Search

The main mechanism of virulence in Klebsiella pneumoniae is the acquisition of K. pneumoniae virulence plasmids (KpVPs), which include two dominant types, namely, KpVP-1 (carrying iuc1, iro1, rmpA, and rmpA2) and KpVP-2 (carrying iuc2, iro2, and rmpA). Both are non-conjugative and associated with different hypervirulent clones. In contrast to KpVP-1 reported in K1, K2, and other serotypes of K. pneumoniae, KpVP-2 was only reported in K2 strains and rarely characterized. In this study, we identified a conjugative KpVP-2-type virulence plasmid from a clinical hypervirulent K. pneumoniae strain. This plasmid was generated by the integration of conjugative transfer genes into the KpVP-2-type plasmid Kp52.145 II and could be readily conjugated to Escherichia coli strain EC600 and K. pneumoniae strains of various types which are clinically existing, mediating hypervirulence. Furthermore, this kind of conjugative KpVP-2-type virulence plasmid has been disseminated in clinical settings in Hong Kong and other regions of the world. The generation of conjugative virulence plasmid may promote its transmission and explain the evolution of this type of virulence plasmid.

We recently reported the recovery of a novel IncI1 type conjugative helper plasmid which could target mobile genetic elements (MGE) located in non-conjugative plasmid and form a fusion conjugative plasmid to mediate the horizontal transfer of the non-conjugative plasmid. In this study, interactions between the helper plasmid pSa42-91k and two common MGEs, ISEcp1 and IS15DI, which were cloned into a pBackZero-T vector, were monitored during the conjugation process to depict the molecular mechanisms underlying the plasmid fusion process mediated by insertion sequence (IS) elements. The MinION single-molecule long-read sequencing technology can dynamically reveal the plasmid recombination events and produce valuable information on genetic polymorphism and plasmid heterogeneity in different multidrug resistance (MDR) encoding bacteria. Such data would facilitate the development of new strategies to control evolution and dissemination of MDR plasmids.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains have been increasingly reported, and it is important to understand the evolutionary mechanisms of these highly pathogenic and resistant bacterial pathogens. In this study, we characterized a ST11 carbapenem-resistant K. pneumoniae strain which harbored an IncFIB/IncHI1B type virulence plasmid and an IncFII/IncR type blaKPC–2-bearing plasmid. The virulence plasmid was found to be conjugative and harbored a 35-kbp fragment including aerobactin encoding cluster from virulence plasmid pLVPK and multiple resistance genes, resulting in a mosaic multi-drug resistance and virulence plasmid. This virulence plasmid could be transferred via conjugation to Escherichia coli and K. pneumoniae strains alone as well as together with the blaKPC–2-bearing plasmid. Co-transmission of virulence and blaKPC–2-bearing plasmids would directly convert a classic K. pneumoniae strain into CR-HvKP strain, leading to a sharp increase in the prevalence of CR-HvKP in clinical settings, which poses a great threat to human health.

Resistance to ciprofloxacin, a treatment choice for Salmonella infections, has increased dramatically in recent years in particular in serotype Salmonella Derby with most of strains carrying chromosome-encoded multiple plasmid-mediated quinolone resistance (PMQR) genes. In this work, we discovered a conjugative plasmid, pSa64-96kb, in a Salmonella Derby isolate, namely Sa64, which could extract and fuse to a multiple drug resistance (MDR) DNA fragment containing two PMQR genes, aac(6’)-Ib-cr, and qnrS2 located on the chromosome of the Salmonella strain. This process led to the formation of a new 188 kb fusion plasmid, which could be then subsequently transmitted to recipient strain Escherichia coli J53. The chromosomal MDR DNA fragment was shown to be flanked by one copy of IS26 element at each end and could be excised from the chromosome to form circular intermediate, which was then fused to pSa64-96kb and form a single plasmid through IS26 mediated homologous recombination. The role of IS26 on enhancing the efficacy of fusion and transmission of this chromosomal MDR DNA fragment was further proven in other Salmonella strains. These findings showed that dynamic interaction between specific chromosomal fragment and plasmids may significantly enhance resistance development and transferability of mobile resistance-encoding elements among bacterial pathogens.

Acinetobacter baumannii is an important clinical pathogen which often causes fatal infections among seriously ill patients. Treatment options for managing infections caused by this organism have become limited as a result of emergence of carbapenem resistant strains. In the current study, whole genome sequencing, gene expression studies and enzyme kinetics analyses were performed to investigate the underlying carbapenem resistance mechanisms in fourteen clinical A. baumannii strains isolated from two hospitals, one each in Hong Kong and Henan Province, People’s Republic of China. A large majority of the A. baumannii strains (11/14) were found to belong to the International Clone II (IC-II), among which six were ST208. Twelve of these strains were carbapenem resistant and found to either harbor blaOXA–23/blaOXA–72, or exhibit over-expression of the blaOXA–51 gene upon ISAba1 insertion. Enzymatic assay confirmed that the OXA variants, including those of blaOXA–51, exhibited strong carbapenem-degrading activities. In terms of other intrinsic mechanisms, a weak correlation was observed between reduced production of outer membrane porin CarO/expression resistance-nodulation-division (RND) efflux AdeB and phenotypic resistance. This finding implied that over-production of carbapenem-hydrolyzing-class D-ß-lactamases (CHDLs), including the intrinsic blaOXA–51 gene and the acquired blaOXA–23 and blaOXA–24 elements, is the key mechanism of carbapenem resistance in A. baumannii. This view is confirmed by testing the effect of NaCl, a known blaOXA inhibitor, which was found to cause reduction in carbapenem MIC by twofolds to eightfolds, suggesting that inhibiting OXA type carbapenemases represents the most effective strategy to control phenotypic carbapenem resistance in A. baumannii.

This study aims to investigate and compare the complete nucleotide sequences of the multidrug resistance plasmids pVb0267 and pVb0499, which were recovered from foodborne Vibrio parahaemolyticus isolates, and analyze the genetic environment of blaCTX–M–55 to provide insight into the dissemination mechanisms of this resistance element. Analysis of the sequences of plasmids pVb0267 (166,467 bp) and pVb0499 (192,739 bp) revealed that the backbones of these two plasmids exhibited a high degree of similarity with pR148, a recognized type 1a IncC plasmid recovered from Aeromonas hydrophila (99% identity). The resistance genes, found in both plasmids, included qacH, aadB, arr2, blaOXA–10, aadA1, sul1, tet(A), and blaCTX–M–55, which were mostly arranged in a specific region designated ARI-A. Plasmid pVb0499 was found to possess a larger size of ARI-A than pVb0267, which lacked a mer determination region, a qnr A segment, an aacC3 gene and several mobility-encoding genes. Comparative analysis of resistance island (RI) of these plasmids and others revealed the potential evolution route of these RI sequences. In conclusion, plasmids harboring the blaCTX–M–55 gene has been recovered in Vibrio parahaemolyticus strains of food origin. It is alarming to find that IncC plasmids harboring resistance islands are disseminating in aquatic bacterial strains. The continuous evolution of resistance genes in conjugative plasmid in aquatic bacteria could be due to bacterial adaption to aquaculture environment, where antibiotics were increasingly used.

This study reports the complete sequence of pE80, a conjugative IncFII plasmid recovered from an E. coli strain isolated from chicken meat. This plasmid harbors multiple resistance determinants including oqxAB, fosA3, blaCTX-M-55 and blaTEM-1, and is a close variant of the recently reported p42-2 element, which was recovered from E. coli of veterinary source. Recovery of pE80 constitutes evidence that evolution or genetic re-arrangement of IncFII type plasmids residing in animal-borne organisms is an active event, which involves acquisition and integration of foreign resistance elements into the plasmid backbone. Dissemination of these plasmids may further compromise the effectiveness of current antimicrobial strategies.

A PCR-based assay was developed for more accurate identification of Vibrio parahaemolyticus through targeting the blaCARB-17 like element, an intrinsic β-lactamase gene that may also be regarded as a novel species-specific genetic marker of this organism. Phylogenetic analysis showed that blaCARB-17 like genes were more conservative than the tlh, toxR and atpA genes, the genetic markers commonly used as detection targets in identification of V. parahaemolyticus. Our data showed that this blaCARB-17-specific PCR-based detection approach consistently achieved 100% specificity, whereas PCR targeting the tlh, toxR and atpA genes occasionally produced false positive results. Furthermore, a positive result of this test is consistently associated with an intrinsic ampicillin resistance phenotype of the test organism, presumably conferred by the products of blaCARB-17 like genes. We envision that combined analysis of the unique genetic and phenotypic characteristics conferred by blaCARB-17 shall further enhance the detection specificity of this novel yet easy-to-use detection approach to a level superior to the conventional methods used in V. parahaemolyticus detection and identification.

Hypervirulent strains of Klebsiella pneumoniae (hvKP) are genetic variants of Klebsiella pneumoniae which can cause life-threatening community-acquired infection in healthy individuals. Currently, methods for efficient differentiation between classic K. pneumoniae (cKP) and hvKP strains are not available, often causing delay in diagnosis and treatment of hvKP infections. To address this issue, we devised a Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) approach for rapid identification of K1 hvKP strains. Four standard algorithms, genetic algorithm (GA), support vector machine (SVM), supervised neural network (SNN), and quick classifier (QC), were tested for their power to differentiate between K1 and non-K1 strains, among which SVM was the most reliable algorithm. Analysis of the receiver operating characteristic curves of the interest peaks generated by the SVM model was found to confer highly accurate detection sensitivity and specificity, consistently producing distinguishable profiles for K1 hvKP and non-K1 strains. Of the 43 K. pneumoniae modeling strains tested by this approach, all were correctly identified as K1 hvKP and non-K1 capsule type. Of the 20 non-K1 and 17 K1 hvKP validation isolates, the accuracy of K1 hvKP and non-K1 identification was 94.1% and 90.0% respectively according to the SVM model. In summary, the MALDI-TOF MS approach can be applied alongside the conventional genotyping techniques to provide rapid and accurate diagnosis, and hence prompt treatment of infections caused by hvKP.

This study investigated the transmission characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains collected from a hospital setting in China, in which consistent emergence of CRE strains were observable during the period of May 2013 to February 2014. Among the 45 carbapenem-resistant Enterobacteriaceae (CRE) isolates tested, 21 (47%) strains were found to harbour the blaNDM-1 element, and the rest of 24 CRE strains were all positive for blaKPC-2. The 21 blaNDM-1 –borne strains were found to comprise multiple Enterobacteriaceae species including 9 E. cloacae, 3 E. coli, 3 C. freundii, 2 K. pneumoniae, 2 K. oxytoca and 2 M. morganii strains, indicating that cross-species transmission of blaNDM-1 is a common event. Genetic analyses by PFGE and MLST showed that, with the exception of E. coli and E. cloacae, strains belonging to the same species were often genetically unrelated. In addition to blaNDM-1, several CRE strains were also found to harbour the blaKPC-2, blaVIM-1, and blaIMP-4 elements. Conjugations experiments confirmed that the majority of carbapenem resistance determinants were transferable. Taken together, our findings suggest that transmission of mobile resistance elements among members of Enterobacteriaceae and clonal spread of CRE strains may contribute synergistically to a rapid increase in the population of CRE in clinical settings, prompting a need to implement more rigorous infection control measures to arrest such vicious transmission cycle in CRE-prevalent areas.

Metagenome assembly is a core yet methodologically challenging step for taxonomic classification and functional annotation of a microbiome. This study aims to generate the high-resolution human gut metagenome using both Illumina and Nanopore platforms. Assembly was achieved using four assemblers, including Flye (Nanopore), metaSPAdes (Illumina), hybridSPAdes (Illumina and Nanopore), and OPERA-MS (Illumina and Nanopore). Hybrid metagenome assembly was shown to generate contigs with almost same sizes comparable to those produced using Illumina reads alone, but was more contiguous, informative, and longer compared with those assembled with Illumina reads only. In addition, hybrid metagenome assembly enables us to obtain complete plasmid sequences and much more AMR gene-encoding contigs than the Illumina method. Most importantly, using our workflow, 58 novel high-quality metagenome bins were obtained from four assembly algorithms, particularly hybrid assembly (47/58), although metaSPAdes could provide 11 high-quality bins independently. Among them, 29 bins were currently uncultured bacterial metagenome-assembled genomes. These findings were highly consistent and supported by mock community data tested. In the analysis of biosynthetic gene clusters (BGCs), the number of BGCs in the contigs from hybridSPAdes (241) is higher than that of contigs from metaSPAdes (233). In conclusion, hybrid metagenome assembly could significantly enhance the efficiency of contig assembly, taxonomic binning, and genome construction compared with procedures using Illumina short-read data alone, indicating that nanopore long reads are highly useful in metagenomic applications. This technique could be used to create high-resolution references for future human metagenome studies.