Co-production of Tet(X) and MCR-1, two resistance enzymes by a single plasmid.

Tigecycline and colistin are few of 'last-resort' antibiotic defences used in anti-infection therapies against carbapenem-resistant bacterial pathogens. The successive emergence of plasmid-borne tet(X) tigecycline resistance mechanism and mobile colistin resistance (mcr) determinant, renders them clinically useless. Here, we report that co-carriage of tet(X6) and mcr-1 gives co-resistance to both classes of antibiotics by a single plasmid in Escherichia coli. Tet(X6), the new tigecycline resistance enzyme is functionally defined. Both Tet(X6) and MCR-1 robustly interfere accumulation of antibiotic-induced reactive oxygen species (ROS). Unlike that mcr-1 exerts fitness cost in E. coli, tet(X6) does not. In the tet(X6)-positive strain that co-harbors mcr-1, tigecycline resistance is independently of colistin resistance caused by MCR-1-mediated lipid A remodelling, and vice versa. In general consistency with that of MCR-1, Tet(X6) leads to the failure of tigecycline treatment in the infection model of G. mellonella. Taken together, the co-production of Tet(X) and MCR-1 appears as a major clinic/public health concern.

The catalytic process of poly-silicate-ferric (PSF) and generation mechanism of hydroxyl radical based on photo-Fenton system.

Poly-silicate-ferric (PSF) was developed as an heterogeneous UV-Fenton catalyst, which was characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), X-ray fluorescence (XRF), UV-vis diffuse reflectance spectroscopy (DRS), Brunauer-Emmett-Teller (BET) and scanning electron microscopy (SEM). The catalytic process of PSF and generation mechanism of hydroxyl radical based on photo-Fenton system were studied in detail. In the heterogeneous UV-Fenton system, the kapp value of Orange II degradation was as high as 0.268 min-1, which was 1.5 times compared to that with α-FeOOH as catalyst. As a result, the Orange II decolouration and mineralization rates were as high as 99.9% and 92.5% after 40 min treatment, respectively. Moreover, the hydroxyl radical concentration would increase to a peak value of 13.4 µmol/L at about 15 min. The fundamental cause of the high hydroxyl radical generation lay in the high release ability of iron ions from PSF. The peak concentrations of total iron ions and ferrous ions could increase to 4.53 mg/L and 1.57 mg/L at 20 min and 10 min, respectively. After treatment, the re-adsorption of iron ions on the surface of PSF could avoid the additional pollution caused by iron ions. The results confirmed that PSF was a high activity catalyst for an heterogeneous UV-Fenton system.

An IncR Plasmid Harbored by a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Strain Possesses Five Tandem Repeats of the blaKPC-2::NTEKPC-Id Fragment.

Completed sequences of three plasmids from a carbapenem-resistant hypervirulent Klebsiella pneumoniae isolate, SH9, were obtained. In addition to the pLVPK-like virulence-conferring plasmid (pVir-CR-HvKP_SH9), the two multidrug-resistant plasmids (pKPC-CR-HvKP4_SH9 and pCTX-M-CR-HvKP4_SH9) were predicted to originate from a single pKPC-CR-HvKP4-like multireplicon plasmid through homologous recombination. Interestingly, the blaKPC-2 gene was detectable in five tandem repeats exhibiting the format of an NTEKPC-Id-like transposon (IS26-ΔTn3-ISKpn8-blaKPC-2-ΔISKpn6-korC-orf-IS26). The data suggest an important role of DNA recombination in mediating active plasmid evolution.

Identification and Characterization of Conjugative Plasmids That Encode Ciprofloxacin Resistance in Salmonella.

This study aimed to characterize novel conjugative plasmids that encode transferable ciprofloxacin resistance in Salmonella In this study, 157 nonduplicated Salmonella isolates were recovered from food products, of which 55 were found to be resistant to ciprofloxacin. Interestingly, 37 of the 55 CiprSalmonella isolates (67%) did not harbor any mutations in the quinolone resistance-determining regions (QRDR). Six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer the ciprofloxacin resistance phenotype to Escherichia coli J53 (azithromycin resistant [Azir]). The first type of conjugative plasmid belonged to the ∼110-kb IncFIB-type conjugative plasmids carrying qnrB-bearing and aac(6')-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli and Salmonella could confer a ciprofloxacin MIC of 1 to 2 µg/ml. The second type of conjugative plasmid belonged to ∼240-kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS Importantly, this type of conjugative ciprofloxacin resistance plasmid could be detected in clinical Salmonella isolates. The dissemination of these conjugative plasmids that confer ciprofloxacin resistance poses serious challenges to public health and Salmonella infection control.

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.

Dissemination of IncI2 Plasmids That Harbor the blaCTX-M Element among Clinical Salmonella Isolates.

The extended-spectrum-ß-lactamase (ESBL) determinant CTX-M-55 is increasingly prevalent in Escherichia coli but remains extremely rare in Salmonella. This study reports the isolation of a plasmid harboring the blaCTX-M-55 element in a clinical Salmonella enterica serotype Typhimurium strain resistant to multiple antibiotics. This plasmid is genetically identical to several known IncI2-type elements harbored by E. coli strains recovered from animals. This finding indicates that IncI2 plasmids harboring the blaCTX-M genes may undergo cross-species migration among potential bacterial pathogens, with E. coli as the major source of such elements.

Genomic insight into the distribution and genetic environment of blaIMP-4 in clinical carbapenem-resistant Klebsiella pneumoniae strains in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major threat to public health due to its resistance to almost all antibiotics. It is associated with substantial morbidity and mortality and poses a significant challenge to healthcare systems around the globe. Based on our previous nationwide survey of carbapenem-resistant Enterobacteriaceae (CRE) in China, seven blaIMP-4-carrying CRKP isolates were identified, all exhibiting MDR and epidemiologically linked to four different regions in China. WGS analysis revealed that the seven blaIMP-4 genes were all located on plasmids, of which five blaIMP-4 genes were located on the IncHI5 plasmids and the other two belonged to the IncN and IncFIIK plasmids, respectively. Except for the IncHI5 plasmid, conjugation assays revealed that the IncN and IncFIIK plasmids could be transferred to the recipient strain Escherichia coli J53. This study revealed significant genetic variation and identified numerous resistance factors among blaIMP-4-carrying CRKP strains in China, suggesting that blaIMP-4-carrying CRKP strains evolved via multiple phylogenetic routes and highlighting a need for expanded surveillance and establishment of control measures to prevent dissemination of CRKP strains, and facilitate development of more effective antibiotic stewardship policies and infection control programs.

Chasing the landscape for intrahospital transmission and evolution of hypervirulent carbapenem-resistant Klebsiella pneumoniae.

The spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) is a global health concern. Here, we report the intrahospital colonization and spread of Hv-CRKP isolates in a tertiary hospital from 2017 to 2022. Analyses of 90 nonredundant CRKP isolates from 72 patients indicated that Hv-CRKP transferability relies on the dominant ST11-K64 clone. Whole-genome sequencing of 11 representative isolates gave 31 complete plasmid sequences, including 12 KPC-2 resistance carriers and 10 RmpA virulence vehicles. Apart from the binary vehicles, we detected two types of fusion plasmids, favoring the cotransfer of RmpA virulence and KPC-2 resistance. The detection of ancestry/relic plasmids enabled us to establish genetic mechanisms by which rare fusion plasmids form. Unexpectedly, we found a total of five rmpA promoter variants (P9T-P13T) exhibiting distinct activities and varying markedly in their geographic distributions. CRISPR/Cas9 manipulation confirmed that an active PT11-rmpA regulator is a biomarker for the "high-risk" ST11-K64/CRKP clone. These findings suggest clonal spread and clinical evolution of the prevalent ST11-K64/Hv-CRKP clones. Apart from improved public awareness of Hv-CRKP convergence, our findings might benefit the development of surveillance (and/or intervention) strategies for the dominant ST11-K64 lineage of the Hv-CRKP population in healthcare sectors.

A Small KPC-2-Producing Plasmid in Klebsiella pneumoniae: Implications for Diversified Vehicles of Carbapenem Resistance.

The convergence of hypervirulence to carbapenem-resistant K. pneumoniae (CRKP) in a highly transmissible ST11 clone poses a great challenge to public health and anti-infection therapy. Recently, we revealed that an expanding repertoire of diversified KPC-2-producing plasmids occurs in these high-risk clones. Here, we report a clinical case infected with a rare isolate of ST437 CRKP, K186, which exhibited KPC-2 production. Apart from its 5,322,657-bp long chromosome, whole-genome sequencing of strain K186 elucidated three distinct resistance plasmids (designated pK186_1, pK186_2, and pK186_KPC, respectively). Unlike the prevalently larger form of KPC-2-producing plasmids (~120 to ~170 kb) earlier we observed, pK186_KPC is an IncN-type, small plasmid of 26,012bp in length. Combined with the colinear alignment of plasmid genome, the analyses of insertion sequences further suggested that this carbapenem-resistant pK186_KPC might arise from the cointegration of its ancestral IncN and IncFII plasmids, exclusively relying on IS26-based transposition events. Taken together, the result represents an unusual example of blaKPC-2-bearing small plasmids, and highlights an ongoing arsenal of diversified carriers benefiting the transferability of KPC-2 carbapenem resistance. IMPORTANCE A rare ST437 isolate termed K186 was clinically determined which was unlike ST11, the dominant sequence type of CRKP. Whole-genome sequencing enabled us to discover three distinct resistance plasmids, namely, pK186_1, pK186_2, and pK186_KPC. Among them, pK186_KPC appears as a unique plasmid ~26 kb in size, much smaller than the prevalent forms (~120 to ~170 kb). Intriguingly, genetic analysis suggests that it might originate from Proteus mirabilis. This result constitutes an additional example of differentiated plasmid vehicles dedicated to the emergence and dissemination of KPC-2 carbapenem resistance.

Convergence of carbapenem resistance and hypervirulence in a highly-transmissible ST11 clone of K. pneumoniae: An epidemiological, genomic and functional study.

Co-occurrence of hypervirulence and KPC-2 carbapenem resistant phenotypes in a highly-transmissible ST11 clone ofKlebsiella pneumoniae has elicited deep concerns from public health stand point. To address this puzzle, we conducted a large-scale epidemiological, clinical and genomic study of K. pneumonia ST11 clones with both hypervirulence and carbapenem resistance in two tertiary hospitals in Zhejiang province. Most of the patients (15/23) were diagnosed with exclusively carbapenem-resistant K. pneumoniae (CRKP) infections. Ten death cases were reported, some of which are due to the failure of antibiotic therapies. As a result, we identified one new rare sequence types (ST449) to KPC-2-producing CRKP, in addition to the dominant ST11. These clinical isolates of K. pneumoniae are multi-drug resistant and possess a number of virulence factors. Experimental infections of wax moth larvae revealed the presence of hypervirulence at varied level, suggesting the complexity in bacterial virulence factors. However, plasmid curing assays further suggested that the rmpA2-virulence plasmid is associated with, but not sufficient for neither phenotypic hypermucoviscosity nor virulence of K. pneumoniae. Intriguingly, all the rmpA2 genes were found to be inactive due to genetic deletion. In total, we reported 21 complete plasmid sequences comprising 13 rmpA2-positive virulence plasmids and 8 blaKPC-2-harboring resistance plasmids. In addition to the prevalent pLVKP-like virulence plasmid variants (~178kb), we found an unexpected diversity among KPC-2-producing plasmids whose dominant form is IncFII-IncR type (~120kb), rather than the previously anticipated version of ~170kb. These findings provide an updated snapshot of convergence of hypervirulence and carbapenem resistance in ST11 K. pneumoniae.

Carriage of Distinct mcr-1-Harboring Plasmids by Unusual Serotypes of Salmonella.

Colistin acts as a last-resort antibiotic against lethal infections by carbapenem-resistant Enterobacterial pathogens. Enterobacteriaceae carrying mobile colistin resistance (MCR) genes are rapidly emerging and threatening human health and food safety. Despite mcr-1 being prevalent in Escherichia coli, its dissemination in Salmonella is not well characterized. Herein, two unusual serotypes of colistin-resistant Salmonella isolates are reported first, namely serotype Ngor (ST5399) and Goldcoast (ST2529). Using whole genome sequencing, it is shown that mcr-1 is located on the IncHI2-like plasmid pTB501 (188,527 bp) of strain SSDFZ54 and the IncX4-type plasmid pTB602 (33,303 bp) in strain SSDFZ69, respectively. Furthermore, the backbone, tra- and antimicrobial resistance genes relative variable regions in the mcr-1-bearing IncHI2 plasmids are systematically characterized. Phylogenetic analysis shows that all IncHI2-type plasmids from non-Chinese regions are clustered together, suggesting a possible Chinese origin. Taken together, these findings extend the understanding of Salmonella as a vehicle of mcr-1 carriage and distribution.

Characterisation of a cointegrate plasmid harbouring blaNDM-1 in a clinical Salmonella Lomita strain.

This study aimed to characterise the molecular events underlying formation and evolution of a cointegrate plasmid harbouring blaNDM-1 and blaCMY-2 in a clinical Salmonella Lomita (S. Lomita) strain. The Salmonella strain SL131 was found to harbour two multidrug resistant (MDR) plasmids. One plasmid, pSL131_IncHI2, is a typical IncHI2 plasmid containing blaOXA-1, catB3, arr-3, sul1, qnrB4 and blaDHA-1 in a complex class 1 integron. The other plasmid, pSL131_IncA/C-IncX3, is a blaNDM-1-bearing cointegrate plasmid consisting of IncX3 and IncA/C backbones, the formation of which is mediated by IS26. Stability assay showed that the cointegrate plasmid was highly stable in its natural host - S. Lomita - but would readily resolve into single plasmids upon conjugation, during which the IncX3 blaNDM-1-bearing plasmid could be transferred to Escherichia coli strain EC600. Plasmid evolution through integration of two or more MDR plasmids would not only expand the resistance profile of the resultant plasmid, but also broaden the host spectrum of such resistance-encoding mobile elements. Better understanding of the underlying and triggering mechanisms of cointegration may facilitate development of intervention measures to curb formation and dissemination of such elements.

Action and mechanism of the colistin resistance enzyme MCR-4.

Colistin is the last-resort antibiotic against lethal infections with multidrug-resistant bacterial pathogens. A rainbow coalition of mobile colistin resistance (mcr) genes raises global health concerns. Here, we describe the action and mechanism of colistin resistance imparted by MCR-4, a recently-identified member from the broader MCR family. We found that MCR-4 originates from the silenced variant of Shewanella frigidimarina via progressive evolution and forms a phylogenetically-distinct group from the well-studied MCR-1/2 family. Domain-swapping experiments further confirmed that MCR-1 and MCR-4 transmembrane and catalytic domains are not functionally-interchangeable. However, structural and functional analyses demonstrated that MCR-4 possesses a similar PE lipid substrate-recognizable cavity and exploits an almost-identical ping-pong catalysis mechanism. MCR-4 also can alleviate colistin-triggered accumulation of reactive oxygen species (ROS). Taken together, this finding constitutes a functional proof that MCR-4 proceeds in a distinct evolutionary path to fulfill a consistent molecular mechanism, resulting in phenotypic colistin resistance.

Genome analysis of clinical multilocus sequence Type 11 Klebsiella pneumoniae from China.

The increasing prevalence of KPC-producing Klebsiella pneumoniae strains in clinical settings has been largely attributed to dissemination of organisms of specific multilocus sequence types, such as ST258 and ST11. Compared with the ST258 clone, which is prevalent in North America and Europe, ST11 is common in China but information regarding its genetic features remains scarce. In this study, we performed detailed genetic characterization of ST11 K. pneumoniae strains by analyzing whole-genome sequences of 58 clinical strains collected from diverse geographic locations in China. The ST11 genomes were found to be highly heterogeneous and clustered into at least three major lineages based on the patterns of single-nucleotide polymorphisms. Exhibiting five different capsular types, these ST11 strains were found to harbor multiple resistance and virulence determinants such as the blaKPC-2 gene, which encodes carbapenemase, and the yersiniabactin-associated virulence genes irp, ybt and fyu. Moreover, genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA) were detectable in six genomes, whereas genes encoding salmochelin were found in three genomes. In conclusion, our data indicated that carriage of a wide range of resistance and virulence genes constitutes the underlying basis of the high level of prevalence of ST11 in clinical settings. Such findings provide insight into the development of novel strategies for prevention, diagnosis and treatment of K. pneumoniae infections.

Comparative characterization of nontyphoidal Salmonella isolated from humans and food animals in China, 2003-2011.

Food animals are major reservoirs from which specific pathogenic Salmonella strains emerge periodically. Probing the identity and origin of such organisms is essential for formulation of highly-focused infection control measures and analysis of factors underlying dissemination of such strains. In this work, the genetic and phenotypic features of animal and human clinical isolates collected at different geographical localities in China during the period 2003-2011 were characterized and compared. Animal-specific serotypes were identified, with S. Enteritidis, S. Cremieu and S. Fyris being recovered almost exclusively from chicken, ducks and pigs respectively. Nevertheless, only four serotypes were commonly found to be transmitted among both animal and human clinical isolates: S. Enteritidis, S. Typhimurium, S. Derby and S. Indiana. Strains of the serotypes S. Enteritidis and S. Typhimurium not only accounted for up to 50% of all human clinical isolates tested, but often shared identical genetic profiles with the animal isolates. Using a recently identified mobile efflux gene, oqxAB, as genetic marker for assessing the efficiency of transmission between animal and human isolates, we demonstrated that a newly emerged genetic trait could be simultaneously detectable among both animal and human clinical isolates. Findings in this work show that transmission of Salmonellae between animal and human is highly efficient and serotype dependent.

Nationwide Surveillance of Clinical Carbapenem-resistant Enterobacteriaceae (CRE) Strains in China.

The increasing incidence of carbapenem-resistant Enterobacteriaceae (CRE) - mediated hospital infections in China prompted a need to investigate the genetic basis of emergence of such strains. A nationwide survey was conducted in China covering a total of 1105 CRE strains collected from 25 geographical locales with results showing that acquisition of two carbapenemase genes, blaKPC-2 and blaNDM, was responsible for phenotypic resistance in 90% of the CRE strains tested (58% and 32% respectively), among which several major strain types, such as ST11 of K. pneumoniae and ST131/ST167 of E. coli, were identified, suggesting that dissemination of specific resistant clones is mainly responsible for emergence of new CRE strains. Prevalence of the fosA3 gene which mediates fosfomycin resistance, was high, while the colistin resistance determinant mcr-1 was rarely present in these isolates. Consistently, the majority of the blaNDM-bearing plasmids recoverable from the test strains belonged to IncX3, which contained a common core structure, blaNDM-blaMBL-trpF. Likewise, the core structure of ISKpn27-blaKPC-2-ISKpn2 was observed among plasmids harboring the blaKPC-2 gene, although they were genetically more divergent. In conclusion, the increasing prevalence of CRE strains in China is attributed to dissemination of conservative mobile elements carrying blaNDM or blaKPC-2 on conjugative and non-conjugative plasmids.

Selection of target mutation in rat gastrointestinal tract E. coli by minute dosage of enrofloxacin.

It has been suggested that bacterial resistance is selected within a mutation selection window of antibiotics. More recent studies showed that even extremely low concentration of antibiotic could select resistant bacteria in vitro. Yet little is known about the exact antibiotic concentration range that can effectively select for resistant organisms in animal gastrointestinal (GI) tract. In this study, the effect of different dosages of enrofloxacin on resistance and mutation development in rat GI tract E. coli was investigated by determining the number of resistant E. coli recoverable from rat fecal samples. Our data showed that high dose antibiotic treatment could effectively eliminate E. coli with single gyrA mutation in the early course of treatment, yet the eradication effects diminished upon prolonged treatment. Therapeutic and sub-therapeutic dose (1/10 and 1/100 of therapeutic doses) of enrofloxacin could effectively select for mutation in GI tract E. coli at the later course of enrofloxacin treatment and during the cessation periods. Surprisingly, very low dose of enrofloxacin (1/1000 therapeutic dose) could also select for mutation in GI tract E. coli at the later course of enrofloxacin treatment, only with slightly lower efficiency. No enrofloxacin-resistant E. coli could be selected at all test levels of enrofloxacin during long term treatment and the strength of antibiotic treatment does not alter the overall level of E. coli in rat GI tract. This study demonstrated that long term antibiotic treatment seems to be the major trigger for the development of target mutations in GI tract E. coli, which provided insight into the rational use of antibiotics in animal husbandry.

Characterization of a novel zinc transporter ZnuA acquired by Vibrio parahaemolyticus through horizontal gene transfer.

Vibrio parahaemolyticus is a clinically important foodborne pathogen that causes acute gastroenteritis worldwide. It has been shown that horizontal gene transfer (HGT) contributes significantly to virulence development of V. parahaemolyticus. In this study, we identified a novel znuA homolog (vpa1307) that belongs to a novel subfamily of ZnuA, a bacterial zinc transporter. The vpa1307 gene is located upstream of the V. parahaemolyticus pathogenicity island (Vp-PAIs) in both tdh-positive and trh-positive V. parahaemolyticus strains. Phylogenetic analysis revealed the exogenous origin of vpa1307 with 40% of V. parahaemolyticus clinical isolates possessing this gene. The expression of vpa1307 gene in V. parahaemolyticus clinical strain VP3218 is induced under zinc limitation condition. Gene deletion and complementation assays confirmed that vpa1307 contributes to the growth of VP3218 under zinc depletion condition and that conserved histidine residues of Vpa1307 contribute to its activity. Importantly, vpa1307 contributes to the cytotoxicity of VP3218 in HeLa cells and a certain degree of virulence in murine model. These results suggest that the horizontally acquired znuA subfamily gene, vpa1307, contributes to the fitness and virulence of Vibrio species.