Levofloxacin-induced MexS mutation triggers imipenem-relebactam resistance in a KPC-producing Pseudomonas aeruginosa.

OBJECTIVES: Imipenem-relebactam (IMR), a novel ß-lactam/ß-lactamase inhibitor combination, is recommended for infections caused by difficult-to-treat Pseudomonas aeruginosa. This study aimed to investigate the evolution trajectory of IMR resistance under the selection of levofloxacin in P. aeruginosa. METHODS: Antimicrobial susceptibility testing, complete genome sequencing and gene manipulation experiments were performed. Quantitative reverse transcription PCR for specific genes and porin levels were detected. Evolution trajectory was simulated in vitro by induction assay. RESULTS: P. aeruginosa HS347 and HS355 were isolated from abdominal drainage of two neighbouring patients (S and Z) undergoing surgery of colon carcinoma in Shanghai, China, with the latter patient having received levofloxacin. They were closely related ST16 strains, and both carried blaKPC-2 plasmids highly similar to those of P. aeruginosa endemic clones from Zhejiang province, where patient Z had received enteroscopy before this admission. Acquisition of resistance was observed for both IMR and fluoroquinolones in HS355, likely prompted by treatment with levofloxacin. The T274I substitution in MexS (putative oxidoreductase), upregulated efflux pump operon mexEF-oprN and decreased production of porin OprD leading to cross-resistance to fluoroquinolones and IMR, which was also verified by in vitro mutant selection under levofloxacin selection. CONCLUSIONS: The emergence of a rare blaKPC-2-plasmid-bearing ST16 clone implies the horizonal spread and inter-regional dissemination of a high-risk plasmid-clone combination, representing a public health challenge. Levofloxacin exposure can select for mexS inactivating mutation, which in turn leads to IMR resistance phenotype, implicating the role of an unrelated, widely used antimicrobial agent in insidiously triggering the development of cross resistance to a latest ß-lactam/ß-lactamase inhibitor combination.

Type I BREX system defends against antibiotic-resistant plasmids in Escherichia coli.

The Bacteriophage Exclusion (BREX) system is a novel antiphage defense system identified in Bacillus cereus in 2015. The purpose of this study was to investigate the presence of the BREX system defenses against antibiotic-resistant plasmids such as blaKPC and blaNDM invasion in Escherichia coli. The BREX system was present in 5.4% (23/424) of E. coli clinical isolates and 6.5% (84/1283) of E. coli strains with completely sequenced genomes in the GenBank database. All 23 BREX-positive E. coli clinical isolates were susceptible to carbapenems, while all five isolates carrying blaKPC and 11 carrying blaNDM were BREX-negative. For E. coli strains in the GenBank database, 37 of 38 strains carrying blaKPC and 109 of 111 strains carrying blaNDM were BREX negative. The recognition site sequence of methyltransferase PglX in a clinical E. coli 3756 was 5'-CANCATC-3' using PacBio single-molecular real-time sequencing. The transformation efficiency of plasmid psgRNA-ColAori-target with the PglX recognition site was reduced by 100% compared with the plasmid without the recognition site in E. coli DH5α-pHSG398-BREX. The BREX showed lower defense efficacy against plasmid psgRNA-15Aori-target which had the same plasmid backbone but different surrounding sequences of recognition sites with psgRNA-ColAori-target. The conjugation frequency of the KPC-2 plasmid and NDM-5 plasmid in E. coli 3756-ΔBREX was higher than that in E. coli 3756 clinical isolate (1.0 × 10-6 vs 1.3 × 10-7 and 5.5 × 10-7 vs 1.7 × 10-8, respectively). This study demonstrated that the type I BREX system defends against antibiotic-resistant plasmids in E. coli.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Dissecting the genotypic features of a fluoroquinolone-resistant Pseudomonas aeruginosa ST316 sublineage causing ear infections in Shanghai, China.

Limited information is available regarding the genomic characteristics of P. aeruginosa causing ear infections. Our aim is to characterize the genotypic features of an emerging ST316 sublineage causing aural infections in Shanghai. A total of 199 ear swab isolates were subjected to whole genome sequencing (WGS). Complete genomes for two isolates were resolved. We showed this recently emerged sublineage exhibited high-level resistance to fluoroquinolones (FQs) primarily by accumulation of known mutations in quinolone resistance determining regions (QRDRs). Loss-of-function mutations in mexR and mexCD were frequently detected. Mutations in fusA1 (P166S) and parE (S492F) were resident in this sublinage about 2 years after its emergence. Recombination events might be a key driver of genomic diversity in this sublineage. Convergent evolution events on Multidrug-resistant (MDR) determinants were also observed. We generated predictive machine models and identified biomarkers of resistance to gentamicin, fosfomycin, and cefoperazone-sulbactam in this sublineage. This sublineage tended to be less virulent by loss of a series virulence genes represented by ppkA, rhlI, and iron uptake- and antimicrobial activity-related genes. Specific mutations were detected in pilU and lpxB genes that related to surface structures. Moreover, this sublineage differed from non-ST316 isolates in several ways, including virulence genes related to cell surface structure. Our analysis suggested acquisition of a roughly 390 kbp MDR plasmid carrying qnrVC1 might play an important role in the success of this sublinage. Clonal expansion of this sublineage exhibiting enhanced adaptation to cause ear infections is concerning, which requires urgent control measures to be implemented.

A Novel Anti-CRISPR AcrIE9.2 Is Associated with Dissemination of blaKPC Plasmids in Klebsiella pneumoniae Sequence Type 15.

Sequence type (ST) 15 has become an emerging clone of carbapenem-resistant Klebsiella pneumoniae in which type I-E* CRISPR-Cas usually exists, indicating that the CRISPR-Cas system may not be able to block the transfer of blaKPC plasmids. The purpose of this study was to explore the mechanisms underlying dissemination of blaKPC plasmids in K. pneumoniae ST15. The type I-E* CRISPR-Cas system was present in 98.0% of 612 nonduplicate K. pneumoniae ST15 strains (88 clinical isolates and 524 from the NCBI database). Twelve ST15 clinical isolates were completely sequenced, and self-targeted protospacers were found on blaKPC plasmids flanked by a protospacer adjacent motif (PAM) of AAT in 11 isolates. The type I-E* CRISPR-Cas system was cloned from a clinical isolate and expressed in Escherichia coli BL21(DE3). In BL21(DE3) harboring the CRISPR system, the transformation efficiency of protospacer-bearing plasmids with a PAM of AAT was reduced by 96.2% compared to the empty vector, indicating that the type I-E* CRISPR-Cas system impeded blaKPC plasmid transfer. BLAST for known anti-CRISPR (Acr) amino acid sequences uncovered a novel AcrIE9-like protein with 40.5% to 44.6% sequence identity with AcrIE9 designated AcrIE9.2, which was present in 90.1% (146 of 162) of ST15 strains carrying both blaKPC and the CRISPR-Cas system. When AcrIE9.2 was cloned and expressed in a ST15 clinical isolate, the conjugation frequency of a CRISPR-targeted blaKPC plasmid was increased from 3.96 × 10-6 to 2.01 × 10-4 compared to the AcrIE9.2 absent strain. In conclusion, AcrIE9.2 may be associated with the dissemination of blaKPC in ST15 by repressing CRISPR-Cas activity.

Genomic characterization of colistin resistance in Klebsiella spp. under the pressure of colistin.

Introduction. Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult.Hypothesis/Gap Statement. The molecular mechanisms of colistin resistance in Klebsiella spp. under the pressure of colistin have not been fully investigated.Aim. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible Klebsiella spp. strains under selective pressure of colistin.Methodology. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml-1, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (mgrB, phoP, phoQ, pmrA, pmrB, pmrD, pmrE and pmrK) were measured by quantitative real-time PCR.Results. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in phoQ (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with mgrB (8/18, 44.44 %) was found to be the commonest. Mutation of mgrB led to a significant increase in the MIC for colistin (from 64 to >128 µg ml-1), and a novel mutation C28R in mgrB was first reported in this study.Conclusion. Colistin-resistant Klebsiella spp. could be easily selected under pressure of different concentrations of colistin. Mutations of mgrB, phoP, phoQ and pmrB genes were the main mechanisms leading to chromosomally mediated colistin resistance in Klebsiella spp.

Insertion sequence mediating mrgB disruption is the major mechanism of polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae isolates from China.

OBJECTIVES: Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a huge health challenge worldwide. The aim of this study was to evaluate the incidence of polymyxin resistance in clinical CRKP isolates in China and to characterize the molecular mechanisms underlying these polymyxin-resistant CRKP (PR-CRKP) isolates. METHODS: A total of 493 CRKP clinical isolates from patients were collected from six tertiary-care hospitals in China during 2017-2018. Minimum inhibitory concentrations of polymyxin B and colistin were determined using the broth microdilution method. PR-CRKP isolates were identified and subjected to whole-genome sequencing. Quantitative real-time PCR and structural modelling analysis were also performed. RESULTS: We observed a 2.2% (11/493) polymyxin resistance rate in this multicentre cohort. Polymyxin B MICs ranged from 4 to 64 µg/mL and colistin MICs ranged from 8 to 128 µg/mL in 11 PR-CRKP isolates. Key genetic variations identified in PR-CRKP isolates involved eight disruptions (seven insertional inactivation by an insertion sequence [IS] element, one frameshift deletion) in mgrB, and three missense mutations in pmrA, pmrB, and phoP. ISKpn26 was the predominant IS (4/7), and three of these occurred in nucleotide position 74 in the mgrB gene. In addition, we reported a novel mutation S62R in pmrB that may confer polymyxin resistance in K. pneumoniae. CONCLUSIONS: Our findings highlight the multifaceted molecular mechanisms of polymyxin resistance in CRKP.

Molecular Characterization and Phylogenetic Analysis of the 2019 Dengue Outbreak in Wenzhou, China.

In 2019, a dengue outbreak occurred with 290 confirmed cases in Wenzhou, a coastal city in southeast China. To identify the origin of the dengue virus (DENV) from this outbreak, viral RNA was extracted from four serum samples and sequenced for whole genome analysis. Then, phylogenetic analysis, gene mutation, secondary structure prediction, selection pressure analysis, and recombination analysis were performed. DENV strains Cam-03 and Cam-11 were isolated from patients traveling from Cambodia, while ZJWZ-18 and ZJWZ-62 strains were isolated from local patients without a record of traveling abroad. The whole genome sequence of all four strains was 10,735 nucleotides long. Phylogenetic tree analysis showed that the four strains belonged to genotype 1 of DENV-1, but the local Wenzhou strains and imported strains clustered in different branches. ZJWZ-18 and ZJWZ-62 were closely related to strain MF033254-Singapore-2016, and Cam-03 and Cam-11 were closely related to strain AB608788-China : Taiwan-1994. A comparison of the coding regions between the local strains and the DENV-1 standard strain (EU848545-Hawaii-1944) showed 82 amino acid mutations between the two strains. A total of 55 amino acid mutations were found between the coding regions of the local and imported strains. The overall secondary structure of the 3' UTR of the local strains had changed: apparent changes in the head and tail position were observed when compared to DENV-1 standard strain. Furthermore, selection pressure analysis and recombination detection using the 4 isolates and 41 reference strains showed two credible positive selection sites and eight credible recombination events, which warrant further studies. This study may enhance the understanding of viral replication, infection, evolution, virulence, and pathogenicity of DENV.

Identification of Three Clf-Sdr Subfamily Proteins in Staphylococcus warneri, and Comparative Genomics Analysis of a Locus Encoding CWA Proteins in Staphylococcus Species.

Coagulase-negative Staphylococcus warneri is an opportunistic pathogen that is capable of causing several infections, especially in patients with indwelling medical devices. Here, we determined the complete genome sequence of a clinical S. warneri strain isolated from the blood culture of a 1-year-old nursling patient with acute upper respiratory infection. Genome-wide phylogenetic analysis confirmed the phylogenetic relationships between S. warneri and other Staphylococcus species. Using comparative genomics, we identified three cell wall-anchored (CWA) proteins at the same locus (sdr), named SdrJ, SdrK, and SdrL, on the chromosome sequences of different S. warneri strains. Structural predictions showed that SdrJ/K/L have structural features characteristic of Sdr proteins but exceptionally contained an unusual N-terminal repeat region. However, the C-terminal repetitive (R) region of SdrJ contains a significantly larger proportion of alanine (142/338, 42.01%) than the previously reported SdrI (37.00%). Investigation of the genetic organization revealed that the sdrJ/K/L genes were always followed by one or two glycosyltransferase genes, gtfA and gtfB and were present in an ∼56 kb region bordered by a pair of 8 bp identical direct repeats, named Sw-Sdr. This region was further found to be located on a 160-kb region subtended by a pair of 160-bp direct repeats along with other virulence genes and resistance genes. Sw-Sdr contained a putative integrase that was probably a remnant of a functional integrase. Evidence suggests that Sw-Sdr is improbably an efficient pathogenicity island. A large-scale investigation of Staphylococcus genomes showed that sdr loci were a potential hotspot of insertion sequences (ISs), which could lead to intraspecific diversity at these loci. Our work expanded the repository of Staphylococcus Sdr proteins, and for the first time, we established the connection between sdr loci and phylogenetic relationships and compared the sdr loci in different Staphylococcus species, which provided large insights into the genetic environment of CWA genes in Staphylococcus.

Characterization and identification of SFDC-1, a novel AmpC-type ß-lactamase in Serratia fonticola.

The clinical and environmental infections caused by AmpC ß-lactamases have been increasingly reported recently. In this study, we characterize the novel chromosome-encoded AmpC ß-lactamase SFDC-1 identified in Serratia fonticola strain R28, which was isolated from a rabbit raised on a farm in southern China. SFDC-1 shared the highest amino acid identity of 79.6% with the functionally characterized AmpC ß-lactamase gene blaYRC-1 , although it had highly homologous functionally uncharacterized relatives in the same species from different sources, including some of the clinical significance. The cloned blaSFDC-1 exhibited resistance to a broad spectrum of ß-lactam antibiotics, including most cephalosporins with the highest resistance to ampicillin, cefazolin and ceftazidime, with increased MIC levels ≥128-fold compared with the control strains. The purified SFDC-1 showed catalytic activities against ß-lactams with the highest catalytic activity to cefazolin. The genetic context of blaSFDC-1 and its relatives was conserved in the chromosome, and no mobile genetic elements were found surrounding them.

Identification and characteristics of a novel aminoglycoside phosphotransferase, APH(3')-IId, from an MDR clinical isolate of Brucella intermedia.

OBJECTIVES: To describe a novel chromosomal aminoglycoside phosphotransferase named APH(3')-IId identified in an MDR Brucella intermedia ZJ499 isolate from a cancer patient. METHODS: Species identity was determined by PCR and MALDI-TOF MS analysis. WGS was performed to determine the genetic elements conferring antimicrobial resistance. Gene cloning, transcriptional analysis and targeted gene deletion, as well as protein purification and kinetic analysis, were performed to investigate the mechanism of resistance. RESULTS: APH(3')-IId consists of 266 amino acids and shares the highest identity (48.25%) with the previously known APH(3')-IIb. Expression of aph(3')-IId in Escherichia coli decreased susceptibility to kanamycin, neomycin, paromomycin and ribostamycin. The aph(3')-IId gene in ZJ499 was transcriptionally active under laboratory conditions and the relative abundance of this transcript was unaffected by treatment with the above four antibiotics. However, deletion of aph(3')-IId in ZJ499 results in decreased MICs of these drugs. The purified APH(3')-IId showed phosphotransferase activity against kanamycin, neomycin, paromomycin and ribostamycin, with catalytic efficiencies (kcat/Km) ranging from ∼105 to 107 M-1 s-1. Genetic environment and comparative genomic analyses suggested that aph(3')-IId is probably a ubiquitous gene in Brucella, with no mobile genetic elements detected in its surrounding region. CONCLUSIONS: APH(3')-IId is a novel chromosomal aminoglycoside phosphotransferase and plays an important role in the resistance of B. intermedia ZJ499 to kanamycin, neomycin, paromomycin and ribostamycin. To the best of our knowledge, APH(3')-IId represents the fourth characterized example of an APH(3')-II enzyme.

LncRNA AK087124/miR-224-5p/PTEN axis modulates endothelial cell injury in atherosclerosis through apoptosis and AKT signaling pathway.

Noncoding RNAs (ncRNAs) have been shown to play important roles in atherosclerosis-related endothelial cells dysfunction during atherosclerosis processes. In the study, our purpose was to discover new long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) via competitively interacting each other to regulate the pathogenesis process of atherosclerosis. We investigated the roles of lncRNA AK087124 and miR-224-5p in atherosclerotic pathogenesis and found that AK087124 was up-regulated while miR-224-5p was down-regulated in in the plasma and plaque from atherosclerotic mice compared with normal mice. Ox-LDL was used to establish the mouse aorta endothelial cell (MAEC) injury model. The function study indicated that knockdown of AK087124 inhibited ox-LDL induced endothelial apoptosis and inflammatory response. Bioinformatic prediction combining with luciferase assays indicated that AK087124 could sponge miR-224-5p and enhance the PTEN expression which is a target of miR-224-5p. RNA pull down assays also showed that biotin-miR-224-5p probe could interacted directly with AK087124 and PTEN. Pearson correlation analysis further demonstrated that AK087124 and PTEN expression are negatively correlated with miR-224-5p. Rescue study revealed that miR-224-5p silencing and PTEN overexpression both can reverse the effect of AK087124 on the ox-LDL induced endothelial injury. These data indicated that AK087124 and miR-224-5p could be potential biomarkers and target molecules to treatment and diagnosis for atherosclerosis.

High-Level Aminoglycoside Resistance in Human Clinical Klebsiella pneumoniae Complex Isolates and Characteristics of armA-Carrying IncHI5 Plasmids.

Aminoglycosides are important options for treating life-threatening infections. However, high levels of aminoglycoside resistance (HLAR) among Klebsiella pneumoniae isolates have been observed to be increasing frequently. In this study, a total of 292 isolates of the K. pneumoniae complex from a teaching hospital in China were analyzed. Among these isolates, the percentage of HLAR strains was 13.7% (40/292), and 15 aminoglycoside resistance genes were identified among the HLAR strains, with rmtB being the most dominant resistance gene (70%, 28/40). We also described an armA-carrying Klebsiella variicola strain KP2757 that exhibited a high-level resistance to all aminoglycosides tested. Whole-genome sequencing of KP2757 demonstrated that the strain contained one chromosome and three plasmids, with all the aminoglycoside resistance genes (including two copies of armA and six AME genes) being located on a conjugative plasmid, p2757-346, belonging to type IncHI5. Comparative genomic analysis of eight IncHI5 plasmids showed that six of them carried two copies of the intact armA gene in the complete or truncated Tn1548 transposon. To the best of our knowledge, for the first time, we observed that two copies of armA together with six AME genes coexisted on the same plasmid in a strain of K. variicola with HLAR. Comparative genomic analysis of eight armA-carrying IncHI5 plasmids isolated from humans and sediment was performed, suggesting the potential for dissemination of these plasmids among bacteria from different sources. These results demonstrated the necessity of monitoring the prevalence of IncHI5 plasmids to restrict their worldwide dissemination.

Molecular and Functional Characterization of a Novel Plasmid-Borne bla NDM-Like Gene, bla AFM-1, in a Clinical Strain of Aeromonas hydrophila.

PURPOSE: An increasing frequency of antibiotic resistance has been observed in both clinical and environmental Aeromonas hydrophila isolates in recent years. However, there are still very few in-depth studies regarding the role of plasmids in the antibiotic resistance of A. hydrophila. Hence, we investigated the molecular and functional characterization of a multidrug-resistant plasmid encoding an NDM-like metallo-ß-lactamase, AFM-1, in the clinical A. hydrophila isolate SS332. METHODS: The minimum inhibitory concentrations (MICs) of 24 antibiotics against A. hydrophila SS332 were measured by the agar dilution method. The genome of A. hydrophila SS332 was sequenced with PacBio and Illumina platforms. Six plasmid-borne antimicrobial resistance genes were chosen for cloning, including bla AFM-1, bla OXA-1, msr(E), mph(E), aac(6')-Ib10, and aph(3')-Ia. Phylogenetic analysis, amino acid sequence alignment, and comparative genomic analysis were performed to elucidate the active site requirements and genetic context of the bla AFM-1 gene. RESULTS: A. hydrophila SS332 showed high levels of resistance to 15 antibiotics, especially those with MIC levels at or above 1024 µg/mL, including ampicillin, cefazolin, ceftriaxone, aztreonam, spectinomycin, and roxithromycin. Six plasmid-borne resistance genes from A. hydrophila were verified to be functional in E. coli DH5α. AFM-1 shared 86% amino acid identity with NDM-1 and showed resistance to ampicillin, cefazolin, cefoxitin, and ceftazidime. In addition, the bla AFM-1 gene was associated with three different novel ISCR19-like elements, designated ISCR19-1, ISCR19-2 and ∆ISCR19-3, which may be involved in the acquisition and mobilization of the bla AFM-1 gene. CONCLUSION: Our investigation showed that plasmid-borne resistance genes can contribute to antibiotic resistance in A. hydrophila SS332. A novel bla NDM-like gene, bla AFM-1, was verified to be functional and associated with novel ISCR19-like elements. This fact indicated the risk of spread of bla AFM-1 genes and ISCR19-like elements.

Distribution of ß-Lactamase Genes and Genetic Context of bla KPC-2 in Clinical Carbapenemase-Producing Klebsiella pneumoniae Isolates.

BACKGROUND: This study was designed to characterize the dissemination mechanism and genetic context of Klebsiella pneumoniae carbapenemase (KPC) genes in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates. METHODS: A retrospective analysis was performed on CRKP strains isolated from a teaching hospital of Wenzhou Medical University during 2015-2017. Polymerase chain reaction (PCR)-based amplification and whole-genome sequencing (WGS) were used to analyze the genetic context of the bla KPC-2 gene. Conjugation experiments were performed to evaluate the transferability of bla KPC-2-bearing plasmids. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to investigate the clonal relatedness of bla KPC-2-producing strains. RESULTS: The bla KPC-2 gene was identified from 13.61% (40/294) of clinical K. pneumoniae isolates. Three different sequence types (ST11, ST15 and ST656) and 5 PFGE subtypes (A to E) were classified among them. ST11 was the dominant sequence type (92.50%, 37/40). Plasmid-oriented antibiotic resistance genes, such as extended spectrum-ß-lactamases (ESBLs) and other antimicrobial resistance genes, were also found in KPC-positive K. pneumoniae (KPC-Kp) isolates. Mapping PCR and genomic sequencing revealed that the bla KPC-2-bearing sequence regions, which are related to different mobile elements, including Tn1721- and IS26-based transposons, were mainly located in but not restricted to IncFII-like plasmids and were structurally divergent. CONCLUSION: The bla KPC-2 genes related to divergent mobile genetic elements encoded on transferable plasmids may transfer widely, facilitating the spread of carbapenem resistance among bacteria with different genetic backgrounds. The dissemination of bla KPC-bearing plasmids that collectively carry additional multidrug resistance genes has caused widespread public concern, further limiting the antibiotics available to treat infections caused by KPC-producing pathogens.

Characterization of florfenicol resistance genes in the coagulase-negative Staphylococcus (CoNS) isolates and genomic features of a multidrug-resistant Staphylococcus lentus strain H29.

BACKGROUND: With the wide use of florfenicol to prevent and treat the bacterial infection of domestic animals, the emergence of the florfenicol resistance bacteria is increasingly serious. It is very important to elucidate the molecular mechanism of the bacteria's resistance to florfenicol. METHODS: The minimum inhibitory concentration (MIC) levels were determined by the agar dilution method, and polymerase chain reaction was conducted to analyze the distribution of florfenicol resistance genes in 39 CoNS strains isolated from poultry and livestock animals and seafood. The whole genome sequence of one multidrug resistant strain, Staphylococcus lentus H29, was characterized, and comparative genomics analysis of the resistance gene-related sequences was also performed. RESULTS: As a result, the isolates from the animals showed a higher resistance rate (23/28, 82.1%) and much higher MIC levels to florfenicol than those from seafood. Twenty-seven animal isolates carried 37 florfenicol resistance genes (including 26 fexA, 6 cfr and 5 fexB genes) with one carrying a cfr gene, 16 each harboring a fexA gene, 5 with both a fexA gene and a fexB gene and the other 5 with both a fexA gene and a cfr gene. On the other hand, all 11 isolates from seafood were sensitive to florfenicol, and only 3 carried a fexA gene each. The whole genome sequence of S. lentus H29 was composed of a chromosome and two plasmids (pH29-46, pH29-26) and harbored 11 resistance genes, including 6 genes [cfr, fexA, ant(6)-Ia, aacA-aphD, mecA and mph(C)] encoded on the chromosome, 4 genes [cfr, fexA, aacA-aphD and tcaA] on pH29-46 and 1 gene (fosD) on pH29-26. We found that the S. lentus H29 genome carried two identical copies of the gene arrays of radC-tnpABC-hp-fexA (5671 bp) and IS256-cfr (2690 bp), of which one copy of the two gene arrays was encoded on plasmid pH29-46, while the other was encoded on the chromosome. CONCLUSIONS: The current study revealed the wide distribution of florfenicol resistance genes (cfr, fexA and fexB) in animal bacteria, and to the best of our knowledge, this is the first report that one S. lentus strain carried two identical copies of florfenicol resistance-related gene arrays.

Characterization of an IncR Plasmid with Two Copies of ISCR-Linked qnrB6 from ST968 Klebsiella pneumoniae.

To characterize the molecular structure of IncR plasmid-related sequences, comparative genomic analysis was conducted using 261 IncR plasmid backbone-related sequences. Among the sequences, 257 were IncR plasmids including the multidrug-resistance IncR plasmid pR50-74 from Klebsiella pneumoniae strain R50 of this work, and the other four were from bacterial chromosomes. The IncR plasmids were derived from different bacterial genera or species, mainly Klebsiella pneumoniae (70.82%, 182/257), Escherichia coli (11.28%, 29/257), Enterobacter cloacae (7.00%, 18/257), and Citrobacter freundii (3.50%, 9/257). The bacterial chromosomes carrying IncR plasmid backbone sequences were derived from Proteus mirabilis AOUC-001 and Klebsiella pneumoniae KPN1344, among others. The IncR backbone sequence of P. mirabilis AOUC-001 chromosome shows the highest identity with that of pR50-74. Complex class 1 integrons carrying various copies of ISCR1-sdr-qnrB6-△qacE/sul1 (ISCR1-linked qnrB6 unit) were identified in IncR plasmids. In addition to two consecutive copies of qnrB6-qacE-sul1, the other resistance genes encoded on pR50-74 are all related to mobile genetic elements, such as IS1006, IS26, and the class 1 integron. This study provides a clear understanding of the mobility and plasticity of the IncR plasmid backbone sequence and emphasizes the important role of ISCR in the recruitment of multicopy resistance genes.

Analysis of Resistance to Florfenicol and the Related Mechanism of Dissemination in Different Animal-Derived Bacteria.

Bacterial resistance to antibiotics has become an important concern for public health. This study was aimed to investigate the characteristics and the distribution of the florfenicol-related resistance genes in bacteria isolated from four farms. A total of 106 florfenicol-resistant Gram-negative bacilli were examined for florfenicol-related resistance genes, and the positive isolates were further characterized. The antimicrobial sensitivity results showed that most of them (100, 94.33%) belonged to multidrug resistance Enterobacteriaceae. About 91.51% of the strains carried floR gene, while 4.72% carried cfr gene. According to the pulsed-field gel electrophoresis results, 34 Escherichia coli were subdivided into 22 profiles, the genetic similarity coefficient of which ranged from 80.3 to 98.0%. The multilocus sequence typing (MLST) results revealed 17 sequence types (STs), with ST10 being the most prevalent. The genome sequencing result showed that the Proteus vulgaris G32 genome consists of a 4.06-Mb chromosome, a 177,911-bp plasmid (pG32-177), and a 51,686-bp plasmid (pG32-51). A floR located in a drug-resistant region on the chromosome of P. vulgaris G32 was with IS91 family transposase, and the other floR gene on the plasmid pG32-177 was with an ISCR2 insertion sequence. The cfr gene was located on the pG32-51 flanked by IS26 element and TnpA26. This study suggested that the mobile genetic elements played an important role in the replication of resistance genes and the horizontal resistance gene transfer.

Characterization of a Novel Chromosomal Class C ß-Lactamase, YOC-1, and Comparative Genomics Analysis of a Multidrug Resistance Plasmid in Yokenella regensburgei W13.

Yokenella regensburgei, a member of the family Enterobacteriaceae, is usually isolated from environmental samples and generally resistant to early generations of cephalosporins. To characterize the resistance mechanism of Y. regensburgei strain W13 isolated from the sewage of an animal farm, whole genome sequencing, comparative genomics analysis and molecular cloning were performed. The results showed that a novel chromosomally encoded class C ß-lactamase gene with the ability to confer resistance to ß-lactam antibiotics, designated bla YOC - 1, was identified in the genome of Y. regensburgei W13. Kinetic analysis revealed that the ß-lactamase YOC-1 has a broad spectrum of substrates, including penicillins, cefazolin, cefoxitin and cefotaxime. The two functionally characterized ß-lactamases with the highest amino acid identities to YOC-1 were CDA-1 (71.69%) and CMY-2 (70.65%). The genetic context of the bla YOC - 1 -ampR-encoding region was unique compared with the sequences in the NCBI nucleotide database. The plasmid pRYW13-125 of Y. regensburgei W13 harbored 11 resistance genes (bla OXA - 10, bla LAP - 2, dfrA14, tetA, tetR, cmlA5, floR, sul2, ant(3″)-IIa, arr-2 and qnrS1) within an ∼34 kb multidrug resistance region; these genes were all related to mobile genetic elements. The multidrug resistance region of pYRW13-125 shared the highest identities with those of two plasmids from clinical Klebsiella pneumoniae isolates, indicating the possibility of horizontal transfer of these resistance genes between bacteria of various origins.