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Klebsiella pneumoniae (Kp) is increasingly recognized as a reservoir for a range of antibiotic resistance genes and a pathogen that frequently causes severe infections in both hospital and community settings. In this study, we have identified a novel mechanism of conjugative transfer of a non-conjugative virulence plasmid through the formation of a fusion plasmid between the virulence plasmid and a novel 59,162 bp IncN- plasmid. This plasmid was found to be a multidrug-resistance (MDR) plasmid and carried a T4SS cluster, which greatly facilitated the efficient horizontal transfer of the fusion plasmid between Kp strains. The fused virulence plasmid conferred the resistance of serum killing and macrophage phagocytosis to the transconjugants. Importantly, this plasmid was shown to be essential for Kp virulence in a mouse model. Mechanistic analysis revealed that the virulence factors encoded by this virulence plasmid contributed to resistance to in vivo clearance and induced a high level of proinflammatory cytokine IL-1β, which acts as an inducer for more neutrophil recruitment. The transmission of the fusion plasmid in Kp has the potential to convert it into both MDR and hypervirulent Kp, accelerating its evolution, and posing a serious threat to human health. The findings of this study provide new insights into the rapid evolution of MDR and hypervirulent Kp in recent years., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The global public health threat of antibiotic resistance continues to escalate, and necessitates the implementation of urgent measures to expand the arsenal of antimicrobial drugs. This study identified a benzoxaborane compound, namely 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2178), which can inhibit the catalytic activity of the Klebsiella pneumoniae carbapenemase (KPC-2) enzyme effectively. The efficacy of AN2718 as an inhibitor for the KPC-2 enzyme was verified through various assays, including enzyme activity assays and isothermal titration calorimetry. Results of multiple biochemical assays, minimum inhibitory concentration assays and time-killing assays also showed that binding of AN2718 to KPC-2 enabled restoration of the bactericidal effect of meropenem. The survival rate of mice infected with carbapenem-resistant, high-virulence strains increased significantly upon treatment with AN2718. Most importantly, the meropenem and AN2718 combination was effective on KPC-2 mutations such as KPC-33, which evolved clinically and exhibited resistance to ceftazidime-avibactam after clinical use for a couple of years. Comprehensive safety tests both in vitro and in vivo, such as cytotoxicity, haemolytic activity and cytochrome P450 inhibition assays, demonstrated that AN2718 was safe for clinical use. These promising data indicate that AN2718 has high potential for approval for the treatment of drug resistant-bacterial infections, including those caused by ceftazidime-avibactam-resistant strains. AN2718 can be regarded as a valuable addition to the current antimicrobial armamentarium, and a promising tool to combat antimicrobial resistance., (Copyright © 2024. Published by Elsevier Ltd.)

Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Chen Sheng reports financial support was provided by the Research Grant Council of the Government of Hong Kong SAR. YANG Xuemei reports financial support was provided by the Research Grant Council of the Government of Hong Kong SAR. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper

The tigecycline resistance gene tet(X4) has been widely reported in animals and animal products in some Asian countries including China in recent years but only sporadically detected in human. In this study, we investigated the prevalence and genetic features of tet(X4)-positive clinical E. coli strains. A total of 462 fecal samples were collected from patients in four hospitals located in four provinces in China in 2023. Nine tet(X4)-positive E. coli strains were isolated and subjected to characterization of their genetic and phenotypic features by performing antimicrobial susceptibility test, whole-genome sequencing, bioinformatic and phylogenetic analysis. The majority of the test strains were found to exhibit resistance to multiple antimicrobial agents including tigecycline but remained susceptible to colistin and meropenem. A total of seven different sequence types (STs) and an unknown ST type were identified among the nine tet(X4)-positive strains. Notably, the tet(X4) gene in six out of these nine tet(X4)-positive E. coli strains was located in a IncFIA-HI1A-HI1B hybrid plasmid, which was an tet(X4)-bearing epidemic plasmid responsible for dissemination of the tet(X4) gene in China. Furthermore, the tet(X4) gene in four out of nine tet(X4)-positive E. coli isolates could be successfully transferred to E. coli EC600 through conjugation. In conclusion, this study characterized the epidemic tet(X4)-bearing plasmids and tet(X4)-associated genetic environment in clinical E. coli strains, suggested the importance of continuous surveillance of such tet(X4)-bearing plasmids to control the increasingly widespread dissemination of tigecycline-resistant pathogens in clinical settings in China., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a global threat due to its high mortality in clinical patients. However, the specific mechanisms underlying this increased mortality remain unclear. The objective of this study is to investigate how the development of a resistance phenotype contributes to the significantly higher mortality associated with this pathogen. To achieve this, a collection of isogeneic strains was generated. The clinical carbapenem-susceptible K. pneumoniae (CSKP) strain HKU3 served as the control isolate, while HKU3-KPC was created through conjugation with a bla KPC-2 -bearing plasmid and served as clinical CRKP strain. Using a sepsis model, it was demonstrated that both HKU3 and HKU3-KPC exhibited similar levels of virulence. Flow cytometry, RNA-seq, and ELISA analysis were employed to assess immune cell response, M1 macrophage polarization, and cytokine storm induction, revealing that both strains elicited comparable types and levels of these immune responses. Subsequently, meropenem was utilized to treat K. pneumoniae infection, and it was found that meropenem effectively reduced bacterial load, inhibited M1 macrophage polarization, and suppressed serum cytokine production during HKU3 (CSKP) infection. However, these effects were not observed in the case of HKU3-KPC (CRKP) infection. These findings provide evidence that the high mortality associated with CRKP is attributed to its enhanced survival within the host during antibiotic treatment, resulting in a cytokine storm and subsequent host death. The development of an effective therapy for CRKP infections could significantly reduce the mortality caused by this pathogen., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier GmbH.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

Vibrio spp., known as significant marine pathogens, have become more prevalent due to global warming. Antibiotics released into the environment drive Vibrio resistance. The increasing consumption of seafood leads to more interactions between Vibrio and humans. Despite this concerning trend, there remains a lack of large-scale surveillance for Vibrio contamination across various types of food. This study isolated 4027 Vibrio strains, primarily comprising V. parahaemolyticus and V. alginolyticus, in 3581 fresh shrimp and meat products from 2013 to 2022. The Vibrio strains showed increased resistance to important antibiotics, especially β-lactams used to treat foodborne bacterial infections. Whole genome sequencing of 591 randomly chosen strains showed a strong correlation between antibiotic resistance and genotypes in Vibrio. Notably, various ESBL genes have evolved over the past 8 years, with bla VEBs being the most dominant. Additionally, carbapenemase genes, such as bla NDM-1 , have become increasingly prevalent in recent years. Various mobile genetic elements, including IncQ and IncA/C plasmids, recoverable in Vibrio, facilitate the transmission of crucial β-lactamase genes. These data provide insights into the evolutionary traits of antimicrobial resistance in foodborne Vibrio strains over a decade. Policymakers should consider these findings when devising appropriate strategies to combat bacterial antimicrobial resistance and safeguard human health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier GmbH.)

Acinetobacter baumannii (AB) infections have become a global public health concern due to the continued increase in the incidence of infection and the rate of resistance to carbapenems. This study aimed to investigate the genomic features of AB strains recovered from a tertiary hospital and assess the clinical implications of the findings. A total of 217 AB strains were collected between 2016 and 2018 at a tertiary hospital in Guangzhou, with 183 (84.33%) being carbapenem-resistant AB (CRAB), with the main mechanism being the carriage of the bla OXA-23 gene. The overall mortality rate of patients caused by such strains was 15.21% (n = 33). Artificial lung ventilation and the use of meropenem were mortality risk factors in AB-infected patients, while KL2 AB infection was negatively associated. Core genome multilocus sequence typing and clustering analysis were performed on the integrated AB genome collection from the NCBI database and this study to illustrate the population structure among China. The results revealed diverse core genome profiles (n = 17) among AB strains from China, and strains from this single hospital exhibited most of the core genome profiles (n = 13), suggesting genetic variability within the hospital and transmission across the country. These findings show that the high transmission potential of the CRAB strains and meropenem usage that confers a selective advantage of CRAB clinically are two major factors that pose significant challenges to the effective clinical management of AB infections. Understanding the genetic features and transmission patterns of clinical AB strains is crucial for the effective control of infections caused by this pathogen., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Masson SAS.)

This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC, was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum β-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens., Competing Interests: Declaration of Competing Interest We declare that we have no conflicts of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

This study employs a comprehensive approach combining metagenomic analysis and bacterial isolation to elucidate the microbial composition, antibiotic resistance genes (ARGs), and virulence factors (VFGs) present in shrimps from market and supermarket. Metagenomic analysis of shrimps revealed a dominance of Proteobacteria and Bacteroidetes with Firmicutes notably enriched in some samples. On the other hand, the dominant bacteria isolated included Citrobacter portucalensis, Escherichia coli, Salmonella enterica, Vibrio species and Klebsiella pneumonaie. Metagenomic analysis unveiled a diverse spectrum of 23 main types and 380 subtypes of ARGs in shrimp samples including many clinical significant ARGs such as bla KPC , bla NDM , mcr, tet(X4) etc. Genomic analysis of isolated bacterial strains identified 14 ARG types with 109 subtype genes, which complemented the metagenomic data. Genomic analysis also allowed us to identify a rich amount of MDR plasmids, which provided further insights into the dissemination of resistance genes in different species of bacteria in the same samples. Examination of VFGs and mobile genetic elements (MGEs) in both metagenomic and bacterial genomes revealed a complex landscape of factors contributing to bacterial virulence and genetic mobility. Potential co-occurrence patterns of ARGs and VFGs within human pathogenic bacteria underlined the intricate interplay between antibiotic resistance and virulence. In conclusion, this integrated analysis for the first time provides a comprehensive view and sheds new light on the potential hazards associated with shrimp products in the markets. The findings underscore the necessity of ongoing surveillance and intervention strategies to mitigate risks posed by antibiotic-resistant bacteria in the food supply chain using the novel comprehensive approaches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The erm(T) gene encodes the 23 s rRNA methyltransferase and confers erythromycin resistance in Gram-positive bacteria, while has rarely been identified in Gram-negative bacteria. In this study, we identified a small IncQ1 plasmid of 6135 bp harboring the erm(T) gene in a clinical K. pneumoniae strain and confirmed the role of the erm(T) gene in mediating azithromycin resistance. This plasmid was found to be generated by incorporating the erm(T) gene from mobile elements into an IncQ1 plasmid. Our data indicated the spread of the erm(T) gene from Gram-positive bacteria to Gram-negative bacteria and the clonal spread of the ST11-KL47 type K. pneumoniae strains carrying this plasmid. Generation of this kind of multi-host plasmid will promote the dissemination of the erm(T) gene among Gram-negative bacteria and result in failures of azithromycin in treating bacterial infections., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2024. Published by Elsevier GmbH.)

Antibiotic resistance is a global health issue, with Klebsiella pneumoniae (KP) posing a particular threat due to its ability to acquire resistance to multiple drug classes rapidly. OXA-232 is a carbapenemase that confers resistance to carbapenems, a class of antibiotics often used as a last resort for treating severe bacterial infections. The study reports the earliest known identification of six OXA-232-producing KP strains that were isolated in Zhejiang, China, in 2008 and 2009 within a hospital, two years prior to the first reported identification of OXA-232 in France. The four KP strains carry the OXA-232 gene and exhibit hypervirulent loci, suggesting a broader temporal and geographical spread and integration of this resistance and virulence than previously recognized with implications for public health. Global analysis of all OXA-232-bearing KP strains revealed that OXA-232-encoding plasmids are conservative, while the strains were very diverse suggesting the plasmid mediated transmission of this carbapenemase genes. Importantly, a large proportion of the OXA-232-bearing KP strains also carried virulence plasmids, in particular the recent emergence of ST15 type of KP that carried both OXA-232-encoding plasmids and hypervirulent (hv) plasmids in China since 2019, highlighting the importance of the emergence of this type of KP strains in clinical setting. The early detection and investigations of OXA-232 in these strains warrants the retrospective studies to uncover the true timeline of antibiotic resistance spread, which could provide valuable insights for shaping future strategies to tackle the global health crisis., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier GmbH.)

The global transmission of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a significant and grave threat to human health. To investigate the potential relationship between hospital sewage and the transmission of CRAB within healthcare facilities, isolates of Acinetobacter spp. obtained from untreated hospital sewage samples were subjected to antimicrobial susceptibility tests, genome sequencing, and bioinformatic and phylogenetic tree analysis, and that data were matched with those of the clinical isolates. Among the 70 Acinetobacter spp. sewage isolates tested, A. baumannii was the most prevalent and detectable in 5 hospitals, followed by A. nosocomialis and A. gerneri. Worryingly, 57.14 % (40/70) of the isolates were MDR, with 25.71 % (18/70) being resistant to carbapenem. When utilizing the Pasteur scheme, ST2 was the predominant type among these CRAB isolates, with Tn2006 (ΔISAba1-bla OXA-23 -ATPase-yeeB-yeeA- Δ ISAba1) and Tn2009 ( Δ ISAba1-bla OXA-23 -ATPase-hp-parA-yeeC-hp-yeeB- Δ ISAba1) being the key mobile genetic elements that encode carbapenem resistance. Seven A. gerneri isolates which harbored Tn2008 (ISAba1-bla OXA-23 -ATPase) and the bla PER-1 gene were also identified. Besides, an A. soil isolate was found to exhibit high-level of meropenem resistance (MIC ≥128 mg/L) and harbor a bla NDM-1 gene located in a core genetic structure of ISAba125-bla NDM-1 -ble-trpF-dsbC-cutA. To investigate the genetic relatedness between isolates recovered from hospital sewage and those collected from ICUs, a phylogenetic tree was constructed for 242 clinical isolates and 9 sewage isolates. The results revealed the presence of two evolutionary clades, each containing isolates from both ICU and sewage water, suggesting that CRAB isolates in untreated sewage water were also the transmission clones or closely related evolutionary isolates recoverable in hospital settings. Findings in this work confirm that hospital sewage is a potential reservoir of CRAB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The epidemiological features of the Klebsiella pneumoniae causing bloodstream infections in Hong Kong and their potential threats to human health remained unknown., Methods: K. pneumoniae strains collected from four hospitals in Hong Kong during the period of 2009-2018 were subjected to molecular typing, string test, antimicrobial susceptibility testing, whole genome sequencing and analysis. Clinical data of patients from whom these strains were isolated were analyzed retrospectively using univariate and multivariate logistic regression approaches., Findings: The 240 Klebsiella spp. strains belonged to 123 different STs and 63 different capsule loci (KLs), with KL1 and KL2 being the major type. 86 out of 212 BSI-KP (40.6%) carried at least one of the virulence genes iuc, iro, rmpA or rmpA2. Virulence plasmid correlated well with the string test positive result, yet 8 strains without rmp genes were also hypermucoviscous, which was due to wzc mutation. The mortality rate of bloodstream infection patients was 43.0%. Univariant analysis showed that factors including renal replacement therapy (FDR adjusted p = 0.0007), mechanical ventilation (FDR adjusted p < 0.0001) and respiratory sepsis (FDR adjusted p < 0.0001) were found to pose the highest risk of death upon infection by Klebsiella spp., Interpretation: This study revealed the high mortality rate and risk factors associated with bloodstream infections caused by K. pneumoniae in Hong Kong, which warrants immediate action to develop effective solution to tackle this problem., Funding: Theme Based Research Scheme (T11-104/22-R), Research Impact Fund (R5011-18 F) and Postdoctoral Fellowship (PDFS2223-1S09)., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Klebsiella pneumoniae is an important clinical bacterial pathogen that has hypervirulent and multidrug-resistant variants. Uniform Manifold Approximation and Projection (UMAP) was used to cluster genomes of 16 797 K. pneumoniae strains collected, based on core genome distance, in over 100 countries during the period 1937 to 2021. A total of 60 high-density genetic clusters of strains representing the major epidemic strains were identified among these strains. Using UMAP bedding, the relationship between genetic cluster, capsular polysaccharide (KL) types and sequence type (ST) of the strains was clearly demonstrated, with some important STs, such as ST11 and ST258, found to contain multiple clusters. Strains within the same cluster often exhibited significant diverse features, such as originating from different areas and being isolated in different years, as well as carriage of different resistance and virulence genes. These data enable the routes of evolution of the globally prevalent K. pneumoniae strains to be traced. Alarmingly, carbapenem-resistant K. pneumoniae strains accounted for 51.7% of the test strains and worldwide transmission was observed. Carbapenem-resistant and hypervirulent K. pneumoniae strains are mainly reported in China; however, these strains are increasingly reported in other parts of the world. Also identified in this study were several key genetic loci that facilitate development of a new K. pneumoniae typing method to differentiate between high- and low-risk strains. In particular, the acrR, ompK35 and hha genes were predicted to play a key role in expression of the resistance and virulence phenotypes., (Copyright © 2023. Published by Elsevier Ltd.)

Understanding tetracycline resistance in Vibrio parahaemolyticus from food products is crucial for effective control measures against this foodborne pathogen. This study aimed to investigate the prevalence, evolution routes, and mechanism of transmission of tetracycline resistance in Vibrio parahaemolyticus isolates collected from food products in Shenzhen, China. A total of 2342 non-duplicate Vibrio parahaemolyticus were isolated from 3509 food samples during the period 2013-2021. Among these 2342 Vibrio parahaemolyticus strains, 530 (21.37 %) were resistant to tetracycline. These tetracycline-resistant Vibrio parahaemolyticus strains were mainly isolated from shrimp samples, with the highest resistance rate (46.9 %) observed in 2019. Phylogenetic and genomic analyses of 387 isolates carrying the tet genes revealed that five different types of tet genes (tet(34), tet(A), tet(B), tet(M), and tet(E)) were present. The tet(A) gene was the most common (65 % of isolates), while tet(E) and tet(M) genes were only detected in specific years. Although tet(A) is the most commonly detected gene, it only encodes resistance in a low percentage of strains (47/129). On the other hand, the resistance rate is highest in isolates carrying tet(B) (41/55). Interestingly, V. parahaemolyticus carrying the tet genes were not necessarily tetracycline-resistant, and vice versa. A total of six different types of plasmids and two transposable units were found to carry the tet genes. V. parahaemolyticus strains that harbored these plasmids were often resistant to multiple antibiotics, indicating that horizontal transfer of antibiotic resistance genes is common among V. parahaemolyticus strains. Our findings suggest a high prevalence of tetracycline resistance in Vibrio parahaemolyticus strains recovered from food products in Shenzhen, China. These results provide valuable insight into the evolution and transmission of tetracycline resistance in foodborne Vibrio parahaemolyticus isolates and highlight the need for effective control measures to prevent the spread of antibiotic resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

BackgroundIn China, the bla NDM gene has been recovered from human bacterial isolates since 2011. After 2014, detections of this gene in animal and food bacterial isolates have increasingly been reported.AimWe aimed to understand how bla NDM -bearing bacteria could spread between humans, animals, and animal-derived food.MethodsA total of 288 non-duplicate Escherichia coli strains, including 130 bla NDM -carrying and 158 bla NDM -negative strains were collected from clinical (humans), food-producing animals (pigs) and food (retail pork) sources between 2015 and 2017. The strains were whole genome sequenced. Core-genome-multilocus-sequence-typing was conducted. To investigate if sequence types (STs) found in human, animal or food samples could have a prior origin in a clinical, animal or food-borne animal reservoir, discriminant analysis of principal components (DAPC) was used. Plasmids bearing bla NDM were characterised.ResultsThe 130 bla NDM -carrying E. coli strains comprised a total of 60 STs, with ST167 (10/51), ST77 (6/33) and ST48 (6/46) being most prevalent in clinical, animal and food sources, respectively. Some ST10 and ST167 strains were respectively found among all three sources sampled, suggesting they might enable transfer of bla NDM between sources. DAPC analysis indicated possible transmissions of ST167 from humans to animals and ST10 from animals to human. In 114 of 130 bla NDM -carrying isolates, bla NDM was located on an IncX3 plasmid.ConclusionThis study in a Chinese context suggests that cross-species transmission of certain STs of E. coli harbouring bla NDM on mobile elements, may facilitate the spread of carbapenem-resistant Enterobacteriaceae. Stringent monitoring of bla NDM -bearing E. coli in ecosystems is important.

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., Competing Interests: The authors declare no conflict of interest.

Expression of the hypermucoviscosity (HMV) phenotype and capsular polysaccharide (CPS) biosynthesis in Klebsiella pneumoniae were reported to be encoded by genes located in the chromosomal rmp locus. However, the functions of the rmp locus in the virulence plasmid remained unclear, and most of the rmp loci in clinical K. pneumoniae are plasmid carried. In this study, we investigated the functional characteristics of plasmid-borne rmp homologues in clinical hypervirulent K. pneumoniae (hvKP) strains by cloning and introducing such gene homologues into K. pneumoniae strains of different capsule types, followed by the evaluation of phenotypic changes in these strains. Acquisition of the plasmid-borne p rmpADC and p rmpA2D2 loci were found to result in an increase in mucoviscosity and CPS production in K1 and K2 K. pneumoniae, while only the p rmpA2D2 locus contributed to phenotypic changes in the ST11/KL64 strain. Consistently, both rmpD and rmpD2 increased HMV in K1 and K2 K. pneumoniae, while only rmpD2 contributed to HMV in the ST11/KL64 strain; rmpC contributed to CPS overproduction in K1 and K2 strains but not in the ST11/KL64 strain. Furthermore, we proposed a logistic molecular basis of the HMV phenotype of K. pneumoniae on which p rmpD2 -mediated HMV is attributed to the increase of cell-free CPS production. Our data confirm that the rmp homologues carried by the virulence plasmid play a key role in virulence expression in K. pneumoniae, but the phenotype is highly dependent on the genetic background of the host strain and explained why most of the clinical ST11 strains carry only the p rmpA2D2 locus. IMPORTANCE Klebsiella pneumoniae has become the most frequently isolated bacterial pathogen in hospital settings, with a very high mortality rate worldwide. Factors contributing to the virulence of K. pneumoniae are the overproduction of capsular polysaccharide (CPS) as well as the hypermucoviscosity (HMV) phenotype. These two phenotypes were reported to be regulated by rmpA/A2 homologues, which are often carried by virulence plasmids. Here, we determined the functional role of two plasmid-borne rmpA in mediating expression of the HMV phenotype and CPS production in K. pneumoniae. Different capsule types exhibited differences in the expression of HMV and CPS production although they harbored an identical plasmid-borne rmpA or rmpA2 locus, indicating that these virulence-related phenotypes are strongly related to the genetic background of the host strains. Our study provides a novel understanding of the regulation of virulence-related phenotypes and clinical management of K. pneumoniae infections., Competing Interests: The authors declare no conflict of interest.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a bacterial pathogen that may cause serious drug-resistant infections that are potentially fatal. To investigate the genetic characteristics of these organisms, we tested 416 P. aeruginosa strains recovered from 12 types of clinical samples collected in 29 different hospital wards in 10 hospitals in Guangdong Province, China, from 2017 to 2020. These strains were found to belong to 149 known sequence types (STs) and 72 novel STs, indicating that transmission of these strains involved multiple routes. A high rate of resistance to imipenem (89.4%) and meropenem (79.4%) and a high prevalence of pathogenic serotypes (76.4%) were observed among these strains. Six STs of global high-risk clones (HiRiCs) and a novel HiRiC strains, ST1971, which exhibited extensive drug resistance, were identified. Importantly, ST1971 HiRiC, which was unique in China, also exhibited high virulence, which alarmed the further surveillance on this highly virulent and highly resistant clone. Inactivation of the oprD gene and overexpression of efflux systems were found to be mainly responsible for carbapenem resistance in these strains; carriage of metallo-β-lactamase (MBL)-encoding genes was less common. Interestingly, frameshift mutations (49.0%) and introduction of a stop codon (22.4%) into the oprD genes were the major mechanisms of imipenem resistance. On the other hand, expression of the MexAB-OprM efflux pump and MBL-encoding genes were mechanisms of resistance in >70% of meropenem-resistant strains. The findings presented here provide insights into the development of effective strategies for control of worldwide dissemination of CRPA. IMPORTANCE Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major concern in clinical settings worldwide, yet few genetic and epidemiological studies on CRPA strains have been performed in China. Here, we sequence and analyze the genomes of 416 P. aeruginosa strains from hospitals in China to elucidate the genetic, phenotypic, and transmission characteristics of CRPA strains and to identify the molecular signatures responsible for the observed increase in the prevalence of CRPA infections in China. These findings may provide new insight into the development of effective strategies for worldwide control of CRPA and minimize the occurrence of untreatable infections in clinical settings., Competing Interests: The authors declare no conflict of interest.

Bacterial antimicrobial resistance, especially phenotypic resistance to multiple drugs (MDR), has posed a serious threat to public health worldwide. To clarify the mechanism of transmission of multidrug resistance encoding plasmids in Enterobacterales , all seven plasmids of an Escherichia coli (E. coli) strain 1108 obtained from a chicken meat sample were extracted and sequenced by Illumina Nextseq 500 and MinION platforms. Plasmids in strain 1108 possessed 16 known antimicrobial resistance genes, with p1108-NDM (~97K) being the most variable plasmid. The multidrug resistance region of p1108-NDM was punctuated by eight IS 26 insertion sequences; thus, four MDR regions were found in the backbone of this plasmid. The plasmid p1108-MCR (~65K) was found to lack the IS Apl1 element and harbor the bla CTX-M-64 -IS Ecp1 transposition unit. Moreover, the IS Ecp1 - bla CMY-2 transposition unit was found in plasmid p1108-CMY2 (~98K), whereas plasmid p1108-emrB (~102K) was associated with resistance to erythromycin ( emrB ) and streptomycin ( aadA22 ). p1108-IncY (96K) was a phage P1-like plasmid, while p1108-IncFIB (~194K) was found to harbor a virulence region similar to ColV plasmids, and they were found to encode a conserved conjugative transfer protein but harbor no resistance genes. Finally, no mobile element and resistant genes were found in p1108-ColV (~2K). Carriage of mcr-1 -encoding elements in carbapenemase-producing Escherichia coli will potentially render all antimicrobial treatment regimens ineffective. Enhanced surveillance and effective intervention strategies are urgently needed to control the transmission of such multidrug resistance plasmids. IMPORTANCE Antimicrobial resistance (AMR) has been increasingly prevalent in agricultural and clinical fields. Understanding the genetic environment involved in AMR genes is important for preventing transmission and developing mitigation strategies. In this study, we investigated the genetic features of an E. coli strain (1108) isolated from food product and harboring 16 AMR genes, including bla NDM-1 and mcr-1 genes encoding resistance to last line antibiotics, meropenem, and colistin. Moreover, this strain also carried virulence genes such as iroBCDEN , iucABCD , and iutA . Our findings confirmed that multiple conjugative plasmids that were formed through active recombination and translocation events were associated with transmission of AMR determinants. Our data warrant the continuous monitoring of emergence and further transmission of these important MDR pathogens.

Phenotypic resistance to fluoroquinolones due to mutational changes in the gyrA and parC genes is common among clinical Vibrio strains; the plasmid-mediated quinolone resistance (PMQR) qnrVC genes were also suggested to play a role in enhancing resistance development. This study investigated the prevalence of qnrVC genes in foodborne Vibrio strains collected in Shenzhen, China, during the period August 2015 and April 2017. A total of 1811 foodborne Vibrio strains were collected, mostly (73.8%) from shrimp samples and 20.2% of these strains were resistant to ciprofloxacin. Investigation of resistance mechanisms showed that mutations in the gyrA and parC genes were commonly associated with ciprofloxacin resistance. The presence of qnrVC genes was shown to enhance ciprofloxacin MIC in Vibrio strains and 69.7% of Vibrio strains that harbored target mutations also carried qnrVC genes, yet only 27.5% of the isolates not harboring such mutations carried the qnrVC genes. A total of 141 strains were found to carry the qnrVC alleles, with qnrVC5 and qnrVC1 being the most common types. Fourteen qnrVC variant genes that contained novel mutations were detectable, with 12 (85.7%) involving qnrVC5-like alleles. For the first time, we found a variant that was likely formed by the recombination of qnrVC1 and qnrVC5. The genetic context of the qnrVC genes found in this study was highly variable, with most being accompanied by mobile genetic elements and other resistance genes. The increasing prevalence of qnrVC genes in Vibrio and its contribution on mediating the development of ciprofloxacin resistance need to be further investigated., (Copyright © 2022. Published by Elsevier GmbH.)

Colistin resistance mediated by mcr-1-bearing plasmids poses a new challenge to treatment of Salmonella infections. To probe the scale of the problem that colistin resistance mediated by mcr-1 plasmids among Salmonella, the prevalence of mcr-1 in foodborne Salmonella recovered from 2014 to 2017 in Shenzhen, China and genetic profile of mcr-1 positive isolates were investigated. All mcr-1 positives Salmonella strains were collected from food products, characterized by PCR and MALDI-TOF, and subjected to antimicrobial susceptibility testing, whole-genome sequencing, bioinformatics analysis, and conjugation. Twenty-eight mcr-1-positive Salmonella strains were recovered from pork. The rate of recovery displayed an increasing trend and was often accompanied by multidrug resistance. Salmonella Typhimurium was the most prevalent serotypes. Comparative genomic analysis indicated that the mcr-1 gene was located on the transferable IncX4 plasmids, as well as the IncHI2 plasmids, in which the gene was associated with ISApl1. All two types of plasmids were often detected in zoonotic pathogen. Transferable 251K mcr-1-bearing IncHI2 type plasmids were frequently reported in human and food-producing animals, but this is first time to detect a certain number in food. These findings show that dissemination of these two types of plasmids is responsible for the increase in the prevalence of colistin resistance in Salmonella strains in recent years, leading to rapid emergence of MDR Salmonella upon acquisition of these two mcr-1-bearing plasmids. Transmission of IncX4 and IncHI2 plasmids in Salmonella would cause huge public health concerns in controlling foodborne infections caused by Salmonella., Competing Interests: Transparency declarations None to declare., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

The epidemiological features of the newly emerged carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and its potential threat to human health are currently unknown. In this study, a total of 784 bla KPC-2 -bearing CRKP strains collected from three hospitals located at different geographical locales in China during 2014-2017 were subjected to molecular typing, screening of virulence plasmid, string test and WGS (367/784 strains). The proportion of CRKP among all clinical K. pneumoniae strains increased sharply in China during 2014-2017. A large proportion (58%) of these CRKP strains were found to harbour a virulence-encoding plasmid, while only 13% of such strains exhibited a hypervirulent phenotype by string test and neutrophil assay. The lack of hypervirulent phenotype in virulent plasmid-bearing CRKP strains was found to be due to the mutation's presence on rmpA and rmpA2 genes, which rendered them non-functional, while some strains carrying wild type rmpA did not exhibit hypervirulent phenotype either suggesting that other factors might also contribute to the hypervirulence of CRKP. Phylogenetic and SNP analysis indicated that the transmission of these CRKP strains in China likely involved several major clones of ST11. Carriage of IncFII pSWU01-like, bla KPC-2 -bearing plasmid was found to be the major mechanism of carbapenem resistance in these CRKP strains. In conclusion, our data indicated that the prevalence of CRKP strains carrying the virulence plasmid has rapidly increased in China, while genetic markers were not correlated well with the hypervirulent phenotypes, which call for a better definition and screening for these truly hypervirulent CR-HvKP strains in clinical settings.

Acinetobacter baumannii is one of the key Gram-negative pathogens that can cause serious nosocomial infections. In China, a large proportion of clinical A. baumannii strains are multidrug resistant, among which strains resistant to carbapenems are particularly worrisome, as infections caused by such strains may limit the choice of existing antibiotics. We conducted a nationwide and genome-based surveillance on the prevalence and antibiotic susceptibility profile of carbapenem-resistant A. baumannii (CRAB) strains collected from intensive care units (ICUs) in hospitals in different provinces and investigated the routes of transmission and mechanism of resistance by whole-genome sequencing and phylogenetic analysis. We found that CRAB strains were prevalent in 71.4% (55/77) of the ICUs surveyed. Clonal spread of CRAB was found in 37.6% (29/77) of ICUs and a total of 22 different clones were identified. Most clones were transmissible within one ICU, but up to six clones could be detected in at least three hospitals. In addition, carbapenem-hydrolysing class D β-lactamases (CHDL) were found to be mainly responsible for carbapenem-resistance in A. baumannii and the ST2 global-clone is the predominant type of CRAB in China. Importantly, we found that CRAB isolates currently exhibited an extremely low rate of resistance to colistin (0.4%) and tigecycline (2.5%), but a high rate of resistance to ceftazidime-avibactam (70.2%). Findings in this work shall facilitate development of appropriate antimicrobial regimens for treatment of CRAB infections. Further surveillance and research on the evolutionary and epidemiological features of clinical CRAB strains are necessary.

With the development of multidrug resistance in Salmonella spp. in recent years, ciprofloxacin, ceftriaxone, and azithromycin have become the principal antimicrobial agents used for the treatment of Salmonella infections. The underlying mechanisms of plasmid-mediated ciprofloxacin and ceftriaxone resistance have attracted extensive research interest, but not much is focused on azithromycin resistance in Salmonella . In this study, we investigated the genetic features of two conjugative plasmids and a non-transferable virulence plasmid that encode azithromycin resistance in food-borne Salmonella strains. We showed that the azithromycin resistance phenotype of these strains was conferred by erm(B) gene and/or the complete genetic structure IS 26-mph(A)-mrx-mphR- IS 6100. Comparative genetic analysis showed that these conjugative plasmids might originate from Escherichia coli and play a role in the rapid dissemination of azithromycin resistance in Salmonella . These conjugative plasmids may also serve as a reservoir of antimicrobial resistance (AMR) genes in Salmonella in which these AMR genes may be acquired by the virulence plasmids of Salmonella via genetic transposition events. Importantly, the formation of a novel macrolide-resistance and virulence-encoding plasmid, namely pS1380-118 kb, was observed in this study. This plasmid was found to exhibit transmission potential and pose a serious health threat as the extensive transmission of azithromycin resistant and virulent Salmonella strains would further compromise the effectiveness of treatment for salmonellosis. Further surveillance and research on the dissemination and evolution routes of pS1380-118kb-like plasmids in potential human pathogens of the family of Enterobacteriaceae are necessary.

Introduction: Azithromycin resistance in bacterial pathogens has increased worldwide, and Klebsiella pneumoniae (K. pneumoniae) carries a variety of azithromycin resistance encoding genes., Methods: Genomic DNA of K. pneumoniae strain 16HN-12 was subjected to whole-plasmid sequencing using both the 150-bp paired-end Illumina NextSeq 500 platform and the long-read Oxford Nanopore Technologies MinION platform. Transferability of the azithromycin-resistance plasmid and the virulence plasmid was assessed by performing the conjugation assay., Results: This study identified an IncB/O/K/Z conjugative plasmid that harboured erm(B) and mph(A) genes from a clinical K. pneumoniae strain. The plasmid was readily able to conjugate to Escherichia coli (E. coli) strain J53 and Salmonella enterica subsp. enterica serovar Typhimurium strain PY1 and promoted phenotypic resistance to azithromycin. Furthermore, the virulence plasmid harboured by this K. pneumoniae strain could be conjugated to E. coli strain EC600 and K. pneumoniae strain WZ1-2 via the help of this resistance plasmid through formation of a fusion plasmid. The fusion process was generated by homologous recombination through a homologous region located in both the virulence plasmid and resistance plasmid., Conclusions: Generation of this kind of conjugative plasmid simultaneously carrying virulence and resistance determinants could accelerate dissemination of these determinants and generate bacterial pathogens encoding these phenotypes. These data provide more information about transmission of azithromycin resistance and virulence determinants and call for action to further investigate and prevent such an evolutionary trend., Competing Interests: Competing Interests None to declare., (Copyright © 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Background: Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health., Objectives: To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio., Methods: A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains., Results: In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts., Conclusions: The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Enterobacter cloacae complex (ECC) is among the most common carbapenem-resistant Enterobacteriaceae (CRE) in China. The emergence of mcr has rendered CRE strains resistant to the last-line antibiotic colistin. This study investigated the prevalence of mcr-9 and mcr-10 in carbapenem-resistant ECC (CRECC) and carbapenem-susceptible ECC (CSECC) in China., Methods: The CRECC and CSECC strains were collected from different regions of China. Antimicrobial susceptibility tests, conjugation experiments, whole genome sequencing, bioinformatic analysis, and quantitative RT-PCR were performed to understand the mechanisms of resistance and transmission of mcr in ECC., Results: A total of 534 ECC were collected, among which 57 (10.7%) and 23 (4.3%) were positive for mcr-9 and mcr-10, respectively. The prevalence of mcr-9 in CRECC was significantly higher than that in CSECC (31.8% vs. 3.7%; P < 0.001), while the prevalence of mcr-10 in CRECC was significantly lower (0.8% vs. 5.5%; P < 0.05). Most mcr-9-positive strains (n = 45, 78.9%) exhibited multidrug-resistant phenotype, and four (17.4%) of the mcr-10-positive strains exhibited multi-drug resistance. Coexistence of mcr and carbapenemase genes was commonly observed, including 41 (71.9%) mcr-9-positive strains and one (4.3%) mcr-10-positive strain, and the possibility of co-transfer was confirmed by conjugation experiments. The mcr-positive ECC were highly diverse, while most mcr genes were plasmid-encoded, indicating the important role of plasmids in the transmission of mcr in ECC. Furthermore, the expression of mcr-9 was increased after induction by colistin., Conclusions: The widespread mcr genes and co-transfer with carbapenemase genes among ECC strains pose an urgent threat to public health., (Copyright © 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

The continuous emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great challenge to human health owing to the associated extremely high morbidity and mortality. Klebsiella quasipneumoniae is a newly described bacterial species that is often misidentified as K. pneumoniae. Clinical K. quasipneumoniae strains have been reported worldwide, among which multidrug-resistant lineages have become a severe health problem, while less has been understood on this important pathogen. In this study, we characterised three clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates belonging to sequence type 367 (ST367) and capsular type K1 and containing several virulence genes, including salmochelin (iroBCDN), aerobactin (iucABCDiutA) and regulator of mucoid phenotype (rmpA/A2), as well as some resistance genes, including bla KPC-2 , bla TEM-1 , bla OKP-B-9 and oqxAB. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains containing virulence genes exhibited a higher level of virulence and serum resistance than a classical K. pneumoniae strain, while their virulence levels were slightly lower compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reports for the first time the genetic and virulence characterisation of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained bla KPC-2 and virulence genes, contributing to a better understanding of their resistance and pathogenicity as well as for epidemic surveillance worldwide., (Copyright © 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

The main mechanism of virulence in Klebsiella pneumoniae is the acquisition of K. pneumoniae virulence plasmids (KpVPs), which include two dominant types, namely, KpVP-1 (carrying iuc 1, iro 1, rmpA , and rmpA2 ) and KpVP-2 (carrying iuc 2, iro 2, and rmpA ). Both are non-conjugative and associated with different hypervirulent clones. In contrast to KpVP-1 reported in K1, K2, and other serotypes of K. pneumoniae , KpVP-2 was only reported in K2 strains and rarely characterized. In this study, we identified a conjugative KpVP-2-type virulence plasmid from a clinical hypervirulent K. pneumoniae strain. This plasmid was generated by the integration of conjugative transfer genes into the KpVP-2-type plasmid Kp52.145 II and could be readily conjugated to Escherichia coli strain EC600 and K. pneumoniae strains of various types which are clinically existing, mediating hypervirulence. Furthermore, this kind of conjugative KpVP-2-type virulence plasmid has been disseminated in clinical settings in Hong Kong and other regions of the world. The generation of conjugative virulence plasmid may promote its transmission and explain the evolution of this type of virulence plasmid., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflictof interest., (Copyright © 2022 Yang, Liu, Xu, Yang, Chan, Shum and Chen.)

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&#95;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&#95;Vir was found to be evolved into p15WZ-82&#95;int, which underwent homologous recombination with a plasmid encoding a carbapenemase gene, bla NDM-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&#95;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&#95;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.

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.

Colistin is a last-line antibiotic which acts by causing membrane permeabilization in Gram-negative bacteria. However, its clinical value has been limited by its toxicity and the emergence of resistant organisms. In this study, we showed that econazole and colistin can act synergistically to produce a strong antimicrobial effect sufficient for eradication of starvation-induced tolerant and multidrug-resistant populations of Acinetobacter baumannii, a notorious pathogen causing recalcitrant infections, both in vitro and in mouse infection models. Investigation of the underlying mechanism showed that, while colistin disrupts the membrane structure, econazole causes the dissipation of proton motive force, eliciting a vicious cycle of membrane structural damages and disruption of membrane protein functions, and eventually cell death. This drug combination therefore achieves our goal of using a much smaller dosage of colistin to produce a much stronger antimicrobial effect to tackle the problems of toxicity and resistance associated with colistin usage. IMPORTANCE Findings described in this study constitute concrete evidence that it is possible to significantly enhance the antimicrobial activity of colistin by using an antifungal drug, econazole, as a colistin adjuvant. We showed that this drug combination can kill not only multidrug-resistant A. baumannii but also the tolerant subpopulation of such strains known as persisters, which may cause chronic and recurrent infections in clinical settings. The synergistic killing effect of the econazole and colistin combination was also observable in mouse infection models at a very low concentration, suggesting that such a drug combination has high potential to be used clinically. Findings in this study therefore have important implications for enhancing its clinical application potential as well as developing new approaches to enhance treatment effectiveness and reduce suffering in patients.

Colistin is the last-line antibiotic against Gram-negative pathogens. Here we identify an FDA-approved drug, Otilonium bromide (Ob), which restores the activity of colistin against colistin-resistant Gram-negative bacteria in vitro and in a mouse infection model. Ob also reduces the colistin dosage required for effective treatment of infections caused by colistin-susceptible bacteria, thereby reducing the toxicity of the drug regimen. Furthermore, Ob acts synergistically with colistin in eradicating multidrug-tolerant persisters of Gram-negative bacteria in vitro. Functional studies and microscopy assays confirm that the synergistic antimicrobial effect exhibited by the Ob and colistin involves permeabilizing the bacterial cell membrane, dissipating proton motive force and suppressing efflux pumps, resulting in membrane damages, cytosol leakage and eventually bacterial cell death. Our findings suggest that Ob is a colistin adjuvant which can restore the clinical value of colistin in combating life-threatening, multidrug resistant Gram-negative pathogens., (© 2022. The Author(s).)

Recently, many TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. In this study, we identified essential residues including 329, 339, 340, 350, and 351 in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into TetX-A class, TetX-B class and TetX-C class. We further found that TetX-A class variants originated from Bacteroidetes, with some variants further evolving to TetX-C class and acquired by Enterobacteriaceae. On the other hand, our data showed that some variants genes belonging to TetX-A class evolved directly to TetX-B class, which was further transmitted to Acinetobacter spp. This new classification system may facilitate better clinical management of patients infected by TetX-producing strains., (© 2022. The Author(s).)

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 IS 26 and IS 903B . 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., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ye, Dong, Xiong, Li, Li, Heng, Chan and Chen.)

Members of the genus Klebsiella have rapidly evolved within the past decade, generating organisms that simultaneously exhibit both multidrug resistance and hypervirulence (MDR-hv) phenotypes; such organisms are associated with severe hospital- and community-acquired infections. Carbapenem-resistant infections with unknown optimal treatment regime were of particular concern among the MDR-hv Klebsiella strains. Recent studies have revealed the molecular features and the mobile resistance elements they harbour, allowing identification of genetic loci responsible for transmission, stable inheritance, and expression of mobile resistance or virulence-encoding elements that confer the new phenotypic characteristics of MDR-hv Klebsiella spp. Here, we provide a comprehensive review on the taxonomic position, species composition and different phylotypes of Klebsiella spp., describing the diversity and worldwide distribution of the MDR-hv clones, the genetic mutation and horizontal gene transfer events that drive the evolution of such clones, and the potential impact of MDR-hv infections on human health., Competing Interests: Declaration of interests All authors declare that they have no competing interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Coexistence of oqxAB and aac ( 6')-Ib-cr is often associated with the expression of fluoroquinolone resistance in Salmonella. The actual role of the plasmid-borne oqxAB gene and its regulatory mechanism compared to its chromosomally encoded counterpart in Klebsiella pneumoniae remain unclear We found that cloning of oqxAB gene only or chromosomally encoded oqxABR (ABRc) locus did not lead to an increase of ciprofloxacin (CIP) minimum inhibitory concentration (MIC) in S. Typhimurium, while cloning of the plasmid-encoded oqxABR (ABRp) locus led to a 4-fold increase in CIP MIC, reaching 0.0065 μg/mL. The co-carriage of these constructs with aac(6')-Ib-cr further increased the CIP MIC to 0.25 μg/mL in S. Typhimurium carrying aac(6')-Ib-cr and ABRp. Analysis of the transcription start site sequences showed that the expression level of suppressor protein gene, oqxR , in strains carrying ABRp was lower than that of its chromosomal counterpart due to the truncated promoter region in ABRp. The lower expression of OqxR in ABRp led to the overexpression of OqxAB, which elevated CIP MIC and exhibited a synergistic antimicrobial effect with the aac(6')-Ib-cr gene product to confer intermediate CIP (MIC = 0.25 μg/mL) in S. Typhimurium. Global transcriptional regulators in S. Typhimurium did not seem to play a role in regulating the plasmid-borne oqxAB genes. In conclusion, findings in this work showed that neither aac(6')-Ib-cr nor oqxABRp , but the combination of both genes, could mediate intermediated resistance to fluoroquinolone in Salmonella. The truncated promoter region in the oqxR gene of the plasmid-encoded locus led to the constituted expression of oqxAB genes. IMPORTANCE The transferable mechanisms of quinolone resistance (TMQR) gene, oqxAB , has been widely detected in Salmonella and is commonly associated with aac(6')-Ib-cr . It is thought to be associated with fluoroquinolone resistance, while its ancestor gene from K. pneumoniae is not. This study evaluated the actual role of the plasmid-borne oqxAB genes in Salmonella and showed that it was not able to mediate intermediated resistance to fluoroquinolone and only did so when it coexisted with aac(6')-Ib-cr . Chromosomally encoded oqxABRc from K. pneumoniae was not able to mediate enhanced CIP MIC due to tight regulation by the suppressor oqxR . However, plasmid-encoded oqxABRp enabled oqxAB to be expressed constitutionally due to the truncated promoter region of oqxR , leading to lower expression of the suppressor oqxR . This study clarified the roles of oqxAB and aac(6')-Ib-cr in mediating fluoroquinolone resistance in Salmonella and provides insights into the regulation of plasmid-encoded TMQR determinant, oqxAB .

Plasmid-mediated, transmissible, tigecycline-inactivating enzyme Tet(X) has attracted considerable public attention. However, so far studies have not addressed its impact on public health and the ecosystem. Herein, we report the prevalence and molecular epidemiology of tet(X)-positive bacteria (TPB) from diverse sources, investigate the host-specificity of TPB and the transferability of tet(X). Sample collection was conducted between 2018 and 2020 in 30 provinces in China. PCR screening suggested tet(X) was prevalent among freshwater fishes (24.7%, 95% CI 19.4-30.7%), followed by chickens (23.6%, 21.2-26.2%), cattle (19.3%, 16.4-22.5%), healthy individuals (6.2%, 5.4-7.1%), and patients (0.3%, 0.0-1.1%). Soil and freshwater samples all tested negative for tet(X). A total of 289 TPB were isolated from 7516 samples (120/1181 chicken, 82/669 cattle, 68/3229 healthy individual, 17/239 freshwater fish and 2/2121 clinical samples). TPB distributed in six major families of bacteria including Moraxellaceae (n = 99, 34.3%), Flavobacteriaceae (n = 95, 32.9%), Enterobacteriaceae (n = 83, 28.7%), Pseudomonadaceae (n = 9, 3.1%), Sphingobacteriaceae (n = 2, 0.7%) and unclassified Gammaproteobacteria (n = 1, 0.3%). Diverse tet(X) genes including tet(X2), tet(X3), tet(X4), tet(X5) and tet(X6) were identified from different TPB. The tet(X)-positive bacteria were highly diverse, with ST10 complex belonging to the dominant E. coli clone. Novel hosts of tet(X) including Enterobacter hormaechei, Ignatzschineria indica and Oblitimonas alkaliphila were identified. Isolates from different families exhibited different antimicrobial resistance profiles. Co-existence of tet(X) with other resistance genes such as floR (66.8%) and carbapenemase genes (33.2%) was commonly observed. tet(X) could be transferred among E. coli isolates at frequencies from 10 -4 to 10 -10 . Species other than E. coli failed to transfer tet(X) gene to the E. coli recipient via conjugation. Discriminant analysis of principal components analysis suggested inter-host transmission of tet(X)-positive E. coli among diverse hosts was not observed. Future studies are needed to monitor the transmission trend as well as the impact of this resistance gene in clinical infection control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Colistin is one of the few antibiotics that exhibit bactericidal effect on carbapenemase-producing Klebsiella pneumoniae strains. In recent years, however, colistin resistance is increasingly being reported among clinical carbapenem-resistant K. pneumoniae strains worldwide, posing serious challenge to treatment of infections caused by these organisms. In this study, we investigated one colistin-susceptible (YJH4) and one colistin-resistant (YJH15) K. pneumoniae strain, which were collected from a patient before and after colistin treatment, respectively. We characterized the effects of mgrB inactivation-induced colistin resistance on the physiological fitness and virulence in ST11 carbapenem-resistant K. pneumoniae both in vitro and in vivo . The colistin-resistant strain YJH15 was found to exhibit increased fitness and biofilm formation potential in vitro , and increased survival rate in the presence of normal human serum. Interestingly, YJH15 exhibited reduced virulence in the mouse infection model but enhanced virulence in Galleria mellonella infection model when compared to the colistin-susceptible parental strain YJH4. Infection with YJH15 was also found to result in lower expression level of inflammatory cytokine IL-1β in blood and significantly decreased bacterial loads in heart, liver, spleen, lung, kidney and blood. These results demonstrated that mgrB inactivation-induced colistin resistance has significant effects on multiple fitness and virulence-associated traits in K. pneumoniae ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xie, Chen, Dong, Xu, Chan, Zhang and Chen.)

We recently reported the recovery of a novel IncI1 type conjugative helper plasmid which could target mobile genetic elements (MGE) located in non-conjugative plasmid and form a fusion conjugative plasmid to mediate the horizontal transfer of the non-conjugative plasmid. In this study, interactions between the helper plasmid pSa42-91k and two common MGEs, IS Ecp1 and IS 15DI , which were cloned into a pBackZero-T vector, were monitored during the conjugation process to depict the molecular mechanisms underlying the plasmid fusion process mediated by insertion sequence (IS) elements. The MinION single-molecule long-read sequencing technology can dynamically reveal the plasmid recombination events and produce valuable information on genetic polymorphism and plasmid heterogeneity in different multidrug resistance (MDR) encoding bacteria. Such data would facilitate the development of new strategies to control evolution and dissemination of MDR plasmids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chen, Xie, Chan and Chen.)

Objectives: Bacterial antibiotic tolerance is responsible for the recalcitrance of chronic infections. This study aims to investigate a potential drug that can effectively kill antibiotic-tolerant bacteria and evaluate the ability of this drug on the eradication of tolerant cells both in vitro and in vivo., Methods: The in vitro effect of econazole on eradicating starvation-induced tolerant bacterial populations was studied by testing the amount of survival bacteria in the presence of econazole combining conventional antibiotics. Proton motive force (PMF) was determined after econazole treatment by DiOC2(3). Finally, mouse infection models were used to detect the ability of econazole on killing the tolerant populations in vivo., Results: Econazole eradicated starvation-induced tolerant cells of various bacterial species within 24 or 96 h when used in combination with conventional antibiotics. Moreover, mouse survival rate drastically increased along with the decrease of in vivo bacterial count after treatment of infected mice with the econazole and ceftazidime combination for 72 h. PMF was found to have dissipated almost completely in econazole-treated cells., Conclusions: Econazole could act in combination with conventional antibiotics to effectively eradicate bacterial tolerant cells. The combined use of econazole and ceftazidime was shown to be effective for eradicating tolerant cells in a mouse infection model. The ability of econazole to eradicate tolerant cells was due to its ability to cause dissipation of bacterial transmembrane PMF. Econazole-mediated PMF disruption is a feasible strategy for the treatment of chronic and recurrent bacterial infections., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)