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The emergence of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) poses grave threats to human health. These strains increased dramatically in clinical settings in China in the past few years but not in other parts of the world. Four isogenic K. pneumoniae strains, including classical K. pneumoniae, carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKP) and CR-hvKP, were created and subjected to phenotypic characterization, competition assays, mouse sepsis model and rat colonization tests to investigate the mechanisms underlying the widespread nature of CR-hvKP in China. Acquisition of virulence plasmid led to reduced fitness and abolishment of colonization in the gastrointestinal tract, which may explain why hvKP is not clinically prevalent after its emergence for a long time. However, tigecycline treatment facilitated the colonization of hvKP and CR-hvKP and reduced the population of Lactobacillus spp. in animal gut microbiome. Feeding with Lactobacillus spp. could significantly reduce the colonization of hvKP and CR-hvKP in the animal gastrointestinal tract. Our data implied that the clinical use of tigecycline to treat carbapenem-resistant K. pneumoniae infections facilitated the high spread of CR-hvKP in clinical settings in China and demonstrated that Lactobacillus spp. was a potential candidate for anticolonization strategy against CR-hvKP.

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.

Aging, an independent risk factor for cardiometabolic diseases, refers to a progressive deterioration in physiological function, characterized by 12 established hallmarks. Vascular aging is driven by endothelial dysfunction, telomere dysfunction, oxidative stress, and vascular inflammation. This study investigated whether aged gut microbiome promotes vascular aging and metabolic impairment. Fecal microbiome transfer (FMT) was conducted from aged (>75 weeks old) to young C57BL/6 mice (8 weeks old) for 6 weeks. Wire myography was used to evaluate endothelial function in aortas and mesenteric arteries. ROS levels were measured by dihydroethidium (DHE) staining and lucigenin-enhanced chemiluminescence. Vascular and intestinal telomere function, in terms of relative telomere length, telomerase reverse transcriptase expression and telomerase activity, were measured. Systemic inflammation, endotoxemia and intestinal integrity of mice were assessed. Gut microbiome profiles were studied by 16S rRNA sequencing. Some middle-aged mice (40–42 weeks old) were subjected to chronic metformin treatment and exercise training for 4 weeks to evaluate their anti-aging benefits. Six-week FMT impaired glucose homeostasis and caused vascular dysfunction in aortas and mesenteric arteries in young mice. FMT triggered vascular inflammation and oxidative stress, along with declined telomerase activity and shorter telomere length in aortas. Additionally, FMT impaired intestinal integrity, and triggered AMPK inactivation and telomere dysfunction in intestines, potentially attributed to the altered gut microbial profiles. Metformin treatment and moderate exercise improved integrity, AMPK activation and telomere function in mouse intestines. Our data highlight aged microbiome as a mechanism that accelerates intestinal and vascular aging, suggesting the gut-vascular connection as a potential intervention target against cardiovascular aging and complications.

ABSTRACT In this work, we collected foodborne Salmonella strains in Shenzhen, China, during 2014–2017 and investigated the genetic profile of all cefotaxime-resistant isolates in the collection. The strains were subjected to antimicrobial susceptibility tests, whole-genome sequencing, bioinformatics analysis, and conjugation studies. A total of 79 cefotaxime-resistant Salmonella were identified and found to exhibit multidrug resistance. Resistance rate recorded during the study period increased from 1.9% to 9.1%. Salmonella Typhimurium was the predominant serovar, and CTX-M family genes were dominant among the ESBLs genes detected. Notably, CTX-M-bearing plasmids or transposons often contain other drug resistance genes. Furthermore, a combination of CTX-M-55 and CTX-M-65 genes was detected for the first time in foodborne Salmonella strains. Our findings reveal the prevalence and molecular characteristics of cefotaxime-resistant foodborne Salmonella strains in southern China. IMPORTANCE Cefotaxime-resistant Salmonella strains pose an increasing threat to human health by causing infections with limited treatment options. It is therefore necessary to undertake a surveillance on the prevalence of such strains and investigate the resistance and transmission mechanisms. In this work, various ESBL genes flanked by different IS located in different mobile genetic elements were detectable among cefotaxime-resistant Salmonella strains. These data show that the high prevalence and genotypic diversity of cefotaxime-resistant foodborne Salmonella strains in China are possibly attributed to the evolution and transmission of a wide range of multidrug resistance-encoding mobile genetic elements.

ABSTRACT Plasmid-mediated quinolone resistance (PMQR) determinants, such as qnrVC genes, have been widely reported in Vibrio spp. while other types of PMQR genes were rarely reported in these bacteria. This study characterized the phenotypic and genotypic features of foodborne Vibrio spp. carrying qnrS, a key PMQR gene in Enterobacteriaceae. Among a total of 1,811 foodborne Vibrio isolates tested, 34 (1.88%) were found to harbor the qnrS gene. The allele qnrS2 was the most prevalent, but coexistence with other qnr alleles was common. Missense mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes were only found in 11 of the 34 qnrS-bearing isolates. Antimicrobial susceptibility tests showed that all 34 qnrS-bearing isolates were resistant to ampicillin and that a high percentage also exhibited resistance to cefotaxime, ceftriaxone, and trimethoprim-sulfamethoxazole. Genetic analysis showed that these phenotypes were attributed to a diverse range of resistance elements that the qnrS-bearing isolates harbored. The qnrS2 gene could be found in both the chromosome and plasmids; the plasmid-borne qnrS2 genes could be found on both conjugative and nonconjugative plasmids. pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of phenotypic resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would speed up the emergence of multidrug-resistant (MDR) pathogens that are resistant to the most important antibiotics used in treatment of Vibrio infections, suggesting that close monitoring of emergence and dissemination of MDR Vibrio spp. in both food samples and clinical settings is necessary. IMPORTANCE Vibrio spp. used to be very susceptible to antibiotics. However, resistance to clinically important antibiotics, such as cephalosporins and fluoroquinolones, among clinically isolated Vibrio strains is increasingly common. In this study, we found that plasmid-mediated quinolone resistance (PMQR) genes, such as qnrS, that have not been previously reported in Vibrio spp. can now be detected in food isolates. The qnrS2 gene alone could mediate expression of ciprofloxacin resistance in Vibrio spp.; importantly, this gene could be found in both the chromosome and plasmids. The plasmids that harbor the qnrS2 gene could be both conjugative and nonconjugative, among which the pAQU-type qnrS2-bearing conjugative plasmids were able to mediate expression of resistance to both ciprofloxacin and cephalosporins. Transmission of this plasmid among Vibrio spp. would accelerate the emergence of multidrug-resistant pathogens.

Carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains are emerging. Here Xie et al. show that these phenotypes are carried on a plasmid formed from the fusion of a virulence plasmid with a conjugative plasmid.

Summary: We previously reported the recovery of five ST11 carbapenem resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains that harbored pLVPK-like virulence plasmids, yet molecular mechanisms underlying acquisition of virulence plasmid by ST11 K. pneumoniae have not been characterized. In this study, we showed that virulence plasmids in these CR-HvKP strains could be transferred to Escherichia coli strain EC600 via conjugation. Transmission of the virulence plasmids was found to involve formation of fusion plasmids with an Incl1 type conjugative plasmid and a small ColRNAI plasmid through homologous recombination and by insertion sequences IS26 and IS903B. The conjugative fusion event would transform different ST types of K. pneumoniae, in particular, the clinically prevalent ST11 or ST258 CRKP into CR-HvKP. Clinical factors that promote or suppress the occurrence of this fusion process should be further investigated to devise new approaches to halt such bacterial evolution trends.

ABSTRACT A plasmid that harbored the virulence factors highly like those of the virulence plasmid commonly found in clinical hypervirulent Klebsiella pneumoniae strains was detected in a foodborne Escherichia coli strain EC1108 and designated p1108-IncFIB. This virulent-like plasmid was found to be common in E. coli from various sources. To understand the contribution of this plasmid to the virulence of E. coli, plasmid p1108-IncFIB in strain EC1108 was first cured to generate strain EC1108-PC. The virulence plasmid p15WZ-82_Vir in Klebsiella pneumoniae strain 15WZ-82 was then transmitted to EC1108-PC to produce the transconjugant, EC1108-PC-TC to assess the contribution of this virulence plasmid to the virulence level of E. coli. During the process of conjugation, p15WZ-82_Vir was found to be evolved into p15WZ-82_int, which underwent homologous recombination with a plasmid encoding a carbapenemase gene, blaNDM-1, p1108-NDM, in EC1108-PC. Comparison between the level of virulence in the EC1108, EC1108-PC-TC, and EC1108-PC through serum and macrophage resistance assay, as well as animal experiments, confirmed that plasmid p1108-IncFIB encoded a high level of virulence in E. coli, yet the fusion plasmid derived from p15WZ-82_Vir did not encode virulence but instead imposed a high fitness cost in the E. coli strain EC1108-PC-TC. These findings indicate that E. coli strains carrying the virulence plasmid p1108-IncFIB in multidrug-resistant (MDR) strains may also impose serious public health threats like that of hypervirulent Klebsiella pneumoniae strains harboring the p15WZ-82_Vir plasmid. IMPORTANCE Acquisition of pLVPK-like virulence plasmid by Klebsiella pneumoniae converts it to hypervirulent K. pneumoniae (HvKP), which has become one of the most important clinical bacterial pathogens. The potential of transmission of this virulence plasmid and its contribution to the virulence of other Enterobacteriaceae, such as E. coli, are not clear yet. In this study, we showed that pLVPK-like virulence plasmid exhibited fitness costs and did not contribute to the virulence in E. coli. However, we identified a novel virulence plasmid, p1108-IncFIB, that encodes similar siderophore genes as those of pLVPK from a foodborne E. coli strain and showed that p1108-IncFIB encoded a high level of virulence in E. coli. BLAST of E. coli genomes from GenBank showed that these siderophore genes were widespread in clinical E. coli strains. Further studies are warranted to understand the impact of this plasmid in the control of clinical infections caused by E. coli.

ABSTRACT Mechanisms of azithromycin resistance have rarely been reported. In this study, an IncFIB/IncHI1B plasmid that confers resistance to azithromycin was recovered from a clinical Klebsiella pneumoniae strain. This plasmid could be efficiently disseminated to Escherichia coli, Salmonella, and other Gram-negative bacterial pathogens through conjugation. This plasmid was shown to carry three macrolide resistance genes: erm(B), a novel erm(42) gene, and mph(A). The functions of erm(42) were confirmed by direct cloning of this gene and determination of the MIC of azithromycin in strains of various bacterial species which have acquired this gene. Of particular concern is the potential transmission of azithromycin-resistance to extensively drug-resistant (XDR) Salmonella, which causes infections for which treatment options are extremely limited. Monitoring and preventing dissemination of this azithromycin resistance-encoding conjugative plasmid in Enterobacteriaceae is of utmost importance. IMPORTANCE In this study, we identified a conjugative plasmid carrying a novel azithromycin resistance gene, erm(42), from a clinical K. pneumoniae strain. Conjugation of this plasmid into Salmonella conjugants conferred resistance to azithromycin, which is considered a choice for treating Salmonella infections. Of particular concern is the dissemination of this type of azithromycin resistance-encoding conjugative plasmid to extensively drug-resistant (XDR) Salmonella. The study shows that further monitoring of the dissemination of this plasmid in clinical strains of Salmonella spp. is warranted.

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.

Objective: The emergence of multidrug-resistant (MDR) and hypervirulent Klebsiella pneumoniae (hvKP) facilitates simultaneous dissemination of virulence and resistance in a single event, which poses serious threat to public health. Methods: This study characterized the multidrug-resistant and moderately virulent ST11 K64 K. pneumoniae strain HB25-1 from a clinical case with microbiological and genomic approaches. Plasmids from strain HB25-1 were subjected to whole plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. Klebsiella pneumoniae HB25-1 was subjected to a conjugation experiment and Galleria mellonella infection model to evaluate the transmission and virulence potential. Results: We report the emergence of an ST11, serotype K64 K. pneumoniae isolate, which is resistant to third-generation cephalosporin and exhibited a moderate level of virulence. WGS revealed that this strain harboured a plasmid, pHB25-1, which carried multidrug resistance genes (blaDHA-1, qnrB4, dfrA12, aadA2, sul1, aac(3)-lld, blaTEM-1, mph(E)) and virulence-encoding genes (the regulator of mucoid phenotype A gene rmpA2 and the aerobactin gene cluster iutAiucABCD). Genomic analysis indicated that pHB25-1 was formed through co-integration of structural regions located in two different plasmids, enabling it to encode both resistance and virulent phenotypes. Conclusion: Findings in this study provide evidence of active plasmid evolution in K. pneumoniae and suggest that surveillance of multidrug-resistant and hypervirulent K. pneumoniae is urgently needed.

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.

Acinetobacter sp. is among the ESKAPE organisms which represent the major nosocomial pathogens that exhibited a high resistance rate. A. pittii, frequently associated with antimicrobial resistance particularly to carbapenems, is one of the most common Acinetobacter species causing invasive infection. Pandrug resistant A. pittii has rarely been reported. Here, we report the case of a patient with acute exacerbations of chronic obstructive pulmonary disease three years after double lung transplantation and developed severe pneumonia associated with pandrug resistant A. pittii infection. Phenotypic and genomic characteristics of this pandrug resistant isolate (17-84) was identified, and the mechanisms underlying its resistance phenotypes were analyzed. Isolate 17-84 belonged to ST63, carried a non-typable and non-transferable plasmid encoding multiple acquired resistance genes including carbapenemase gene blaOXA-58. Point mutations and acquired resistance genes were identified which were associated with different drug resistance phenotypes. To our knowledge, this is the first detailed phenotypic and genomic characterization of PDR A. pittii causing severe infections in clinical settings. Findings from us and others indicate that A. pittii could serve as a reservoir for carbapenem determinants. The emergence of such a superbug could pose a serious threat to public health. Further surveillance of PDR A. pittii strains and implementation of stricter control measures are needed to prevent this emerging pathogen from further disseminating in hospital settings and the community.

ABSTRACT Our objective was to characterize the genetic features of plasmids harbored by two genetically related, MCR-1 and NDM-5-producing Escherichia coli strains recovered from a chicken meat sample. The genetic profiles of all plasmids harbored by the two test strains, namely, 1106 and 1107, were determined by whole-genome sequencing, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and bioinformatics analysis. The transferability of plasmids harbored by the two strains was assessed by filter mating assay. Strains 1106 and 1107 were resistant to almost all the antibiotics, including colistin and fosfomycin, but remained susceptible to amikacin and tigecycline. The plasmids of p1107-NDM-5 and p1106-NDM-5 both contain a class I integron which lacks the ISAba125 element. The backbone of p1106-IncFII exhibited a high degree of similarity with that of p1106-NDM-5 and p1107-NDM-5, implying that events of plasmid fusion and resolution were involved in the formation of the two plasmids. The plasmids p1106-IncHI2MCR and p1107-IncHI2MCR belong to an IncHI2 replicon type, with three copies of ISApl1 being observed in p1106-IncHI2MCR, implying that the mcr-1 gene was transferable among bacteria that reside in the same food matrix. In this study, p1106-IncFIB, p1107-99K, p1107-111K, and p1107-118K were all found to be phage-like plasmids, with p1106-IncFIB and p1107-118K containing several virulence genes, including iroBCDEN, iucABCD, sitABCD, hlyF, and iss. Surprisingly, resistance genes such as aph(3′)-Ia, sul3, and aac(3′)-IId could also be found in p1107-118K, but resistance genes were not detected in other phage-like plasmids. In conclusion, enhanced surveillance is required to monitor and control the dissemination of various resistance determinants among foodborne pathogens. IMPORTANCE Carbapenem and colistin are last-resort antibiotics used to treat serious clinical infections caused by multidrug-resistant (MDR) bacterial pathogens. Plasmids encoding resistance to carbapenems and colistin have been reported in clinical pathogens in recent years, and yet few studies reported cocarriage of mcr and blaNDM genes in Escherichia coli strains of food origin. How plasmids encoding these two important resistance determinants are being evolved and transmitted in bacterial pathogens is not well understood. In this study, we investigated the genetic features of plasmids harbored by two nonclonal, mcr-1- and blaNDM-5-bearing E. coli strains (1106 and 1107) recovered from a fresh chicken meat sample to understand and provide evidence of the level and dynamics of MDR plasmid transmission. Our data confirmed that active plasmid fusion and resolution events were involved in the formation of plasmids that harbor multiple resistance genes, which provide insights into the further control of plasmid evolution in bacterial pathogens.

ABSTRACT Klebsiella pneumoniae is increasingly being regarded as a reservoir of diverse antibiotic resistance genes and a pathogen that causes severe infections in both the hospital and the community. In this study, we performed molecular characterization of a carbapenem-resistant and highly virulent K. pneumoniae strain recovered from a hospital patient. The virulence-encoding genes rmpA2 and iucABCDiutA were found to be located in the chromosome and are flanked by IS26 elements. These chromosomally located virulence genes, if not incurring fitness costs, are expected to be much more stable than plasmid-located genes. Detailed analysis of the fragment in which these virulence genes are located showed that the fragment can readily form a circular intermediate that may promote integration of this virulence-encoding fragment into various plasmid backbones and other chromosomal regions. Findings in this work provide new insights into mechanisms of transmission of virulence-encoding genes in K. pneumoniae. IMPORTANCE This study reported for the first time and characterized in detail the genetic features of a mobile virulence-encoding fragment located in the chromosome of a clinical virulent K. pneumoniae strain and revealed the occurrence of a transposition event mediated by IS26. This genetic structure could mediate the transposition of the virulence-encoding fragment into various plasmid backbones and chromosomes through formation of a circular intermediate. Therefore, findings in this work provide important insights into the transmission mechanisms of mobile virulence profiles in K. pneumoniae strains and lay the foundation for devising effective intervention approaches aimed at preventing the dissemination of these virulence-encoding elements.

ABSTRACT The incidence of ciprofloxacin resistance in Salmonella has increased dramatically in the past decade. To track the evolutionary trend of ciprofloxacin resistance-encoding genetic elements during this period, we surveyed the prevalence of Salmonella in food products in Shenzhen, China, during the period of 2012 to 2017 and performed whole-genome sequencing and genetic analysis of 566 ciprofloxacin-resistant clinical Salmonella strains collected during this survey. We observed that target gene mutations have become much less common, with single gyrA mutation currently detectable in Salmonella enterica serovar Typhimurium only. Multiple plasmid-mediated quinolone resistance (PMQR) genes located in the chromosome and plasmids are now frequently detectable in ciprofloxacin-resistant Salmonella strains of various serotypes. Among them, the qnrS1 gene was often harbored by multiple plasmids, with p10k-like plasmids being the most dominant. Importantly, p10k-like plasmids initially were not conjugative but became transmissible with the help of a helper plasmid. Ciprofloxacin resistance due to combined effect of carriage of the qnrS1 gene and other resistance mechanisms is common. In S. Typhimurium, carriage of qnrS1 is often associated with a single gyrA mutation; in other serotypes, combination of qnrS1 and other PMQR genes located in the chromosomal fragment or plasmid is observed. Another major mechanism of ciprofloxacin resistance, mainly observable in S. Derby, involves a chromosomal fragment harboring the qnrS2–aac(6′)lb-cr–oqxAB elements. Intriguingly, this chromosomal fragment, flanked by IS26, could form a circular intermediate and became transferrable. To conclude, the increase in the incidence of various PMQR mobile genetic elements and their interactions with other resistance mechanism contribute to a sharp increase in the prevalence of ciprofloxacin-resistant clinical Salmonella strains in recent years. IMPORTANCE Resistance of nontyphoidal Salmonella to fluoroquinolones such as ciprofloxacin is known to be mediated by target mutations. This study surveyed the prevalence of Salmonella strains recovered from 2,989 food products in Shenzhen, China, during the period 2012 to 2017 and characterized the genetic features of several PMQR gene-bearing plasmids and ciprofloxacin resistance-encoding DNA fragments. The emergence of such genetic elements has caused a shift in the genetic location of ciprofloxacin resistance determinants from the chromosomal mutations to various mobile genetic elements. The distribution of these PMQR plasmids showed that they exhibited high serotype specificity, except for the p10k-like plasmids, which can be widely detected and efficiently transmitted among Salmonella strains of various serotypes by fusing to a new conjugative helper plasmid. The sharp increase in the prevalence of ciprofloxacin resistance in recent years may cause a predisposition to the emergence of multidrug-resistant Salmonella strains and pose huge challenges to public health and infection control efforts.

The human gut microbiome is the presumed site in which the emergence and evolution of antibiotic-resistant organisms constantly take place. To delineate the genetic basis of resistance formation in gut microbiome strains, we investigated the changes in the subpopulation structure of Escherichia coli in rat intestine before and after antimicrobial treatment. We observed that antibiotic treatment was selected for an originally minor subpopulation E. coli carrying the biofilm-forming genetic locus pgaABCD and the toxin-antitoxin system HipAB. Such strains possessed dramatically enhanced ability to withstand the detrimental effects of antibiotics, becoming a dominant subspecies upon antibiotic treatment and eventually evolving into resistant mutants. In contrast, E. coli strains that did not carry pgaABCD and HipAB were eradicated upon antibiotic treatment. Our findings, therefore, suggested that genes encoding biofilm-forming ability played an important role in conferring specific gut E. coli strains the ability to evolve into resistant strains upon a prolonged antibiotic treatment, and that such strains may therefore be considered bacterial antibiotic resistance progenitor cells in the gut microbiome.

ABSTRACT We recently reported the recovery of a conjugative virulence plasmid, p15WZ-82_Vir, from a clinical Klebsiella variicola strain. In this study, we found that several new plasmid types were generated due to genetic rearrangement. Partial integration of plasmid p15WZ-82_Vir with existing plasmids such as resistance plasmids by different homologous recombination events was observable in three recipient strains. Such recombination events enable the formation of various types of mosaic plasmids simultaneously carrying virulence-encoding and antibiotic resistance-encoding genes as well as genes involved in plasmid conjugation, which promote transmission of various virulence-encoding and resistance-encoding elements among pathogens. Our data also suggest that these conjugative events may play an integral role in the development of novel mosaic plasmids, which is vital for plasmid evolution. IMPORTANCE Although they are often nonconjugative, large virulence plasmids are increasingly detected in clinical K. pneumoniae and contribute to the hypervirulence phenotype of this organism. In this study, we demonstrated that the virulence-encoding region that originated from virulence plasmid pLVPK actively interacted with different types of plasmids via homologous recombination to generate new conjugative plasmids. This report provides insights into the evolution of self-transmissible plasmids carrying genetic elements encoding both hypervirulent and multidrug-resistant phenotypes, which facilitate the rapid development of clinical K. pneumoniae strains that are hypervirulent and multidrug resistant.

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.

Background: A pandemic outbreak caused by a novel coronavirus, 2019-nCoV, has originated from Wuhan, China and spread to many countries around the world. The outbreak has led to around 45 thousand cases and over one thousand death so far. Methods: Phylogenetic analysis and sequence alignment were used to align the whole genome sequence of 2019-nCoV with other over 200 sequences of coronaviruses to predict the origin of this novel virus. In addition, protein modeling and analysis were performed to access the potential binding of the spike protein of 2019-nCoV with human cell receptor, angiotensin-converting enzyme 2 (ACE2). Results: Detailed genomic and structure-based analysis of a new coronavirus, namely 2019-nCoV, showed that the new virus is a new type of bat coronavirus and is genetically fairly distant from the human SARS coronavirus. Structure analysis of the spike (S) protein of this new virus showed that its S protein only binds much weaker to the ACE2 receptor on human cells whereas the human SARS coronavirus exhibits strongly affinity to the ACE receptor. Conclusions: These findings suggest that the new virus should theoretically not be able to cause very serious human infection when compared to human SARS virus. However, the lower pathogenicity of this new virus may lead to longer incubation time and better adaption to human, which may favor its efficient transmission in human. These data are important to guide design of infection control policy and inform the public on the nature of threat imposed by 2019-nCov. Most importantly, using the analysis platform that we have developed, we should be able to predict whether the new mutations could lead to the increase of infectivity of the mutated virus in a very short time.

Background: A pandemic outbreak caused by a novel coronavirus, 2019-nCoV, has originated from Wuhan, China and spread to many countries around the world. The outbreak has led to around 45 thousand cases and over one thousand death so far. Methods: Phylogenetic analysis and sequence alignment were used to align the whole genome sequence of 2019-nCoV with other over 200 sequences of coronaviruses to predict the origin of this novel virus. In addition, protein modeling and analysis were performed to access the potential binding of the spike protein of 2019-nCoV with human cell receptor, angiotensin-converting enzyme 2 (ACE2). Results: Detailed genomic and structure-based analysis of a new coronavirus, namely 2019-nCoV, showed that the new virus is a new type of bat coronavirus and is genetically fairly distant from the human SARS coronavirus. Structure analysis of the spike (S) protein of this new virus showed that its S protein only binds much weaker to the ACE2 receptor on human cells whereas the human SARS coronavirus exhibits strongly affinity to the ACE receptor. Conclusions: These findings suggest that the new virus should theoretically not be able to cause very serious human infection when compared to human SARS virus. However, the lower pathogenicity of this new virus may lead to longer incubation time and better adaption to human, which may favor its efficient transmission in human. These data are important to guide design of infection control policy and inform the public on the nature of threat imposed by 2019-nCov. Most importantly, using the analysis platform that we have developed, we should be able to predict whether the new mutations could lead to the increase of infectivity of the mutated virus in a very short time.

Objectives: To investigate a set of MDR conjugative plasmids found in Vibrio species and characterize the underlying evolution process.Methods: pAQU-type plasmids from Vibrio species were sequenced using both Illumina and PacBio platforms. Bioinformatics tools were utilized to analyse the typical MDR regions and core genes in the plasmids.Results: The nine pAQU-type plasmids ranged from ∼160 to 206 kb in size and were found to harbour as many as 111 core genes encoding conjugative, replication and maintenance functions. Eight plasmids were found to carry a typical MDR region, which contained various accessory and resistance genes, including ISCR1-blaPER-1-bearing complex class 1 integrons, ISCR2-floR, ISCR2-tet(D)-tetR-ISCR2, qnrVC6, a Tn10-like structure and others associated with mobile elements. Comparison between a plasmid without resistance genes and different MDR plasmids showed that integration of different mobile elements, such as IS26, ISCR1, ISCR2, IS10 and IS6100, into the plasmid backbone was the key mechanism by which foreign resistance genes were acquired during the evolution process.Conclusions: This study identified pAQU-type plasmids as emerging MDR conjugative plasmids among important pathogens from different origins in Asia. These findings suggest that aquatic bacteria constitute a major reservoir of resistance genes, which may be transmissible to other human pathogens during food production and processing. [ABSTRACT FROM AUTHOR]