An inhibitory mechanism of AasS, an exogenous fatty acid scavenger: Implications for re-sensitization of FAS II antimicrobials.

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 µM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation of anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors.

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.

Construction and application of the conditionally essential gene knockdown library in Klebsiella pneumoniae to screen potential antimicrobial targets and virulence genes via Mobile-CRISPRi-seq.

Klebsiella pneumoniae is a ubiquitous human pathogen, and its clinical treatment faces two major challenges: multidrug resistance and the pathogenesis of hypervirulent K. pneumoniae. The discovery and study of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern due to their restriction in the development of novel antibiotics. However, the lack of essential functional genomic data has hampered the study of the mechanisms of essential genes related to antimicrobial susceptibility. In this study, we developed a pooled CE genes mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference screening method (Mobile-CRISPRi-seq) for K. pneumoniae to identify genes that play critical roles in antimicrobial fitness in vitro and host immunity in vivo. Targeting 870 predicted CE genes in K. pneumoniae, Mobile-CRISPRi-seq uncovered the depletion of tetrahydrofolate synthesis pathway genes folB and folP under trimethoprim pressure. Our screening also identified genes waaE and fldA related to polymyxin and ß-lactam susceptibility by applying a screening strategy based on Mobile-CRISPRi-seq and comparative genomics. Furthermore, using a mouse infection model and Mobile-CRISPRi-seq, multiple virulence genes were identified, and among these genes, pal, yciS, and ribB were demonstrated to contribute to the pathogenesis of K. pneumoniae. This study provides a simple, rapid, and effective platform for screening potential antimicrobial targets and virulence genes in K. pneumoniae, and this broadly applicable system can be expanded for high-throughput functional gene study in multiple pathogenic bacteria, especially in gram-negative bacteria. IMPORTANCE The discovery and investigation of conditionally essential (CE) genes that can function as potential antimicrobial targets has always been a research concern because of the restriction of antimicrobial targets in the development of novel antibiotics. In this study, we developed a pooled CE gene-wide mobile clustered regularly interspaced short palindromic repeat (CRISPR) interference sequencing (Mobile-CRISPRi-seq) strategy in Klebsiella pneumoniae to identify genes that play critical roles in the fitness of antimicrobials in vitro and host immunity in vivo. The data suggest a robust tool to screen for loss-of-function phenotypes in a pooled gene knockdown library in K. pneumoniae, and Mobile-CRISPRi-seq may be expanded to multiple bacteria for screening and identification of genes with crucial roles in the fitness of antimicrobials and hosts.

An updated atlas of antibody evasion by SARS-CoV-2 Omicron sub-variants including BQ.1.1 and XBB.

Emerging Omicron sub-variants are causing global concerns, and their immune evasion should be monitored continuously. We previously evaluated the escape of Omicron BA.1, BA.1.1, BA.2, and BA.3 from an atlas of 50 monoclonal antibodies (mAbs), covering seven epitope classes of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor-binding domain (RBD). Here, we update the atlas of totally 77 mAbs against emerging sub-variants including BQ.1.1 and XBB and find that BA.4/5, BQ.1.1, and XBB display further evasion. Besides, investigation into the correlation of binding and neutralization of mAbs reveals the important role of antigenic conformation in mAb functioning. Moreover, the complex structures of BA.2 RBD/BD-604/S304 and BA.4/5 RBD/BD-604/S304/S309 further elucidate the molecular mechanism of antibody evasion by these sub-variants. By focusing on the identified broadly potent mAbs, we find a general hotspot epitope on the RBD, which could guide the design of vaccines and calls for new broad-spectrum countermeasures against COVID-19.

Carriage and Transmission of mcr-1 in Salmonella Typhimurium and Its Monophasic 1,4,[5],12:i:- Variants from Diarrheal Outpatients: a 10-Year Genomic Epidemiology in Guangdong, Southern China.

The banning of colistin as a feed additive for food-producing animals in mainland China in 2017 caused the decline in the prevalence of Escherichia coli-mobilized colistin resistance (mcr-1) in China. Salmonella Typhimurium and its monophasic 1,4,[5],12:i:- variants are also the main species associated with the spread of mcr-1; however, the evidence of the prevalence and transmission of mcr-1 among Salmonella is lacking. Herein, the 5,354 Salmonella isolates recovered from fecal samples of diarrheal patients in Guangdong, Southern China, from 2009 to 2019 were screened for colistin resistance and mcr-1, and mcr-1-positive isolates were characterized based on whole-genome sequencing (WGS) data. Relatively high prevalence rates of colistin resistance and mcr-1 (4.05%/4.50%) were identified, and more importantly, the prevalence trends of colistin-resistant and mcr-1-positive Salmonella isolates had a similar dynamic profile, i.e., both were first detected in 2012 and rapidly increased during 2013 to 2016, followed by a sharp decrease since 2017. WGS and phylogenetic analysis indicate that, whether before or after the ban, the persistence and cross-hospital transmission of mcr-1 are primarily determined by IncHI2 plasmids with similar backbones and sequence type 34 (ST34) Salmonella in specific clades that are associated with a high prevalence of IncHI2 plasmids and clinically important antimicrobial resistance genes, including blaCTX-M-14-fosA3-oqxAB-floR genotypes. Our work reveals the difference in the prevalence rate of mcr-1 in clinical Salmonella before and after the Chinese colistin ban, whereas mcr-1 transmission was closely linked to multidrug-resistant IncHI2 plasmid and ST34 Salmonella across diverse hospitals over 10 years. Continued surveillance is required to explore the factors related to a sharp decrease in mcr-1 after the recent ban and determine whether the ban has affected the carriage of mcr-1 in Salmonella circulating in the health care system. IMPORTANCE Colistin is one of the last-line antibiotics for the clinical treatment of Enterobacteriaceae. However, the emergence of the mobilized colistin resistance (mcr-1) gene has spread throughout the entire human health system and largely threatens the usage of colistin in the clinical setting. In this study, we investigated the existence of mcr-1 in clinical Salmonella from a 10-year continuous surveillance and genomic study. Overall, the colistin resistance rate and mcr-1 carriage of Salmonella in tertiary hospitals in Guangdong (2009 to 2019) were relatively high and, importantly, rapidly increased from 2013 to 2016 and significantly decreased after the Chinese colistin withdrawal. However, before or after the ban, the MDR IncHI2 plasmid with a similar backbone and ST34 Salmonella were the main vectors involved in the spread of mcr-1. Interestingly, these Chinese mcr-1-carrying Salmonella obtain phylogenetically and phylogeographically distinct patterns compared with those from other continents and are frequently associated with clinically important ARGs including the extended-spectrum ß-lactamases. Our data confirmed that the national stewardship intervention seems to be successful in blocking antibiotic resistance determinants and that continued surveillance of colistin resistance in clinical settings, farm animals, and related products is necessary.

The opportunistic pathogen Pseudomonas aeruginosa exploits bacterial biotin synthesis pathway to benefit its infectivity.

Pseudomonas aeruginosa is an opportunistic pathogen that predominantly causes nosocomial and community-acquired lung infections. As a member of ESKAPE pathogens, carbapenem-resistant P. aeruginosa (CRPA) compromises the limited therapeutic options, raising an urgent demand for the development of lead compounds against previously-unrecognized drug targets. Biotin is an important cofactor, of which the de novo synthesis is an attractive antimicrobial target in certain recalcitrant infections. Here we report genetic and biochemical definition of P. aeruginosa BioH (PA0502) that functions as a gatekeeper enzyme allowing the product pimeloyl-ACP to exit from fatty acid synthesis cycle and to enter the late stage of biotin synthesis pathway. In relative to Escherichia coli, P. aeruginosa physiologically requires 3-fold higher level of cytosolic biotin, which can be attributed to the occurrence of multiple biotinylated enzymes. The BioH protein enables the in vitro reconstitution of biotin synthesis. The repertoire of biotin abundance is assigned to different mouse tissues and/or organ contents, and the plasma biotin level of mouse is around 6-fold higher than that of human. Removal of bioH renders P. aeruginosa biotin auxotrophic and impairs its intra-phagosome persistence. Based on a model of CD-1 mice mimicking the human environment, lung challenge combined with systemic infection suggested that BioH is necessary for the full virulence of P. aeruginosa. As expected, the biotin synthesis inhibitor MAC13772 is capable of dampening the viability of CRPA. Notably, MAC13772 interferes the production of pyocyanin, an important virulence factor of P. aeruginosa. Our data expands our understanding of P. aeruginosa biotin synthesis relevant to bacterial infectivity. In particular, this study represents the first example of an extracellular pathogen P. aeruginosa that exploits biotin cofactor as a fitness determinant, raising the possibility of biotin synthesis as an anti-CRPA target.

Structure and mechanism for streptococcal fatty acid kinase (Fak) system dedicated to host fatty acid scavenging.

Staphylococcus and Streptococcus, two groups of major human pathogens, are equipped with a fatty acid kinase (Fak) machinery to scavenge host fatty acids. The Fak complex is contains an ATP-binding subunit FakA, which interacts with varied FakB isoforms, and synthesizes acyl-phosphate from extracellular fatty acids. However, how FakA recognizes its FakB partners and then activates different fatty acids is poorly understood. Here, we systematically describe the Fak system from the zoonotic pathogen, Streptococcus suis. The crystal structure of SsFakA complexed with SsFakB2 was determined at 2.6 Å resolution. An in vitro system of Fak-PlsX (phosphate: acyl-ACP transacylase) was developed to track acyl-phosphate intermediate and its final product acyl-ACP. Structure-guided mutagenesis enabled us to characterize a mechanism for streptococcal FakA working with FakB partners engaged in host fatty acid scavenging. These findings offer a comprehensive description of the Fak kinase machinery, thus advancing the discovery of attractive targets against deadly infections with Streptococcus.

Three enigmatic BioH isoenzymes are programmed in the early stage of mycobacterial biotin synthesis, an attractive anti-TB drug target.

Tuberculosis (TB) is one of the leading infectious diseases of global concern, and one quarter of the world's population are TB carriers. Biotin metabolism appears to be an attractive anti-TB drug target. However, the first-stage of mycobacterial biotin synthesis is fragmentarily understood. Here we report that three evolutionarily-distinct BioH isoenzymes (BioH1 to BioH3) are programmed in biotin synthesis of Mycobacterium smegmatis. Expression of an individual bioH isoform is sufficient to allow the growth of an Escherichia coli ΔbioH mutant on the non-permissive condition lacking biotin. The enzymatic activity in vitro combined with biotin bioassay in vivo reveals that BioH2 and BioH3 are capable of removing methyl moiety from pimeloyl-ACP methyl ester to give pimeloyl-ACP, a cognate precursor for biotin synthesis. In particular, we determine the crystal structure of dimeric BioH3 at 2.27Å, featuring a unique lid domain. Apart from its catalytic triad, we also dissect the substrate recognition of BioH3 by pimeloyl-ACP methyl ester. The removal of triple bioH isoforms (ΔbioH1/2/3) renders M. smegmatis biotin auxotrophic. Along with the newly-identified Tam/BioC, the discovery of three unusual BioH isoforms defines an atypical 'BioC-BioH(3)' paradigm for the first-stage of mycobacterial biotin synthesis. This study solves a long-standing puzzle in mycobacterial nutritional immunity, providing an alternative anti-TB drug target.

Low-Level Cefepime Exposure Induces High-Level Resistance in Environmental Bacteria: Molecular Mechanism and Evolutionary Dynamics.

Antibiotics exert selective pressures on clinically relevant antibiotic resistance. It is critical to understand how antibiotic resistance evolves in environmental microbes exposed to subinhibitory concentrations of antibiotics and whether evolutionary dynamics and emergence of resistance are predictable. In this study, Comamonas testosteroni isolated from wastewater activated sludge were subcultured in a medium containing 10 ng/mL cefepime for 40 days (∼300 generations). Stepwise mutations were accumulated, leading to an ultimate 200-fold increase in the minimum inhibitory concentration (MIC) of cefepime. Early stage mutation in DNA polymerase-encoding gene dnaE2 played an important role in antibiotic resistance evolution. Diverse resistance mechanisms were employed and validated experimentally, including increased efflux, biofilm formation, reduced antibiotic uptake, and drug inactivation. The cefepime minimal selective concentrations (MSCs) and relative fitness of susceptible, intermediate, and resistant mutants were determined. Agent-based modeling of the modified Moran process enabled simulations of resistance evolution and predictions of the emergence time and frequency of resistant mutants. The unraveled cefepime resistance mechanisms could be employed by broader bacteria, and the newly developed model is applicable to the predictions of general resistance evolution. The improved knowledge facilitates the assessment, prediction, and mitigation of antibiotic resistance progression in antibiotic-polluted environments.

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.

Transferability of tigecycline resistance: Characterization of the expanding Tet(X) family.

Tetracycline and its derivative tigecycline are clinical options against Gram-negative bacterial infections. The emergence of mobile Tet(X) enzymes that destruct tetracycline-type antibiotics is posing a big challenge to antibacterial therapy and food/environmental securities. Here, we present an update on a growing number of Tet(X) variants. We describe structure and action of Tet(X) enzyme, and discuss the evolutional origin. In addition, potential Tet(X) inhibitors are given. This mini-review might benefit better understanding of Tet(X)-mediated tigecycline resistance. This article is categorized under: Infectious Diseases > Genetics/Genomics/Epigenetics Infectious Diseases > Environmental Factors Infectious Diseases > Molecular and Cellular Physiology.

Effects of sub-lethal antimicrobial photodynamic therapy mediated by haematoporphyrin monomethyl ether on polymyxin-resistant Escherichia coli clinical isolate.

BACKGROUND AND AIM: It is generally believed that bacteria can not develop resistance to antimicrobial photodynamic therapy (aPDT). This work employed a polymyxin-resistant Escherichia coli clinical isolate (E15017) to study whether it could become resistant to aPDT mediated by haematoporphyrin monomethyl ether (HMME) via consecutive photodynamic treatments at sub-lethal condition. METHODS: The sub-lethal and lethal photodynamic treatment conditions for E15017 were determined by colony forming units (CFU) assay. Bacterial cells of E15017 were treated with 20 cycles of repeated sub-lethal HMME-mediated aPDT, and subsequently subjected to aPDT at lethal condition. The antibiotic susceptibility, zeta-potential and membrane integrity of sub-lethal aPDT treated E15017 cells were also investigated. RESULTS: After 20 cycles of repeated HMME-mediated aPDT treatments at sub-lethal condition, E15017 cells didn't become more resistant to aPDT. Sub-lethal HMME-mediated aPDT decreased the MIC values of E15017 to ceftazidime and polymyxin E by 4 and 2-fold, respectively, and increased the electronegativity of bacterial surface and affected the bacterial membrane integrity. CONCLUSIONS: The results obtained in this study confirmed that antibiotic-resistant bacteria could not develop resistance to aPDT, and HMME-mediated aPDT is an attractive potential treatment for MDR E. coli caused infections.

The poultry pathogen Riemerella anatipestifer appears as a reservoir for Tet(X) tigecycline resistance.

The transferability of bacterial resistance to tigecycline, the 'last-resort' antibiotic, is an emerging challenge of global health concern. The plasmid-borne tet(X) that encodes a flavin-dependent monooxygenase represents a new mechanism for tigecycline resistance. Natural source for an ongoing family of Tet(X) resistance determinants is poorly understood. Here, we report the discovery of 26 new variants [tet(X18) to tet(X44)] from the poultry pathogen Riemerella anatipestifer, which expands extensively the current Tet(X) family. R. anatipestifer appears as a natural reservoir for tet(X), of which the chromosome harbours varied copies of tet(X) progenitors. Despite that an inactive ancestor rarely occurs, the action and mechanism of Tet(X2/4)-P, a putative Tet(X) progenitor, was comprehensively characterized, giving an intermediate level of tigecycline resistance. The potential pattern of Tet(X) dissemination from ducks to other animals and humans was raised, in the viewpoint of ecological niches. Therefore, this finding defines a large pool of natural sources for Tet(X) tigecycline resistance, heightening the need of efficient approaches to manage the inter-species transmission of tet(X) resistance determinants.

The MCR-3 inside linker appears as a facilitator of colistin resistance.

An evolving family of mobile colistin resistance (MCR) enzymes is threatening public health. However, the molecular mechanism by which the MCR enzyme as a rare member of lipid A-phosphoethanolamine (PEA) transferases gains the ability to confer phenotypic colistin resistance remains enigmatic. Here, we report an unusual example that genetic duplication and amplification produce a functional variant (Ah762) of MCR-3 in certain Aeromonas species. The lipid A-binding cavity of Ah762 is functionally defined. Intriguingly, we locate a hinge linker of Ah762 (termed Linker 59) that determines the MCR. Genetic and biochemical characterization reveals that Linker 59 behaves as a facilitator to render inactive MCR variants to regain the ability of colistin resistance. Along with molecular dynamics (MD) simulation, isothermal titration calorimetry (ITC) suggests that this facilitator guarantees the formation of substrate phosphatidylethanolamine (PE)-accessible pocket within MCR-3-like enzymes. Therefore, our finding defines an MCR-3 inside facilitator for colistin resistance.

Biochemical and structural characterization of the BioZ enzyme engaged in bacterial biotin synthesis pathway.

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of ß-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.

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.

Coexistence and characterization of Tet(X5) and NDM-3 in the MDR-Acinetobacter indicus of duck origin.

Tigecycline and carbapenem are last-resort antibiotics for serious infections caused by pathogens with multi-drug resistance (MDR). Whereas, bacterial pathogens with co-resistance to tigecycline and carbapenem are poorly addressed. Here we report a tigecycline- and carbapenem-resistant Acinetobacter indicus strain HY20 of duck origin, which co-produces Tet(X5) and NDM-3. Tet(X5) is harbored by a novel plasmid pAI01 (116,992 bp long), which carries 10 antimicrobial resistance genes (AMRs), and heavy metal resistance system cobalt-zinc-cadmium (czc) gene cluster. Unlike that tet(X5) is located in the res-tet(X5)-xerD segment of plasmid, the chromosomal blaNDM-3 is flanked by insertion ISAba125. Collectively, our result represents an example of co-carriage of tet(X5) and blaNDM-3, heightening the importance of AMR surveillance needed in poultry production.

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.