in p1_SCLZS63 is located in a conserved structure of ISCR29-ΔgroL-blaAFM-1-ble-ΔtrpF-ΔISCR27-msrB-msrA-yfcG-corA. Comprehensive analysis of the blaAFM-bearing sequences revealed important roles of ISCR29 and ΔISCR27 in the mobilization and truncation of the core module of blaAFM alleles, respectively. The diverse passenger contents of class 1 integrons flanking the blaAFM core module make the complexity of genetic contexts for blaAFM. In conclusion, this study reveals that Comamonas may act as an important reservoir for antibiotics-resistance genes and plasmids in the environment. Continuous monitoring for the environmental emergence of antimicrobial-resistant bacteria is needed to control the spread of antimicrobial resistance.

Whole genome sequencing of multidrug-resistant Proteus mirabilis strain PM1162 recovered from a urinary tract infection in China

Proteus mirabilis is an important opportunistic Gram-negative pathogen. This study reports the whole genome sequence of multidrug-resistant (MDR) P. mirabilis PM1162 and explores its antibiotic resistance genes (ARGs) and their genetic environments. P. mirabilis PM1162 was isolated from a urinary tract infection in China. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed. ARGs, insertion sequence (IS) elements, and prophages were identified using ResFinder, ISfinder, and PHASTER software, respectively. Sequence comparisons and map generation were performed using BLAST and Easyfig, respectively. On its chromosome, P. mirabilis PM1162 harboured 15 ARGs, including cat, tet(J), blaCTX-M-14 (three copies), aph(3')-Ia, qnrB4, blaDHA-1, qacE, sul1, armA, msr(E), mph(E), aadA1, and dfrA1. We focused our analysis on the four related MDR regions: (1) genetic contexts associated with blaCTX-M-14; (2) the prophage containing blaDHA-1, qnrB4, and aph(3')-Ia; (3) genetic environments associated with mph(E), msr(E), armA, sul, and qacE; and (4) the class II integron harbouring dfrA1, sat2, and aadA1. This study reported the whole genome sequence of MDR P. mirabilis PM1162 and the genetic context of its ARGs. This comprehensive genomic analysis of MDR P. mirabilis PM1162 provides a deeper understanding of its MDR mechanism and elucidates the horizontal spread of its ARGs, thus providing a basis for the containment and treatment of the bacteria.

Characterization and comparative genomics analysis of RepA_N multi-resistance plasmids carrying optrA from Enterococcus faecalis.

This research aimed to investigate the antibiotic resistance of Enterococcus faecalis from swine farms in Zhejiang Province and the prevalence and transmission mechanism of oxazolidone resistance gene optrA. A total of 226 Enterococcus faecalis were isolated and their resistance to 14 antibiotics was detected by broth microdilution. The resistance genes were detected by PCR. The antibiotic resistance rate of 226 isolates to nearly 57% (8/14) of commonly used antibiotics was higher than 50%. The resistance rate of tiamulin was highest (98.23%), that of tilmicosin, erythromycin, tetracycline and florfenicol was higher than 80%, and that of oxazolidone antibiotic linezolid was 38.49%. The overall antibiotics resistance in Hangzhou, Quzhou and Jinhua was more serious than that in the coastal cities of Ningbo and Wenzhou. The result of PCR showed that optrA was the main oxazolidinone and phenicols resistance gene, with a detection rate of 71.68%, and optrA often coexisted with fexA in the isolates. Through multi-locus sequence typing, conjugation transfer, and replicon typing experiments, it was found that the horizontal transmission mediated by RepA_N plasmid was the main mechanism of optrA resistance gene transmission in E. faecalis from Zhejiang Province. Two conjugative multi-resistance plasmids carrying optrA, RepA_N plasmid pHZ318-optrA from Hangzhou and Rep3 plasmid from Ningbo, were sequenced and analyzed. pHZ318-optrA contain two multidrug resistance regions (MDR), which contributed to the MDR profile of the strains. optrA and fexA resistance genes coexisted in IS1216E-fexA-optrA-ferr-erm(A)-IS1216E complex transposon, and there was a partial sequence of Tn554 transposon downstream. However, pNB304-optrA only contain optrA, fexA and an insertion sequence ISVlu1. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterococcus through inter-replicon gene transfer. This study can provide theoretical basis for ensuring the quality and safety of food of animal origin, and provide scientific guidance for slowing down the development of multi-antibiotic resistant Enterococcus.

Conjugation of plasmid harboring bla NDM-1 in a clinical Providencia rettgeri strain through the formation of a fusion plasmid.

Providencia rettgeri has recently gained increased importance owing to the New Delhi metallo-β-lactamase (NDM) and other β-lactamases produced by its clinical isolates. These enzymes reduce the efficiency of antimicrobial therapy. Herein, we reported the findings of whole-genome sequence analysis and a comprehensive pan-genome analysis performed on a multidrug-resistant P. rettgeri 18004577 clinical strain recovered from the urine of a hospitalized patient in Shandong, China, in 2018. Providencia rettgeri 18004577 was found to have a genome assembly size of 4.6 Mb with a G + C content of 41%; a circular plasmid p18004577_NDM of 273.3 Kb, harboring an accessory multidrug-resistant region; and a circular, stable IncT plasmid p18004577_Rts of 146.2 Kb. Additionally, various resistance genes were identified in its genome, including bla NDM-1, bla OXA-10, bla PER-4, aph(3')-VI, ant(2'')-Ia, ant(3')-Ia, sul1, catB8, catA1, mph(E), and tet. Conjugation experiments and whole-genome sequencing revealed that the bla NDM-1 gene could be transferred to the transconjugant via the formation of pJ18004577_NDM, a novel hybrid plasmid. Based on the genetic comparison, the main possible formation process for pJ18004577_NDM was the insertion of the [ΔISKox2-IS26-ΔISKox2]-aph(3')-VI-bla NDM-1 translocatable unit module from p18004577_NDM into plasmid p18004577_Rts in the Russian doll insertion structure (ΔISKox2-IS26-ΔISKox2), which played a role similar to that of IS26 using the "copy-in" route in the mobilization of [aph(3')-VI]-bla NDM-1. The array, multiplicity, and diversity of the resistance and virulence genes in this strain necessitate stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of the resistance genes. Roary analysis based on 30 P. rettgeri strains pan genome identified 415 core, 756 soft core, 5,744 shell, and 12,967 cloud genes, highlighting the "close" nature of P. rettgeri pan-genome. After a comprehensive pan-genome analysis, representative biological information was revealed that included phylogenetic distances, presence or absence of genes across the P. rettgeri bacteria clade, and functional distribution of proteins. Moreover, pan-genome analysis has been shown to be an effective approach to better understand P. rettgeri bacteria because it helps develop various tailored therapeutic strategies based on their biological similarities and differences.

Different threats posed by two major mobilized colistin resistance genes — mcr-1.1 and mcr-3.1 — revealed through comparative genomic analysis

Global spread of mobilized colistin resistance gene (mcr)-carrying Escherichia coli poses serious threats to public health. This study aimed to provide insights into different threats posed by two major mcr variants: mcr-1.1 and mcr-3.1. Genetic backgrounds and characteristics of mobile genetic elements carrying mcr-1.1 or mcr-3.1 in 74 (mcr)-carrying E. coli isolated from swine farms were analysed, and comparative genomic analysis was performed with the public sequence database. The mcr-1.1 showed high horizontal transferability (6.30 logCFU/ml). Genetic background of mcr-1.1, including genetic cassette/plasmid, was transferred without insertion sequences (ISs) and/or multi-drug resistance (MDR) and highly shared across strains. The major mcr-1.1-cassette was "mcr-1.1-pap2", mainly encoded in IncI2 and IncX4. Mcr-3.1 exhibited relatively lower conjugation frequency (0.97 logCFU/ml). The mcr-3.1-cassette was flanked by IS26 and was highly variable across strains because of the insertion, deletion, or truncation of IS6100, IS4321, or IS5075. Near the mcr-3.1 cassette, MDR regions consisting of antimicrobial/heavy metal resistance genes were identified, which varied across strains. From the MCR3-E13 strain, a mcr-3.1-carrying IncHI2-fragment was integrated into the bacterial chromosome via IS26-mediated co-integration. To our knowledge, this was the first study to describe that a mcr-3.1-carrying plasmid could be inserted into the bacterial chromosome. Based on high horizontal transferability, mcr-1.1 could play a major role on colistin resistance propagation. On the other hand, mcr-3.1 could be transmitted with MDR and have dual pathways mediated by plasmid transfer (horizontal transmission) and chromosomal insertion (vertical transmission), enabling it to proliferate stably despite its lower horizontal transferability.

Characterization and Identification of a novel chromosome-encoded metallo-β-lactamase WUS-1 in Myroides albus P34.

In this study, we identified and characterized a novel chromosomally-encoded class B metallo-β-lactamase (MBL) gene designated bla WUS-1 in a carbapenem-resistant isolate Myroides albus P34 isolated from sewage discharged from an animal farm. Comparative analysis of the deduced amino acid sequence revealed that WUS-1 shares the highest amino acid similarities with the function-characterized MBLs MUS-1 (AAN63647.1; 70.73%) and TUS-1 (AAN63648.1; 70.32%). The recombinant carrying bla WUS-1 exhibited increased MICs levels against a number of β-lactam antimicrobials such as carbenicillin, ampicillin and imipenem, and β-lactamase inhibitors (clavulanic acid and tazobactam). The metallo-β-lactamase WUS-1 could also hydrolyze these antimicrobials and the hydrolytic activities could be inhibited by EDTA. Genetic context analysis of bla WUS-1 revealed that no mobile genetic element was found in its surrounding region. The plasmid pMA84474 of Myroides albus P34 harbored 6 resistance genes (bla OXA-347, aadS, bla MYO-1, ereD, sul2 and ermF) within an approximately 17 kb multidrug resistance (MDR) region. These genes, however, were all related to mobile genetic elements.

Characterization and identification of a novel chromosomal class C β-lactamase, LAQ-1, and comparative genomic analysis of a multidrug resistance plasmid in Lelliottia amnigena P13.

Lelliottia amnigena, a bacterium usually isolated from natural environments, may cause human infections and has been suggested to be naturally resistant to second- and third-generation cephalosporins. In this study, we determined the whole-genome sequence of an isolate, L. Amnigena P13, isolated from animal farm sewage. On the basis of genome sequence analysis, susceptibility testing, molecular cloning, and enzyme kinetic parameter analysis, we identified a novel chromosome-encoded AmpC β-lactamase, LAQ-1. bla LAQ-1 is resistant to penicillin G, ampicillin, and several first- to fourth-generation cephalosporins, such as cefazolin, cefoxitin and cefepime. The MIC levels of some β-lactams, such as cefoxitin, cefepime, aztreonam and cefazolin, for the recombinant clone (pUCP24-bla LAQ-1/DH5α) increased by approximately 4- to 64-fold compared with those of the control strain (pUCP24/DH5α). The kinetic properties of LAQ-1, with the highest catalytic activity observed toward piperacillin, were basically the same as those of typical class C β-lactamases, and avibactam had a strong inhibitory effect on its hydrolytic activity. The genetic background of bla LAQ-1 was relatively conserved, and no mobile genetic element (MGE) was found around it. The plasmid pP13-67 of L. amnigena P13 harbored 12 resistance genes [qnrS1, aph(6)-Id, aadA2, sul1, sul2, bla TEM-1, qacEΔ1, dfrA12, tetA and floR] related to different mobile genetic elements within an ~22 kb multidrug resistance region. The multidrug resistance region shared the highest nucleotide sequence similarities with those of the chromosomes or plasmids of different bacterial species, indicating the possibility of horizontal transfer of these resistance genes among different bacterial species.

Emergence of an Extensive Drug Resistant Pseudomonas aeruginosa Strain of Chicken Origin Carrying blaIMP-45, tet(X6), and tmexCD3-toprJ3 on an IncpRBL16 Plasmid.

This study reports an extensively drug resistant Pseudomonas aeruginosa strain PA166-2 which was of chicken origin and carrying blaIMP-45, tet(X6) and tmexCD3-toprJ3 on a single plasmid. The strain was characterized by antimicrobial susceptibility testing, resistance gene screening, conjugation assay, whole-genome sequencing, and bioinformatics analysis. Strain PA166-2 was resistant to tigecycline and carbapenems. It belonged to ST313 and carried a plasmid pPA166-2-MDR, which belongs to the incompatibility group IncpRBL16. pPA166-2-MDR harbored a 78 Kb multidrug resistance (MDR) region carrying an array of antimicrobial resistance genes, including blaIMP-45, tet(X6), and tmexCD3-toprJ3. The gene blaIMP-45 was inserted into the backbone of plasmid pPA166-2-MDR within a class 1 integron, In786. tmexCD3-toprJ3 in plasmid pPA166-2-MDR was inserted in umuC, constituting the genetic context of ISCfr1-tnfxB3-tmexC3-tmexD3-toprJ3-△umuC. The genetic context of tet(X6) in this plasmid was identical to that of other reported plasmid-borne tet(X) variants, namely, tet(X6)-abh-guaA-ISVsa3. To the best of our knowledge, this is the first report of the cooccurrence of blaIMP-45, tet(X6), and tmexCD3-toprJ3 in one plasmid in Pseudomonas sp. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes from chickens expanded the global transmission of vital resistance genes. Findings from us and from others indicate that plasmids of the incompatibility group IncpRBL16 may serve as a reservoir for carbapenem and tigecycline resistance determinants. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes infections that are difficult to treat. This study reported, for the first time, the occurrence of last-resort antibiotic resistance determinants blaIMP-45, tet(X6), and tmexCD3-toprJ3 on a single plasmid in P. aeruginosa from chickens. The P. aeruginosa strain belonged to ST313 and was resistant to last-line antibiotics, namely, carbapenems and tigecycline. The plasmid carrying the last-line resistance genes belonged to the incompatibility group IncpRBL16, which was reported to contain different profiles of accessory modules and thus carried diverse collections of resistance genes. The emergence of plasmid-mediated tigecycline resistance genes tmexCD3-toprJ3 and tet(X6), as well as carbapenemase genes, from chickens expanded the global transmission of vital resistance genes. The results in this study highlighted that IncpRBL16 plasmids may serve as a reservoir for the dissemination of resistance genes. Control measures should be implemented to prevent the further dissemination of such strains.

Molecular epidemiology and population genomics of tet(X4), blaNDM or mcr-1 positive Escherichia coli from migratory birds in southeast coast of China.

The emergence of multidrug-resistant (MDR) bacteria harboring tet(X4), blaNDM or mcr-1 posed a serious threat to public health. Wild birds, especially migratory birds, were considered as one of important transmission vectors for antibiotic resistance genes (ARGs) globally, however, few studies were performed on the genomic epidemiology of critical resistance genes among them. Isolates harboring tet(X4), mcr-1 or blaNDM from migratory birds were identified and characterized by PCR, antimicrobial susceptibility testing, conjugation assays, whole genome sequencing and bioinformatics analysis. A total of 14 tet(X4)-bearing E. coli, 4 blaNDM-bearing E. coli and 23 mcr-1-bearing E. coli isolates were recovered from 1060 fecal samples of migratory birds. All isolates were MDR bacteria and most plasmids carrying tet(X4), blaNDM or mcr-1 were conjugative. We first identified an E. coli of migratory bird origin carrying blaNDM-4, which was located on a conjugative IncHI2 plasmid and embedded on a novel MDR region flanked by IS26 that could generate the circular intermediate. The emergency of E. coli isolates co-harboring mcr-1 and blaNDM-5 in migratory birds indicated the coexistence of ARGs in migratory birds was a novel threat. This study revealed the prevalence and molecular characteristics of three important ARGs in migratory birds, provided evidence that migratory birds were potential vectors of novel resistance genes and highlighted the monitoring of ARGs in migratory birds should be strengthened to prevent the spread of ARGs in a One Health strategy.

Characterization of a genomic Island carrying the tet(X4) gene in porcine Acinetobacter towneri co-harboring plasmid-borne bla NDM-1 and bla OXA-58 genes.

Tigecycline and carbapenems are last-resort antimicrobial agents to treat serious infections caused by multi-drug resistant bacterial pathogens. However, the co-occurrence of tigecycline and carbapenem resistance determinants challenges the clinical efficacy of these antimicrobial agents. In this study, we report the co-existence of tet(X4), blaNDM−1 and blaOXA−58 genes in the porcine Acinetobacter towneri isolate 19110F47. Sequence analysis revealed that tet(X4) gene, along with the florfenicol resistance gene floR, was flanked by three copies of IS91-like elements, which can form three different translocatable units (TUs), and were located in a 41,098-bp multidrug resistance region (MDRR) within a novel 100,354-bp genomic island (GI) region. TUs comprising floR-virD2-ISVsa3, hp-abh-tet(X4)-ISVsa3 and virD2-floR-ISVsa3-hp-abh-tet(X4)-ISVsa3 can be looped out from the chromosomal DNA and facilitate the transfer of the TU-based resistance genes into other plasmidic or chromosomal sites. In addition, the carbapenemase genes blaNDM−1 and blaOXA−58 were found on different non-conjugative multiresistance plasmids in this isolate, with the genetic contexts ISAba125-blaNDM−1-bleMBL-tnpR and ΔISAba3-blaOXA−58-ISAba3, respectively. The simultaneous occurrence of tet(X4), blaNDM−1 and blaOXA−58 in the same porcine A. towneri isolate emphasizes the importance of antimicrobial resistance surveillance in food-producing animals.

Emergence of Extensively Drug-Resistant ST170 Citrobacter portucalensis with Plasmids pK218-KPC, pK218-NDM, and pK218-SHV from a Tertiary Hospital, China.

ABSTRACTThe objective of this study is to characterize the molecular mechanism of a clinical carbapenem-resistant Citrobacter portucalensis strain K218, which coproduces KPC and NDM carbapenemases. K218 was isolated from a patient's blood sample in a Chinese tertiary hospital. Carbapenemases were detected by the immunocolloidal gold technique. The MIC values were determined by VITEK2. Whole-genome sequencing was performed on K218 and sequence data were analyzed using phylogenetics and extensive genomic comparison. This study reveals that K218 contains a single 5.08 Mb chromosome (51.8% GC content) and four plasmids, pK218-KPC (106 Kb), pK218-NDM (111 Kb), pK218-SHV (191 Kb), and pK218-NR (5 Kb). Twenty-nine types of antibiotic resistance genes were carried on K218, including blaKPC-2 harbored on pK218-KPC and blaNDM-1 harbored on pK218-NDM. Detailed comparison of related plasmids of pK218-KPC, pK218-NDM, and pK218-SHV showed that they shared similar conserved backbone regions, respectively. Comprehensive annotation revealed large accessory modules were recombined on the genome of K218. Further analysis speculated that mobile genetic elements bearing abundant resistance genes facilitated the formation of these accessory modules. In conclusion, this study provides an in-depth understanding of the genomic characterization of K218, an extensively drug-resistant C. portucalensis strain coproducing NDM and KPC carbapenemase. To the best of our knowledge, this is the first report of C. portucalensis strain coharboring blaKPC-2 and blaNDM-1 from the clinical setting.IMPORTANCE This is the first report of extensively drug-resistant C. portucalensis harboring both blaKPC-2 and blaNDM-1. This study will not only extend the understanding of the structural dissection of plasmids and chromosomes carried in C. portucalensis, but also expand knowledge of the genetic environment of the blaKPC-2 and blaNDM-1 genes. blaKPC-2 and blaNDM-1 genes have been suggested to facilitate the propagation and persistence of their host bacteria under different antimicrobial selection pressures. Large accessory regions carrying blaKPC-2 and blaNDM-1 genes have become hot spots for transposition and integration, and their structural variation and evolution should receive attention. The multidrug-resistant plasmids pK218-KPC, pK218-NDM, and pK218-SHV with several multidrug resistance regions and the chromosome cK218 with two novel transposons Tn7410 and Tn7411 contribute to the formation of extensively drug-resistant C. portucalensis.

Characterization of IncI1/ST71 and IncF18:A−:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate

To characterize IncI1 and IncF18:A−:B1 multidrug-resistance plasmids from an avian Escherichia coli isolate, antibiotic susceptibility testing, conjugation assays, transformation assays, S1-PFGE, and WGS analysis were performed. The 119,457-bp plasmid pEC014–1 with a multidrug-resistance region (MRR) containing four different segments interspersed with six IS26 elements, belonged to incompatibility group I1 and sequence type 71. The 154,516-bp plasmid pEC014–2 with two replicons, typed as FII-18 and FIB-1, carried 14 resistance determinants including blaTEM-1b, blaOXA-1, oqxAB, dfrA17, aac(6′)-Ib-cr, sul1, sul2, tet(A), floR, catB3, hph(aph(4)-Ia), aacC4(aac(3)-IV), aadA5, arr-3, and a merEDACPTR loci in MRR, and additionally encoded three virulence loci: iroNEDCB, sitABCD, and iucABCD-iutA. Plasmid stability assays showed that pEC014–1 and pEC014–2 were stable in recipient E. coli C600 for at least 15 days of passage. Competition assays were carried out to evaluate the fitness impact of pEC014–2 carriage in vitro, revealing a decrease in host fitness. Growth kinetics showed that the growth rate for pEC014–1 or/and pEC014–2 bearing cells was significantly slower than that of the E. coli C600 host strain in the exponential stage (p < 0.01), with only cells carrying pEC014–1 sustaining rapid growth after 6 h of exponential growth. Our findings highlight the mosaic structures of epidemic plasmid IncI1/ST71 and F18:A−:B1 lineages and contribute to a better understanding of the evolution and dissemination of these multidrug resistance and virulence plasmids.

Tigecycline-resistant Escherichia coli ST761 carrying tet(X4) in a pig farm, China.

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC ≥ 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China.

Emergence of extensive multidrug-resistant Staphylococcus aureus carrying novel Sa-MRRlsa(E) in retail food

The aim of this study was to investigate the prevalence and genetic environment of the multidrug resistance gene lsa(E) in food-related Staphyloccocus aureus in China. 1463 S. aureus from retail food products in 39 Chinese cities were investigated to determine the prevalence of lsa(E). Furthermore, antimicrobial susceptibility testing, whole-genome sequencing (WGS), and complete genetic analysis were performed in lsa(E)-positive isolates. Thirty-five isolates (2.4%) were positive for the lsa(E) gene, which had an extensive multidrug-resistance phenotype. ST9-t899 and ST1-t4792 were the common sequence types in positive strains. The lsa(E) genes were located in two different types of novel multidrug resistance region (MRRlsa[E]) on the chromosome. The Sa-MRRlsa(E)-I were inserted into the lctP gene. The Sa-MRRlsa(E)-II were inserted into the crtP gene, and were comprised of seven antibiotic resistance genes (ARGs) interspersed with varieties of insertion sequences (ISs), transposons, and DNA invertase genes, showing a novel arrangement harboring lsa(E). Part of transposon Tn1546 was inserted downstream of lnu(B) in the novel Sa-MRRlsa(E)-II. Both types of Sa-MRRlsa(E) could be excised from the chromosome, indicating that Sa-MRRlsa(E) may be transferable. Our study is the first systematical investigation of lsa(E)-positive S. aureus in retail foods in China. It indicated that the origin of most food-related lsa(E)-positive S. aureus in China might be associated with livestock or poultry breeding farms, and these strains may be transmitted between animals and food. S. aureus carrying novel Sa-MRRlsa(E), especially, serve as reservoirs of antibiotic resistance traits, and warrant further attention.

Genomic characterization of a carbapenem-resistant Citrobacter freundii cocarrying blaKPC-2 and blaNDM-1

Citrobacter freundii is an important opportunistic pathogen, and carbapenem-resistant strains pose a significant challenge to public health. Here we report the genetic features of antimicrobial resistance genes of a carbapenem-resistant C. freundii SCLZS47 from hospital sewage by using whole genome sequencing. Antimicrobial susceptibility was determined by the broth microdilution method. Whole genomic sequences of SCLZS47 were obtained by using the HiSeq 2000 combined with PacBio RSII platforms. Plasmid incompatibility types, resistance genes, and insertion elements were identified using the PlasmidFinder, ResFinder, and ISfinder, respectively. SCLZS47 has a circular chromosome and three resistance plasmids, and it carries 23 known ARGs. Among them, blaCMY-135 and three copies of blaCTX-M-14 are located on the chromosome. Sixteen ARGs are clustered in two accessory modules of a multidrug resistance (MDR) plasmid, and homologous recombination and transposition events contribute to the formation of these MDR regions. Carbapenemase genes blaKPC-2 and blaNDM-1 are carried by a pCKPC18-1-like plasmid and a pNDM-HN380-like plasmid, respectively. Conjugation experiments indicated that both KPC-2- and NDM-1-encoding plasmids are transmissible. Analysis of the genetic features of resistance genes would help to better understand their transmission mechanisms and dynamics in bacterial community, which has significant clinical implications.

Overview of Salmonella Genomic Island 1-Related Elements Among Gamma-Proteobacteria Reveals Their Wide Distribution Among Environmental Species.

The aim of this study was to perform an in silico analysis of the available whole-genome sequencing data to detect syntenic genomic islands (GIs) having homology to Salmonella genomic island 1 (SGI1), analyze the genetic variations of their backbone, and determine their relatedness. Eighty-nine non-redundant SGI1-related elements (SGI1-REs) were identified among gamma-proteobacteria. With the inclusion of the thirty-seven backbones characterized to date, seven clusters were identified based on integrase homology: SGI1, PGI1, PGI2, AGI1 clusters, and clusters 5, 6, and 7 composed of GIs mainly harbored by waterborne or marine bacteria, such as Vibrio, Shewanella, Halomonas, Idiomarina, Marinobacter, and Pseudohongiella. The integrase genes and the backbones of SGI1-REs from clusters 6 and 7, and from PGI1, PGI2, and AGI1 clusters differed significantly from those of the SGI1 cluster, suggesting a different ancestor. All backbones consisted of two parts: the part from attL to the origin of transfer (oriT) harbored the DNA recombination, transfer, and mobilization genes, and the part from oriT to attR differed among the clusters. The diversity of SGI1-REs resulted from the recombination events between GIs of the same or other families. The oriT appeared to be a high recombination site. The multi-drug resistant (MDR) region was located upstream of the resolvase gene. However, most SGI1-REs in Vibrio, Shewanella, and marine bacteria did not harbor any MDR region. These strains could constitute a reservoir of SGI1-REs that could be potential ancestors of SGI1-REs encountered in pathogenic bacteria. Furthermore, four SGI1-REs did not harbor a resolvase gene and therefore could not acquire an integron. The presence of mobilization genes and AcaCD binding sites indicated that their conjugative transfer could occur with helper plasmids. The plasticity of SGI1-REs contributes to bacterial adaptation and evolution. We propose a more relevant classification to categorize SGI1-REs into different clusters based on their integrase gene similarity.

Genetic Characterization of Four Groups of Chromosome-Borne Accessory Genetic Elements Carrying Drug Resistance Genes in Providencia.

PurposeThe aim of this study was to gain a deeper genomics and bioinformatics understanding of diversification of accessory genetic elements (AGEs) in Providencia.MethodsHerein, the complete genome sequences of five Providencia isolates from China were determined, and seven AGEs were identified from the chromosomes. Detailed genetic dissection and sequence comparison were applied to these seven AGEs, together with additional 10 chromosomal ones from GenBank (nine of them came from Providencia).ResultsThese 17 AGEs were divided into four groups: Tn6512 and its six derivatives, Tn6872 and its two derivatives, Tn6875 and its one derivative, and Tn7 and its four derivatives. These AGEs display high-level diversification in modular structures that had complex mosaic natures, and particularly different multidrug resistance (MDR) regions were presented in these AGEs. At least 52 drug resistance genes, involved in resistance to 15 different categories of antimicrobials and heavy metal, were found in 15 of these 17 AGEs.ConclusionIntegration of these AGEs into the Providencia chromosomes would contribute to the accumulation and distribution of drug resistance genes and enhance the ability of Providencia isolates to survive under drug selection pressure.