RfaH contributes to maximal colonization and full virulence of hypervirulent Klebsiella pneumoniae.

Hypervirulent K. pneumoniae (hvKp) have emerged as clinically important pathogens, posing a serious threat to human health. RfaH, a transcriptional elongation factor, has been regarded as implicated in facilitating the transcription of long virulence operons in certain bacterial species. In K. pneumoniae, RfaH plays a vital role in promoting CPS synthesis and hypermucoviscosity, as well as mediating bacterial fitness during lung infection. In this study, we aim to conduct a systematic investigation of the roles of rfaH in the survival, dissemination, and colonization of hvKp through in vitro and in vivo assays. We found that bacterial cells and colonies displayed capsule -deficient phenotypes subsequent to the deletion of rfaH in K. pneumoniae NTUH-K2044. We confirmed that rfaH is required for the synthesis of capsule and lipopolysaccharide (LPS) by positively regulating the expression of CPS and LPS gene clusters. We found that the ΔrfaH mutant led to a significantly decreased mortality of K. pneumoniae in a mouse intraperitoneal infection model. We further demonstrated that the absence of rfaH was associated with slower bacterial growth under conditions of low nutrition or iron limitation. ΔrfaH displayed reduced survival rates in the presence of human serum. Besides, the engulfment of the ΔrfaH mutant was significantly higher than that of NTUH-K2044 by macrophages in vivo, indicating an indispensable role of RfaH in the phagocytosis resistance of hvKp in mice. Both mouse intranasal and intraperitoneal infection models revealed a higher bacterial clearance rate of ΔrfaH in lungs, livers, and spleens of mice compared to its wild type, suggesting an important role of RfaH in the bacterial survival, dissemination, and colonization of hvKp in vivo. Histopathological results supported that RfaH contributes to the pathogenicity of hvKp in mice. In conclusion, our study demonstrates crucial roles of RfaH in the survival, colonization and full virulence of hvKp, which provides several implications for the development of RfaH as an antibacterial target.

Genomic characterisation of a blaKPC-2- and mcr-10-co-harbouring Enterobacter kobei isolate with high-level resistance to colistin and carbapenems.

OBJECTIVES: The emergence and spread of colistin resistance in carbapenem-resistant Enterobacteriaceae pose a serious threat to human and animal health. This work aimed to characterise the genetic features of antimicrobial resistance of the carbapenem- and colistin-resistant Enterobacter kobei strain SCLZS19, isolated from hospital sewage, by using whole genome sequencing. METHODS: Antimicrobial susceptibility tests were performed using the disk diffusion method. Whole genome sequencing of SCLZS19 was carried out on the HiSeq 2000 combined with PacBio RSII platforms. Sequence type, plasmid incompatibility types, resistance genes, and insertion elements were identified using multilocus sequence typing, PlasmidFinder, ResFinder, and ISfinder, respectively. Conjugation assays were performed using both broth- and filter-based methods with the azide-resistant Escherichia coli J53 as the recipient. The function of the mcr-9-like variant was determined by gene cloning. RESULTS: E. kobei SCLZS19 had a 4 862 177-bp circular chromosome and nine circular plasmids ranging in size from 4120 bp to 282 472 bp. It carried 11 antibiotic resistance genes, and 10 of them were located on plasmids. The colistin resistance gene mcr-10 was located on a 118 766-bp non-transferable IncF (Y3:A-:B-) plasmid. The carbapenemase gene blaKPC-2 was carried by a self-transmissible IncP6 plasmid, which is epidemic in China. In addition, SCLZS19 also carried an mcr-9-like variant on a IncHI2 (ST1) plasmid. The cloning assay showed that the mcr-9-like variant did not mediate colistin resistance in E. coli DH5α. CONCLUSION: The findings highlight that carbapenem- and colistin-resistant Enterobacterales from water environments may serve as a reservoir for clinically significant antibiotic resistance genes, and continuous surveillance is required.

Genomic characterization of tigecycline-resistant Escherichia coli and Klebsiella pneumoniae isolates from hospital sewage.

Introduction: The tigecycline-resistant Enterobacterales have emerged as a great public concern, and the mobile tet(X) variants and tmexCD-toprJ efflux pump are mainly responsible for the spread of tigecycline resistance. Hospital sewage is considered as an important reservoir of antimicrobial resistance, while tigecycline resistance in this niche is under-researched. Methods: In this study, five Escherichia coli and six Klebsiella pneumoniae strains were selected from a collection of tigecycline-resistant Enterobacterales for further investigation by antimicrobial susceptibility testing, conjugation, whole-genome sequencing, and bioinformatics analysis. Results: All five E. coli strains harbored tet(X4), which was located on different plasmids, including a novel IncC/IncFIA(HI1)/IncHI1A/IncHI1B(R27) hybrid structure. In addition, tet(X4)-bearing plasmids were able to transfer by conjugation and be stabilized in the recipient in the absence of antibiotics. tmexCD1-toprJ1 was identified in two K. pneumoniae (LZSFT39 and LZSRT3) and it was carried by a novel multidrug-resistance transposon, designated Tn7368, on a novel IncR/IncU hybrid plasmid. In addition, we found that two K. pneumoniae (LZSFZT3 and LZSRT3) showed overexpression of efflux genes acrB and oqxB, respectively, which was most likely to be caused by mutations in ramR and oqxR. Discussion: In conclusion, the findings in this study expand our knowledge of the genetic elements that carry tigecycline resistance genes, which establishes a baseline for investigating the structure diversity and evolutionary trajectories of human, animal, and environmental tigecycline resistomes.

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 Comamonas aquatica 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 ß-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 ß-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.

Isolation and characterization of three novel lytic phages against K54 serotype carbapenem-resistant hypervirulent Klebsiella pneumoniae.

The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has driven us to explore alternative treatments for the limitation of antimicrobial agents. Lytic phages are considered a promising alternative treatment for CR-hvKP infection. In this study, we reported three novel lytic phages, vB_KpnA_SCNJ1-Z, vB_KpnS_SCNJ1-C, and vB_KpnM_SCNJ1-Y, against a CR-hvKP strain SCNJ1, and they possess genomes of double-stranded DNA with a size of 43,428 bp, 46,039 bp, and 50,360 bp, respectively. Phylogenetic analysis demonstrated that vB_KpnA_SCNJ1-Z belongs to the family Autographiviridae within the class Caudoviricetes, while vB_KpnS_SCNJ1-C and vB_KpnM_SCNJ1-Y are unclassified Caudoviricetes. The phages showed a narrow host range only lysing 1 of 50 tested clinical bacterial strains. The one-step growth curves and stability results showed that the phages displayed relatively short latency periods, with broad pH (pH 3-14) and thermal stabilities (20-60°C). The phages showed significant inhibition of the biofilm formation by SCNJ1 and strong antibacterial activity in vitro. In the mouse model, we demonstrated that administration of a single phage or phage cocktail significantly reduced bacteria loads in the lung, liver, and spleen, and effectively rescued mice from the infection of the SCNJ1 strain, with a survival rate of 70-80%. These findings suggested the three phages have great potential as an alternative therapy with favorable stability and strong antibacterial activity both in vivo and in vitro for the treatment of CR-hvKP infection.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Genetic and virulence characteristics of a Raoultella planticola isolate resistant to carbapenem and tigecycline.

Raoultella planticola is an emerging pathogen causing several infections in humans, and its roles in the propagation of antibiotic resistance genes (ARGs) remain uncharacterized. In this study, a carbapenem and tigecycline-resistant R. planticola isolate was recovered from hospital sewage. It carried nine plasmids, bearing 30 ARGs, including one blaKPC-2 and two blaNDM-1. It also contained a plasmid-borne efflux pump gene cluster, tmexCD1-toprJ, conferring resistance to tigecycline. Analysis of plasmid sequences revealed that both blaNDM-1-carrying plasmids were highly similar to those recovered from humans, reinforcing the close relatedness of environmental and clinical isolates. We also identified that plasmid bearing blaNDM-1 or tmexCD1-toprJ1 was transferable, and can be stabilized in the host bacteria, indicating that the R. planticola isolate has a considerable potential in the dissemination of ARGs. Besides, we found that this isolate could produce biofilm and was virulent in a Galleria mellonella infection model. In conclusion, our study shows the convergence of virulence and multidrug resistance in a R. planticola isolate. This potentially virulent superbug may disseminate into its receiving rivers, and finally to humans through cross-contamination during recreation activities or daily use of water, which poses a risk to public health.

Genomic characteristics of clinical multidrug-resistant Proteus isolates from a tertiary care hospital in southwest China.

Multidrug-resistant (MDR) Proteus, especially those strains producing extended-spectrum ß-lactamases (ESBL) and carbapenemases, represents a major public health concern. In the present work, we characterized 27 MDR Proteus clinical isolates, including 23 Proteus mirabilis, three Proteus terrae, and one Proteus faecis, by whole-genome analysis. Among the 27 isolates analyzed, SXT/R391 ICEs were detected in 14 strains, and the complete sequences of nine ICEs were obtained. These ICEs share a common backbone structure but also have different gene contents in hotspots and variable regions. Among them, ICEPmiChn2826, ICEPmiChn2833, ICEPmiChn3105, and ICEPmiChn3725 contain abundant antibiotic resistance genes, including the ESBL gene bla CTX-M-65. The core gene phylogenetic analysis of ICEs showed their genetic diversity, and revealed the cryptic dissemination of them in Proteus strains from food animals and humans on a China-wide scale. One of the isolates, FZP3105, acquired an NDM-1-producing MDR plasmid, designated pNDM_FZP3105, which is a self-transmissible type 1/2 hybrid IncC plasmid. Analysis of the genetic organization showed that pNDM_FZP3105 has two novel antibiotic resistance islands bearing abundant antibiotic resistance genes, among which bla NDM-1 is located in a 9.0 kb ΔTn125 bracketed by two copies of IS26 in the same direction. In isolates FZP2936 and FZP3115, bla KPC-2 was detected on an IncN plasmid, which is identical to the previously reported pT211 in Zhejiang province of China. Besides, a MDR genomic island PmGRI1, a variant of PmGRI1-YN9 from chicken in China, was identified on their chromosome. In conclusion, this study demonstrates abundant genetic diversity of mobile genetic elements carrying antibiotic resistance genes, especially ESBL and carbapenemase genes, in clinical Proteus isolates, and highlights that the continuous monitoring on their transmission and further evolution is needed.

Genomic Characterization of a Proteus sp. Strain of Animal Origin Co-Carrying blaNDM-1 and lnu(G).

The emergence of carbapenem-resistant Proteus represents a serious threat to global public health due to limited antibiotic treatment options. Here, we characterize a Proteus isolate NMG38-2 of swine origin that exhibits extensive drug resistance, including carbapenems. Whole-genome sequencing based on Illumina and MinION platforms showed that NMG38-2 contains 24 acquired antibiotic resistance genes and three plasmids, among which, pNDM_NMG38-2, a pPvSC3-like plasmid, is transferable and co-carries blaNDM-1 and lnu(G). Sequence analysis of pPvSC3-like plasmids showed that they share a conserved backbone but have a diverse accessory module with complex chimera structures bearing abundant resistance genes, which are facilitated by transposons and/or homologous recombination. The acquisition of blaNDM-1 in pNDM_NMG38-2 was due to the ISCR1-mediated integration event. Comprehensive analysis of the lnu(G)-bearing cassettes carried by bacterial plasmids or chromosomes revealed a diversification of its genetic contexts, with Tn6260 and ISPst2 elements being the leading contributors to the dissemination of lnu(G) in Enterococcus and Enterobacteriaceae, respectively. In conclusion, this study provides a better understanding of the genetic features of pPvSC3-like plasmids, which represent a novel plasmid group as a vehicle mediating the dissemination of blaNDM-1 among bacteria species. Moreover, our results highlight the central roles of Tn6260 and ISPst2 in the spread of lnu(G).

Whole-Genomic Analysis of NDM-5-Producing Enterobacteriaceae Recovered from an Urban River in China.

PURPOSE: Three NDM-5-producing Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, and Citrobacter braakii, one each) were isolated during a screening study for the presence of carbapenemase-producing Enterobacteriaceae (CPE) strains in urban rivers in China. The aim of the present study was to characterize these NDM-5-producing isolates by using whole-genome analysis. METHODS: In vitro susceptibility testing was performed using the broth microdilution method. Conjugation assay was carried out to investigate the transferability of bla NDM-5-harboring plasmids. Whole-genome sequencing was performed using an Illumina HiSeq combined with the PacBio RSII system. The genetic characteristics of the bla NDM-5-harboring plasmids were analyzed. Antimicrobial resistance genes and virulence genes were identified from the genome sequences. Phylogenetic analysis was performed based on core genome. RESULTS: Antimicrobial susceptibility testing showed that all three isolates were resistant to carbapenems, cephalosporins, quinolones, and aminoglycosides, and susceptible to colistin. Whole-genome sequencing showed that each isolate carried multiple antibiotic resistance genes mediating multidrug resistance, and harbored numerous virulence genes, some of which were located on plasmids. In these isolates, bla NDM-5 was carried by an IncX3 plasmid in K. pneumoniae and C. braakii, and on an IncR/IncX1 plasmid in E. coli. Conjugation experiments showed that these bla NDM-5-haboring plasmids were successfully transferred to E. coli J53. Phylogenetic analysis revealed that E. coli SCLZR49 was present in a cluster with isolates of different origin, K. pneumoniae SCLZR50 was mainly clustered with clinical isolates, and C. braakii SCLZR53 had closely genetic relationship with environmental isolates. CONCLUSION: This study revealed contamination of the urban river ecosystems by clinically significant carbapenemase gene bla NDM-5, raising the possibility of plasmid transmission into the environmental from humans and highlighting the need for a constant surveillance of CPE in the environment under the "One-Health" perspective.