Synergistic effects of ceftazidime/avibactam combined with meropenem in a murine model of infection with KPC-producing Klebsiella pneumoniae.

OBJECTIVES: The emergence and expansion of carbapenem-resistant Klebsiella pneumoniae infections is a concern due to the lack of 'first-line' antibiotic treatment options. The ceftazidime/avibactam is an important clinical treatment for carbapenem-resistant K. pneumoniae infections but there is an increasing number of cases of treatment failure and drug resistance. Therefore, a potential solution is combination therapies that result in synergistic activity against K. pneumoniae carbapenemase: producing K. pneumoniae (KPC-Kp) isolates and preventing the emergence of KPC mutants resistant to ceftazidime/avibactam are needed in lieu of novel antibiotics. METHODS: To evaluate their synergistic activity, antibiotic combinations were tested against 26 KPC-Kp strains. Antibiotic resistance profiles, molecular characteristics and virulence genes were investigated by susceptibility testing and whole-genome sequencing. Antibiotic synergy was evaluated by in vitro chequerboard experiments, time-killing curves and dose-response assays. The mouse thigh model was used to confirm antibiotic combination activities in vivo. Additionally, antibiotic combinations were evaluated for their ability to prevent the emergence of ceftazidime/avibactam resistant mutations of blaKPC. RESULTS: The combination of ceftazidime/avibactam plus meropenem showed remarkable synergistic activity against 26 strains and restored susceptibility to both the partnering antibiotics. The significant therapeutic effect of ceftazidime/avibactam combined with meropenem was also confirmed in the mouse model and bacterial loads in the thigh muscle of the combination groups were significantly reduced. Furthermore, ceftazidime/avibactam plus meropenem showed significant activity in preventing the occurrence of resistance mutations. CONCLUSIONS: Our results indicated that the combination of ceftazidime/avibactam plus meropenem offers viable therapeutic alternatives in treating serious infections due to KPC-Kp.

SARS-CoV-2 NSP12 utilizes various host splicing factors for replication and splicing regulation.

The RNA-dependent RNA polymerase (RdRp) is a crucial element in the replication and transcription of RNA viruses. Although the RdRps of lethal human coronaviruses severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) have been extensively studied, the molecular mechanism of the catalytic subunit NSP12, which is involved in pathogenesis, remains unclear. In this study, the biochemical and cell biological results demonstrate the interactions between SARS-CoV-2 NSP12 and seven host proteins, including three splicing factors (SLU7, PPIL3, and AKAP8). The entry efficacy of SARS-CoV-2 considerably decreased when SLU7 or PPIL3 was knocked out, indicating that abnormal splicing of the host genome was responsible for this occurrence. Furthermore, the polymerase activity and stability of SARS-CoV-2 RdRp were affected by the three splicing factors to varying degrees. In addition, NSP12 and its homologues from SARS-CoV and MERS-CoV suppressed the alternative splicing of cellular genes, which were influenced by the three splicing factors. Overall, our research illustrates that SARS-CoV-2 NSP12 can engage with various splicing factors, thereby impacting virus entry, replication, and gene splicing. This not only improves our understanding of how viruses cause diseases but also lays the foundation for the development of antiviral therapies.

Characterization of an optrA-harbouring unconventional circularizable structure located on a novel ICESa2603 family-like integrative and conjugative element ICESsuHN38 in Streptococcus suis.

OBJECTIVES: To identify the novel genetic elements involved in the horizontal transfer of the oxazolidinone/phenicol resistance gene optrA in Streptococcus suis. METHODS: Whole-genome DNA of the optrA-positive isolate S. suis HN38 was subjected to WGS via both Illumina HiSeq and Oxford Nanopore platforms. MICs of several antimicrobial agents (erythromycin, linezolid, chloramphenicol, florfenicol, rifampicin and tetracycline) were determined by broth microdilution. PCR assays were performed to identify the circular forms of the novel integrative and conjugative element (ICE) ICESsuHN38, but also the unconventional circularizable structure (UCS) excised from this ICE. The transferability of ICESsuHN38 was evaluated by conjugation assays. RESULTS: S. suis isolate HN38 harboured the oxazolidinone/phenicol resistance gene optrA. The optrA gene was flanked by two copies of erm(B) genes in the same orientation, located on a novel ICESa2603 family-like ICE, designated ICESsuHN38. PCR assays revealed that a novel UCS carrying the optrA and one copy of erm(B) could be excised from ICESsuHN38. Conjugation assays confirmed that ICESsuHN38 was able to successfully transfer into the recipient strain S. suis BAA. CONCLUSIONS: In this work, a novel optrA-carrying mobile genetic element, a UCS, was identified in S. suis. The optrA gene was flanked by copies of erm(B) and its location on the novel ICESsuHN38 will aid its horizontal dissemination.

Mobile Oxazolidinone Resistance Genes in Gram-Positive and Gram-Negative Bacteria.

Seven mobile oxazolidinone resistance genes, including cfr, cfr(B), cfr(C), cfr(D), cfr(E), optrA, and poxtA, have been identified to date. The cfr genes code for 23S rRNA methylases, which confer a multiresistance phenotype that includes resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A compounds. The optrA and poxtA genes code for ABC-F proteins that protect the bacterial ribosomes from the inhibitory effects of oxazolidinones. The optrA gene confers resistance to oxazolidinones and phenicols, while the poxtA gene confers elevated MICs or resistance to oxazolidinones, phenicols, and tetracycline. These oxazolidinone resistance genes are most frequently found on plasmids, but they are also located on transposons, integrative and conjugative elements (ICEs), genomic islands, and prophages. In these mobile genetic elements (MGEs), insertion sequences (IS) most often flanked the cfr, optrA, and poxtA genes and were able to generate translocatable units (TUs) that comprise the oxazolidinone resistance genes and occasionally also other genes. MGEs and TUs play an important role in the dissemination of oxazolidinone resistance genes across strain, species, and genus boundaries. Most frequently, these MGEs also harbor genes that mediate resistance not only to antimicrobial agents of other classes, but also to metals and biocides. Direct selection pressure by the use of antimicrobial agents to which the oxazolidinone resistance genes confer resistance, but also indirect selection pressure by the use of antimicrobial agents, metals, or biocides (the respective resistance genes against which are colocated on cfr-, optrA-, or poxtA-carrying MGEs) may play a role in the coselection and persistence of oxazolidinone resistance genes.

Characterization of an MDR Lactobacillus salivarius isolate harbouring the phenicol-oxazolidinone-tetracycline resistance gene poxtA.

OBJECTIVES: To characterize the oxazolidinone resistance gene poxtA in a Lactobacillus salivarius isolate of pig origin. METHODS: L. salivarius isolate BNS11 was investigated for the presence of mobile oxazolidinone resistance genes by PCR. Antimicrobial susceptibility testing was performed by broth microdilution. Transfer experiments were conducted to assess horizontal transferability of the gene poxtA. WGS was carried out using a combination of Oxford Nanopore MinION/Illumina HiSeq platforms. The presence of translocatable units (TUs) carrying resistance genes was studied by PCR assays and subsequent sequence analysis. RESULTS: L. salivarius isolate BNS11 was positive for poxtA. WGS showed that it harboured two gene copies each of the poxtA and the fexB genes, which were located on the broad-host-range Inc18 plasmid pBNS11-37kb and in the chromosomal DNA, respectively. The plasmid-borne poxtA gene together with the genes fexB, vat(E) and erm(C) were located in an MDR region on plasmid pBNS11-37kb. Analysis of the genetic context showed that an approx. 11 kb poxtA-fexB fragment was integrated into the chromosomal DNA and two novel IS elements ISLasa1 and ISLasa2 were identified in this inserted fragment. PCR assays revealed that five different IS1216E-based TUs carrying the resistance genes poxtA, fexB, vat(E) or erm(C) were formed. CONCLUSIONS: To the best of our knowledge, this is the first report of the transferable oxazolidinone resistance gene poxtA in the genus Lactobacillus. In addition, the presence of IS1216E-based TUs will contribute to the persistence and accelerate the dissemination of resistance genes, including poxtA.

Characterization of the novel optrA-carrying pseudo-compound transposon Tn7363 and an Inc18 plasmid carrying cfr(D) in Vagococcus lutrae.

OBJECTIVES: To investigate the genetic context and transferability of the oxazolidinone resistance genes cfr(D) and optrA in a porcine Vagococcus lutrae isolate. METHODS: V. lutrae isolate BN31 was screened for the presence of known oxazolidinone resistance genes via PCR assays. Conjugation experiments were carried out to assess horizontal transferability of resistance genes. WGS was performed using a combination of Nanopore MinION and Illumina HiSeq platforms. Detection of a translocatable unit (TU) was conducted by PCR. RESULTS: V. lutrae isolate BN31 harboured the oxazolidinone resistance genes cfr(D) and optrA. The optrA gene, together with the phenicol resistance gene fexA, was located on a novel pseudo-compound transposon, designated Tn7363. Tn7363 was bounded by two copies of the new insertion sequence ISVlu1, which represented a new member of the ISL3 family. A TU, comprising one copy of ISVlu1 and the segment between the two IS elements including the optrA gene, was detected. The cfr(D) gene and an erm(B) gene were identified on the broad-host-range Inc18 plasmid pBN31-cfrD, a pAMß1-like plasmid. Similar to plasmid pAMß1, plasmid pBN31-cfrD was conjugative. CONCLUSIONS: To the best of our knowledge, we report the first identification of the cfr(D) and optrA in Vagococcus. Two novel oxazolidinone resistance gene-carrying mobile genetic elements, Tn7363 and pBN31-cfrD, were identified in V. lutrae BN31. Considering their transmission potential, attention should be paid to the risk of transfer of the optrA and cfr(D) genes from V. lutrae to clinically more important bacterial pathogens.

Identification of a novel tetracycline resistance gene, tet(63), located on a multiresistance plasmid from Staphylococcus aureus.

OBJECTIVES: To identify and characterize a novel tetracycline resistance gene on a multiresistance plasmid from Staphylococcus aureus SA01 of chicken origin. METHODS: MICs were determined by broth microdilution according to CLSI recommendations. The whole genome sequence of S. aureus SA01 was determined via Illumina HiSeq and Oxford Nanopore platforms followed by a hybrid assembly. The new tet gene was cloned and expressed in S. aureus. The functionality of the corresponding protein as an efflux pump was tested by efflux pump inhibition assays. RESULTS: A novel tetracycline resistance gene, tet(63), was identified on a plasmid in S. aureus SA01. The cloned tet(63) gene was functionally expressed in S. aureus and shown to confer resistance to tetracycline and doxycycline, and a slightly elevated MIC of minocycline. The tet(63) gene encodes a 459 amino acid efflux protein of the major facilitator superfamily that consists of 14 predicted transmembrane helices. The results of efflux pump inhibitor assays confirmed the function of Tet(63) as an efflux protein. The deduced amino acid sequence of the Tet(63) protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). The plasmid pSA01-tet, on which tet(63) was located, had a size of 25664 bp and also carried the resistance genes aadD, aacA-aphD and erm(C). CONCLUSIONS: A novel tetracycline resistance gene, tet(63), was identified in S. aureus. Its location on a multiresistance plasmid might support the co-selection of tet(63) under the selective pressure imposed by the use of macrolides, lincosamides and aminoglycosides.

Selecting Hub Genes and Predicting Target Genes of microRNAs in Tuberculosis via the Bioinformatics Analysis.

Tuberculosis (TB) is the world's most prevalently infectious disease. Molecular mechanisms behind tuberculosis remain unknown. microRNA (miRNA) is involved in a wide variety of diseases. To validate the significant genes and miRNAs in the current sample, two messenger RNA (mRNA) expression profile datasets and three miRNA expression profile datasets were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed (DE) genes (DEGs) and miRNAs (DE miRNAs) between healthy and TB patients were filtered out. Enrichment analysis was executed, and a protein-protein interaction (PPI) network was developed to understand the enrich pathways and hub genes of TB. Additionally, the target genes of miRNA were predicted and overlapping target genes were identified. We studied a total of 181 DEGs (135 downregulated and 46 upregulated genes) and two DE miRNAs (2 downregulated miRNAs) from two gene profile datasets and three miRNA profile datasets, respectively. 10 hub genes were defined based on high degree of connectivity. A PPI network's top module was constructed. The 23 DEGs identified have a significant relationship with miRNAs. 25 critically significant Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were discovered. The detailed study revealed that, in tuberculosis, the DE miRNA and DEGs form an interaction network. The identification of novel target genes and main pathways would aid with our understanding of miRNA's function in tuberculosis progression.

What Do Social Media Influencers Say about Health? A Theory-Driven Content Analysis of Top Ten Health Influencers' Posts on Sina Weibo.

Social media health influencers play an increasingly important role in disseminating health-related information to the public. To explore how health influencers in China communicate with their followers, we conducted a content analysis of the top ten health influencers' posts (n = 1000) on Sina Weibo guided by the Extended Parallel Processing Model (EPPM) and the transportation theory. These posts were coded in terms of demographic information, topics, message properties (informative, persuasive, and interactive), EPPM variables, and types of evidence (statistical and narrative) used. Results showed that these influencers had a clear emphasis on women's health (OB/GYN diseases and risks related to pregnancy and childcare) and beauty and skincare (in terms of risks and benefits). Overall, they used low fear appeal and high efficacy messages. However, messages containing efficacy information were less likely to be liked. These influencers relied heavily on narrative evidence; however, there was no significant relationship between the use of either narrative or statistical evidence and the number of likes. Differences in the communication strategies in posts about different diseases did exist but were not prevalent.

Basal-Level Effects of (p)ppGpp in the Absence of Branched-Chain Amino Acids in Actinobacillus pleuropneumoniae.

The (p)ppGpp-mediated stringent response (SR) is a highly conserved regulatory mechanism in bacterial pathogens, enabling adaptation to adverse environments, and is linked to pathogenesis. Actinobacillus pleuropneumoniae can cause damage to the lungs of pigs, its only known natural host. Pig lungs are known to have a low concentration of free branched-chain amino acids (BCAAs) compared to the level in plasma. We had investigated the role for (p)ppGpp in viability and biofilm formation of A. pleuropneumoniae Now, we sought to determine whether (p)ppGpp was a trigger signal for the SR in A. pleuropneumoniae in the absence of BCAAs. Combining transcriptome and phenotypic analyses of the wild type (WT) and an relA spoT double mutant [which does not produce (p)ppGpp], we found that (p)ppGpp could repress de novo purine biosynthesis and activate antioxidant pathways. There was a positive correlation between GTP and endogenous hydrogen peroxide content. Furthermore, the growth, viability, morphology, and virulence were altered by the inability to produce (p)ppGpp. Genes involved in the biosynthesis of BCAAs were constitutively upregulated, regardless of the existence of BCAAs, without accumulation of (p)ppGpp beyond a basal level. Collectively, our study shows that the absence of BCAAs was not a sufficient signal to trigger the SR in A. pleuropneumoniae (p)ppGpp-mediated regulation in A. pleuropneumoniae is different from that described for the model organism Escherichia coli Further work will establish whether the (p)ppGpp-dependent SR mechanism in A. pleuropneumoniae is conserved among other veterinary pathogens, especially those in the Pasteurellaceae family.IMPORTANCE (p)ppGpp is a key player in reprogramming transcriptomes to respond to nutritional challenges. Here, we present transcriptional and phenotypic differences of A. pleuropneumoniae grown in different chemically defined media in the absence of (p)ppGpp. We show that the deprivation of branched-chain amino acids (BCAAs) does not elicit a change in the basal-level (p)ppGpp, but this level is sufficient to regulate the expression of BCAA biosynthesis. The mechanism found in A. pleuropneumoniae is different from that of the model organism Escherichia coli but similar to that found in some Gram-positive bacteria. This study not only broadens the research scope of (p)ppGpp but also further validates the complexity and multiplicity of (p)ppGpp regulation in microorganisms that occupy different biological niches.

The cysteine protease ApdS from Streptococcus suis promotes evasion of innate immune defenses by cleaving the antimicrobial peptide cathelicidin LL-37.

Streptococcus suis is a globally distributed zoonotic pathogen associated with meningitis and septicemia in humans, posing a serious threat to public health. To successfully invade and disseminate within its host, this bacterium must overcome the innate immune system. The antimicrobial peptide LL-37 impedes invading pathogens by directly perforating bacterial membranes and stimulating the immune function of neutrophils, which are the major effector cells against S. suis However, little is known about the biological relationship between S. suis and LL-37 and how this bacterium adapts to and evades LL-37-mediated immune responses. In this study by using an array of approaches, including enzyme, chemotaxis, cytokine assays, quantitative RT-PCR, and CD spectroscopy, we found that the cysteine protease ApdS from S. suis cleaves LL-37 and thereby plays a key role in the interaction between S. suis and human neutrophils. S. suis infection stimulated LL-37 production in human neutrophils, and S. suis exposure to LL-37 up-regulated ApdS protease expression in the bacterium. We observed that ApdS targets and rapidly cleaves LL-37, impairing its bactericidal activity against S. suis We attributed this effect to the decreased helical content of the secondary structure in the truncated peptide. Moreover, ApdS rescued S. suis from killing by human neutrophils and neutrophil extracellular traps because LL-37 truncation attenuated neutrophil chemotaxis and inhibited the formation of extracellular traps and the production of reactive oxygen species. Altogether, our findings reveal an immunosuppressive strategy of S. suis whereby the bacterium blunts the innate host defenses via ApdS protease-mediated LL-37 cleavage.

Characterization of a blaNDM-1-carrying IncHI5 plasmid from Enterobacter cloacae complex of food-producing animal origin.

OBJECTIVES: To characterize an NDM-1-encoding multiresistance IncHI5 plasmid from Enterobacter cloacae complex of chicken origin. METHODS: Carbapenemase genes were detected by PCR and Sanger sequencing. The MICs for the E. cloacae complex isolate and its transformant were determined by the agar dilution and broth microdilution methods. Conjugation and electrotransformation were performed to assess the horizontal transferability of the carbapenemase plasmid. Plasmid DNA was isolated from the transformant and fully sequenced using Illumina HiSeq and PacBio platforms. Plasmid stability was investigated by sequential passages on antibiotic-free medium. A circular intermediate was detected by inverse PCR and Sanger sequencing. RESULTS: Plasmid pNDM-1-EC12 carried a conserved IncHI5 backbone and exhibited an MDR phenotype. All antimicrobial resistance genes were clustered in a single MDR region. Genetic environment analysis revealed that the blaNDM-1 gene was in a novel complex integron, In469. Based on sequence analysis, the blaNDM-1-carrying region was thought to be inserted by homologous recombination. Inverse PCR indicated that an ISCR1-mediated circular intermediate can be formed. Plasmid pNDM-1-EC12 was stably maintained both in the parental strain and the transformant without selective pressure. Comprehensive analysis of IncHI5-type plasmids suggested that they may become another key vehicle for rapid transmission of carbapenemase genes. CONCLUSIONS: To the best of our knowledge, this is the first report of a fully sequenced IncHI5 plasmid recovered from an E. cloacae complex strain of food-producing animal origin. Co-occurrence of blaNDM-1 with genes encoding resistance to other antimicrobial agents on the same IncHI5 plasmid may result in the co-selection of blaNDM-1 and facilitates its persistence and rapid dissemination.

Carbon Monoxide Inhibits the Expression of Proteins Associated with Intestinal Mucosal Pyroptosis in a Rat Model of Sepsis Induced by Cecal Ligation and Puncture.

BACKGROUND Carbon monoxide (CO) has anti-inflammatory effects and protects the intestinal mucosal barrier in sepsis. Pyroptosis, or cell death associated with sepsis, is mediated by caspase-1 activation. This study aimed to investigate the role of CO on the expression of proteins associated with intestinal mucosal pyroptosis in a rat model of sepsis induced by cecal ligation and puncture (CLP). MATERIAL AND METHODS The rat model of sepsis was developed using CLP. Male Sprague-Dawley rats (n=120) were divided into six study groups: the sham group (n=20); the CLP group (n=20); the hemin group (treated with ferric chloride and heme) (n=20); the zinc protoporphyrin IX (ZnPPIX) group (n=20); the CO-releasing molecule 2 (CORM-2) group (n=20); and the inactive CORM-2 (iCORM-2) group (n=20). Hemin and CORM-2 were CO donors, and ZnPPIX was a CO inhibitor. In the six groups, the seven-day survival curves, the fluorescein isothiocyanate (FITC)-labeled dextran 4000 Da (FD-4) permeability assay, levels of intestinal pyroptosis proteins caspase-1, caspase-11, and gasdermin D (GSDMD) were measured by confocal fluorescence microscopy. Proinflammatory cytokines interleukin (IL)-18, IL-1ß, and high mobility group box protein 1 (HMGB1) were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). RESULTS CO reduced the mortality rate in rats with sepsis and reduced intestinal mucosal permeability and mucosal damage. CO also reduced the expression levels of IL-18, IL-1ß, and HMGB1, and reduced pyroptosis by preventing the cleavage of caspase-1 and caspase-11. CONCLUSIONS In a rat model of sepsis induced by CLP, CO had a protective role by inhibiting intestinal mucosal pyroptosis.

Differential Abilities of Mammalian Cathelicidins to Inhibit Bacterial Biofilm Formation and Promote Multifaceted Immune Functions of Neutrophils.

Mammalian cathelicidins act as the potent microbicidal molecules for controlling bacterial infection, and are considered promising alternatives to traditional antibiotics. Their ability to modulate host immune responses, as well as their bactericidal activities, is essential for therapeutic interventions. In this study, we compared the bactericidal activities, antibiofilm activities and immune-modulatory properties of cathelicidins BMAP-27, BMAP-34, mCRAMP, and LL-37, and evaluated the therapeutic efficacy of the combination of BMAP-27 and LL-37 using a mouse pulmonary infection model. Our results showed that all of the four cathelicidins effectively killed bacteria via rapid induction of membrane permeabilization, and BMAP-27 exhibited the most excellent bactericidal activity against diverse bacterial pathogens. BMAP-27, mCRAMP, and LL-37 effectively inhibited biofilm formation, while BMAP-34, mCRAMP and LL-37 exerted immunomodulatory functions with varying degrees of efficacy by stimulating the chemotaxis of neutrophils, inducing the production of reactive oxygen species, and facilitating the formation of neutrophil extracellular traps. Of note, the combination of BMAP-27 and LL-37 effectively enhanced the clearance of Pseudomonas aeruginosa and reduced the organ injury in vivo. Together, these findings highlight that identifying the appropriate synergistic combination of mammalian cathelicidins with different beneficial properties may be an effective strategy against bacterial infection.

Genetic characterization of an MDR/virulence genomic element carrying two T6SS gene clusters in a clinical Klebsiella pneumoniae isolate of swine origin.

OBJECTIVES: Multiresistant Klebsiella pneumoniae isolates rarely cause infections in pigs. The aim of this study was to investigate a multiresistant porcine K. pneumoniae isolate for plasmidic and chromosomal antimicrobial resistance and virulence genes and their genetic environment. METHODS: K. pneumoniae strain ZYST1 originated from a pig with pneumonia. Antimicrobial susceptibility testing was performed using broth microdilution. Conjugation experiments were conducted using Escherichia coli J53 as the recipient. The complete sequences of the chromosomal DNA and the plasmids were generated by WGS and analysed for the presence of resistance and virulence genes. RESULTS: The MDR K. pneumoniae ST1 strain ZYST1 contained three plasmids belonging to incompatibility groups IncFIIk5-FIB, IncI1 and IncX4, respectively. The IncFIIk5-FIB plasmid carried the resistance genes aadA2, mph(A), sul1 and aph(3')-Ia, and the IncI1 plasmid carried aadA22 and erm(B). No resistance genes were present on the IncX4 plasmid. Plasmids related to the aforementioned three plasmids were also present in other Enterobacteriaceae species from humans, animals and the environment. Bioinformatic analyses identified a chromosomal 904 kb MDR element flanked by two copies of ISKpn26. This element included virulence factors, such as a type VI secretion system (T6SS) and genes for type 1 fimbriae, the toxin-antitoxin system HipA/HipB, antimicrobial resistance genes, such as blaSHV-187, mdtk, catA and the multiple antibiotic resistance operon marRABC, and heavy metal resistance determinants, such as chrB/chrA and tehA/tehB. CONCLUSIONS: This study reports a novel 904 kb MDR/virulence genomic element and three important plasmids coexisting in a clinical K. pneumoniae isolate of animal origin.

Characterization of a blaIMP-4-carrying plasmid from Enterobacter cloacae of swine origin.

OBJECTIVES: To characterize an MDR blaIMP-4-harbouring plasmid from Enterobacter cloacae EC62 of swine origin in China. METHODS: Plasmid pIMP-4-EC62 from E. cloacae EC62 was transferred by conjugation via filter mating into Escherichia coli J53. Plasmid DNA was extracted from an E. coli J53 transconjugant and sequenced using single-molecule real-time (SMRT) technology. MIC values for both the isolate EC62 and the transconjugant were determined using the broth microdilution and agar dilution methods. Plasmid stability in both the isolate EC62 and the transconjugant was assessed through a series of passages on antibiotic-free media. RESULTS: Plasmid pIMP-4-EC62 is 314351 bp in length, encodes 369 predicted proteins and harbours a novel class 1 integron carrying blaIMP-4 and a group II intron. The blaIMP-4-bearing plasmid belongs to the IncHI2/ST1 incompatibility group. Sequence analysis showed that pIMP-4-EC62 carries four MDR regions and several gene clusters encoding heavy metal resistance. Plasmid pIMP-4-EC62 was stably maintained in both the E. cloacae EC62 isolate and the transconjugant E. coli J53-pIMP-4-EC62 in the absence of selective pressure. Analysis of the evolutionary relatedness of selected IncHI2 plasmids indicates that ST1-type plasmids are key carriers of carbapenemase genes among IncHI2 plasmids. CONCLUSIONS: pIMP-4-EC62 represents the first fully sequenced IncHI2-type blaIMP-4-harbouring plasmid from E. cloacae in China. Co-location of blaIMP-4 with other resistance genes on an MDR plasmid is likely to further accelerate the dissemination of blaIMP-4 by co-selection among bacteria from humans, animals and the environment under the selective pressure of other antimicrobial agents, heavy metals and disinfectants.

Plasmids of Diverse Inc Groups Disseminate the Fosfomycin Resistance Gene fosA3 among Escherichia coli Isolates from Pigs, Chickens, and Dairy Cows in Northeast China.

Thirty-nine fosfomycin-resistant Escherichia coli isolates carrying fosA3 were obtained from pigs, chickens, dairy cows, and staff in four northeastern provinces of China between June 2015 and April 2016. The fosA3 gene was colocated with blaCTX-M genes on conjugative plasmids of the incompatibility groups IncN (n = 12), IncN-F33:A-:B-(n = 2), IncF33:A-:B-(n = 14), IncF14:A-:B-(n = 2), and IncI1/sequence type 136 (ST136) (n = 9). Four different genetic contexts of fosA3 were detected among the 39 E. coli isolates. Three potential epidemic plasmids circulated among E. coli strains from this region.