KpsS1 Mediates the Glycosylation of Pseudaminic Acid in Acinetobacter Baumannii.

Pseudaminic acid (Pse) is found in the polysaccharide structures of the cell surface of various Gram-negative pathogenic bacteria including Acinetobacter baumannii and considered as an important component of cell surface glycans including oligosaccharides and glycoproteins. However, the glycosyltransferase that is responsible for the Pse glycosylation in A. baumannii remains unknown yet. In this study, through comparative genomics analysis of Pse-positive and negative A. baumannii clinical isolates, we identified a potential glycosyltransferase, KpsS1, located right downstream of the Pse biosynthesis genetic locus. Deletion of this gene in an Pse-positive A. baumannii strain, Ab8, impaired the glycosylation of Pse to the surface CPS and proteins, while the gene knockout strain, Ab8ΔkpsS1, could still produce Pse with 2.86 folds higher amount than that of Ab8. Furthermore, impairment of Pse glycosylation affected the morphology and virulence potential of A. baumannii, suggesting the important role of this protein. This study will provide insights into the further understanding of Pse in bacterial physiology and pathogenesis.

Lowering mortality risk in CR-HvKP infection in intestinal immunohistological and microbiota restoration.

Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40 %). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.

Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity.

Class D ß-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this ß-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various ß-lactams were modeled and the potential active site residues that may interact with various ß-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the ß-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type ß-lactamases.

Chromosomal and Plasmid-Borne Tigecycline Resistance Genes tet(X3) and tet(X4) in Dairy Cows on a Chinese Farm.

We isolated 47 Acinetobacter strains carrying tet(X3) and 4 ST767 E. coli strains carrying tet(X4) from 296 rectal swab samples from dairy cows on a Chinese farm. tet(X3) was located on chromosomes or diverse plasmids, and tet(X4) was located on IncFIBκ/FIA(HI1)/X1 nontransferable plasmid. The coexistence of tet(X3) and carbapenemase genes, including blaOXA-58 and blaNDM-1, was detected in 9 Acinetobacter spp. These findings suggested that the use of tetracycline and other antibiotics in food production warrants urgent attention.

Emergence of an Empedobacter falsenii strain harbouring a tet(X)-variant-bearing novel plasmid conferring resistance to tigecycline.

OBJECTIVES: To investigate the genomic and phenotypic characteristics of an MDR Empedobacter falsenii strain isolated from a Chinese patient, which was phenotypically resistant to all last-line antibiotics (carbapenems, colistin and tigecycline). METHODS: Species identity was determined by MALDI-TOF MS analysis. The complete genome sequence of the isolate was determined by WGS and the genetic elements conferring antimicrobial resistance were determined. The origin of this strain was tracked by phylogenetic analysis. RESULTS: The E. falsenii strain was genetically most closely related to an Empedobacter sp. strain isolated from the USA. Members of E. falsenii are speculated to be intrinsically resistant to colistin. The carbapenem resistance of this strain was conferred by a chromosomal blaEBR-2 variant gene. Phylogenetic analysis indicated that the gene encoding the EBR ß-lactamase was widely distributed in Empedobacter spp. Tigecycline resistance was mediated by a tet(X) variant gene encoded by a non-conjugative and non-typeable plasmid. CONCLUSIONS: The MDR phenotype of the E. falsenii isolate was conferred by different mechanisms. Findings from us and others indicate that E. falsenii may serve as a reservoir for carbapenem and tigecycline resistance determinants.

Emergence of OXA-232 Carbapenemase-Producing Klebsiella pneumoniae That Carries a pLVPK-Like Virulence Plasmid among Elderly Patients in China.

This study reported the clonal dissemination of OXA-232-producing sequence type 15 (ST15) carbapenem-resistant Klebsiella pneumoniae among elderly patients in China. All patients were immunocompromised, suffered from multiple underlying diseases, and were hospitalized for a prolonged period; however, they slowly recovered on antimicrobial therapy. The blaOXA-232 gene was in a 6.1-kb ColKP3-type nonconjugative plasmid. The strains displayed a multidrug resistance phenotype and were not hypervirulent despite harboring a virulence plasmid. Active surveillance should be enforced to control further transmission.

Identification and characterization of a conjugative blaVIM-1-bearing plasmid in Vibrio alginolyticus of food origin.

OBJECTIVES: To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978. METHODS: The MICs of V. alginolyticus strain Vb1978 were determined, and the ß-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding plasmid was also obtained using the Illumina and MinION sequencing platforms. RESULTS: V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying conjugative plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The plasmid backbone of pVb1978 was homologous to those of IncP-type plasmids, while its replication region was structurally similar to non-IncP plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon. CONCLUSIONS: A novel blaVIM-1-carrying conjugative plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment.

Characterization of the stability and dynamics of Tn6330 in an Escherichia coli strain by nanopore long reads.

BACKGROUND: The mcr-1 gene has been widely reported in both bacterial chromosomes and plasmids, while its stability in these genetic materials is not well understood. OBJECTIVES: Our aim was to characterize the stability and dynamics of Tn6330 elements in both a plasmid and the chromosome in a single bacterial population. METHODS: Plasmid-borne and chromosomal Tn6330 were characterized by PCR, conjugation, S1-PFGE, stability assay, single-molecule long-read sequencing and bioinformatics analysis. RESULTS: Tn6330 was simultaneously detected in both a plasmid and the chromosome of a clinical Escherichia coli strain. The plasmid was found to comprise the IncFIB replicon and a phage-like replicon, as well as two integrons that harboured various mobile elements and resistance genes including mcr-1, floR, blaTEM-1b and strAB. Both plasmid-borne and chromosomal Tn6330 transposons could be re-organized into a circular intermediate that played a role in transmission of the mcr-1 gene. Tn6330 was found to be very stable in both the plasmid and chromosome after 30 passages of 12 h with or without colistin selective pressure. The decayed structure of Tn6330 in the genuine single DNA molecules of bacterial populations, although occurring at a very low frequency, could be detected for the first time, in which Tn6330 was degraded into a single ISApl1 element. CONCLUSIONS: Long-read sequencing technology is a good tool to study the evolution and stability of genetic elements in bacteria. The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies.

Increased prevalence of Escherichia coli strains from food carrying bla NDM and mcr-1-bearing plasmids that structurally resemble those of clinical strains, China, 2015 to 2017.

IntroductionEmergence of resistance determinants of bla NDM and mcr-1 has undermined the antimicrobial effectiveness of the last line drugs carbapenems and colistin.AimThis work aimed to assess the prevalence of bla NDM and mcr-1 in E. coli strains collected from food in Shenzhen, China, during the period 2015 to 2017.MethodsMultidrug-resistant E. coli strains were isolated from food samples. Plasmids encoding mcr-1 or bla NDM genes were characterised and compared with plasmids found in clinical isolates.ResultsAmong 1,166 non-repeated cephalosporin-resistant E. coli strains isolated from 2,147 food samples, 390 and 42, respectively, were resistant to colistin and meropenem, with five strains being resistant to both agents. The rate of resistance to colistin increased significantly (p < 0.01) from 26% in 2015 to 46% in 2017, and that of meropenem resistance also increased sharply from 0.3% in 2015 to 17% in 2017 (p < 0.01). All meropenem-resistant strains carried a plasmid-borne bla NDM gene. Among the colistin-resistant strains, three types of mcr-1-bearing plasmids were determined. Plasmid sequencing indicated that these mcr-1 and bla NDM-bearing plasmids were structurally similar to those commonly recovered from clinical isolates. Interestingly, both mcr-1-bearing and bla NDM-bearing plasmids were transferrable to E. coli strain J53 under selection by meropenem, yet only mcr-1-bearing plasmids were transferrable under colistin selection.ConclusionThese findings might suggest that mobile elements harbouring mcr-1 and bla NDM have been acquired by animal strains and transmitted to our food products, highlighting a need to prevent a spike in the rate of drug resistant food-borne infections.

Molecular Characterization of qnrVC Genes and Their Novel Alleles in Vibrio spp. Isolated from Food Products in China.

This study reports the prevalences of qnrVC genes in 74 ciprofloxacin-resistant Vibrio sp. isolates. Two novel functional qnrVC alleles, qnrVC8 and qnrVC9, sharing 98% and 99% nucleotide similarity with qnrVC6 and qnrVC7, respectively, were identified. Our findings suggested that carriage of qnrVC alleles, together with target mutations in gyrA and parC genes, may contribute to the development of fluoroquinolone resistance in Vibrio species, posing a serious threat to public health.

Carriage of blaKPC-2 by a virulence plasmid in hypervirulent Klebsiella pneumoniae.

Objectives: To characterize a plasmid in a K1 hypervirulent Klebsiella pneumoniae (HvKP) strain encoding both hypervirulence and carbapenem resistance phenotypes. Methods: Plasmids from HvKP strain KP70-2 were subjected to whole-plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. Results: A hybrid virulence- and resistance-encoding plasmid of 240 kb, harbouring both the virulence gene rmpA2 and the carbapenemase gene blaKPC-2, was recovered from a clinical HvKP strain. Designated pKP70-2, the plasmid was found to be almost structurally identical to various known hypervirulence-encoding plasmids harboured by other HvKP strains, except for an extra MDR-encoding region located within the genetically conserved plasmid backbone. This MDR region was flanked by two copies of IS26 in the same orientation, one at each end and linked to an external 8 bp (CTAAAATT) product of target site duplications, suggesting that an insertion event was responsible for the integration of the MDR region into the virulence plasmid. The MDR region was also found to harbour mobile elements that in turn contain the antibiotic resistance genes dfrA14 and blaKPC-2. Conclusions: Based on the genetic composition of pKP70-2, we postulate that the multiple insertion elements that it harbours were responsible for mediating the plasmid recombination events that underlie continuous emergence and genetic adaptation of novel resistance- and virulence-encoding mobile elements in K. pneumoniae.

IncFII Conjugative Plasmid-Mediated Transmission of blaNDM-1 Elements among Animal-Borne Escherichia coli Strains.

This study aims to investigate the prevalence and transmission dynamics of the blaNDM-1 gene in animal Escherichia coli strains. Two IncFII blaNDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of blaNDM-1 in these strains. The blaNDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

Genetic and Functional Characterization of blaCTX-M-199, a Novel Tazobactam and Sulbactam Resistance-Encoding Gene Located in a Conjugative mcr-1-Bearing IncI2 Plasmid.

We report the genetic and functional characterization of a novel CTX-M-199 ß-lactamase, which was encoded by a blaCTX-M-64 variant gene found in a conjugative mcr-1-bearing IncI2 plasmid and exhibited resistance to ß-lactamase inhibitors, tazobactam, and sulbactam.

Prevalence and Molecular Characterization of mcr-1-Positive Salmonella Strains Recovered from Clinical Specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event.

Molecular Characterization of Escherichia coli Isolates Carrying mcr-1, fosA3, and Extended-Spectrum-ß-Lactamase Genes from Food Samples in China.

This study surveyed the prevalence of mcr-1 in extended-spectrum-ß-lactamase (ESBL)-producing Escherichia coli strains of food origin in China and identified strains that carried mcr-1, fosA3, and ESBL genes, which were carried in various plasmids. The mcr-1 and ESBL genes could be cotransferred by one or more types of plasmids. The presence of these multidrug-resistant E. coli strains in food products might pose a huge threat to public health.

IS26-mediated formation of a virulence and resistance plasmid in Salmonella Enteritidis.

Objectives: To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380. Methods: The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis. Results: Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility. Conclusions: This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains.

Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant.

OBJECTIVES: To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1. METHODS: Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured. RESULTS: Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate. CONCLUSIONS: The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.

Widespread distribution of mcr-1-bearing bacteria in the ecosystem, 2015 to 2016.

The recently discovered colistin resistance-encoding element, mcr-1, adds to the list of mobile resistance genes whose products rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics, but also the last line agents of carbapenems and colistin. The relative prevalence of mcr-1-bearing strains in various ecological niches including 1,371 food samples, 480 animal faecal samples, 150 human faecal samples and 34 water samples was surveyed using a novel in-house method. Bacteria bearing mcr-1 were commonly detected in water (71% of samples), animal faeces (51%), food products (36%), and exhibited stable carriage in 28% of human subjects surveyed. Such strains, which exhibited variable antibiotic susceptibility profiles, belonged to various Enterobacteriaceae species, with Escherichia coli being the most dominant in each specimen type. The mcr-1 gene was detectable in the chromosome as well as plasmids of various sizes. Among these, two conjugative plasmids of sizes ca 33 and ca 60 kb were found to be the key vectors that mediated mcr-1 transmission in organisms residing in various ecological niches. The high mcr-1 carriage rate in humans found in this study highlights the importance of continued vigilance, careful antibiotic stewardship, and the development of new antimicrobials.

IncI1 Plasmids Carrying Various blaCTX-M Genes Contribute to Ceftriaxone Resistance in Salmonella enterica Serovar Enteritidis in China.

Resistance to extended-spectrum ß-lactams in Salmonella, in particular, in serotypes such as Salmonella enterica serovar Enteritidis that are frequently associated with clinical infections, is a serious public health concern. In this study, phenotypic characterization of 433 clinical S. Enteritidis strains obtained from a nationwide collection of the Chinese Center for Disease Control and Prevention during the period from 2005 to 2010 depicted a trend of increasing resistance to ceftriaxone from 2008 onwards. Seventeen (4%) of the strains were found to be resistant to ceftriaxone, 7% were found to be resistant to ciprofloxacin, and 0.7% were found to be resistant to both ciprofloxacin and ceftriaxone. Most of the ceftriaxone-resistant S. Enteritidis strains (15/17) were genetically unrelated and originated from Henan Province. The complete sequence of an IncI1 plasmid, pSE115, which belonged to a novel sequence type, was obtained. This 87,255-bp IncI1 plasmid was found to harbor a blaCTX-M-14 gene in a novel multidrug resistance region (MRR) within the tra locus. Although the majority of strains were also found to contain conjugative IncI1 plasmids with a size similar to that of pSE115 (∼90 kb) and harbor a variety of blaCTX-M group 1 and group 9 elements, the novel MRR site at the tra locus in pSE115 was not detectable in the other IncI1 plasmids. The findings from this study show that cephalosporin resistance in S. Enteritidis strains collected in China was mainly due to the dissemination of IncI1 plasmids carrying blaCTX-M, resembling the situation in which IncI1 plasmids serve as major vectors of blaCTX-M variants in other members of the Enterobacteriaceae.