Prevalence and molecular characteristics of intestinal pathogenic Escherichia coli isolated from diarrheal pigs in Southern China.

Intestinal pathogenic Escherichia coli (InPEC) is one of the most common causes of bacterial diarrhea in farm animals, including profuse neonatal diarrhea and post weaning diarrhea (PWD) in piglets. In this study, we investigated the prevalence of InPEC and associated primary virulence factors among 543 non-duplicate E. coli isolates from diarrheal pigs from 15 swine farms in southern China. Six major virulence genes associated with InPEC were identified among 69 (12.71 %) E. coli isolates and included est (6.62 %), K88 (4.79 %), elt (3.68 %), eae (1.47 %), stx2 (0.92 %) and F18 (0.55 %). Three pathotypes of InPEC were identified including ETEC (8.10 %), EPEC (1.29 %) and STEC/ETEC (0.92 %). In particular, K88 was only found in ETEC from breeding farms, whereas F18 was only present in STEC/ETEC hybrid from finishing farms. Whole genome sequence analysis of 37 E. coli isolates revealed that InPEC strains frequently co-carried multiple antibiotic resistance gene (ARG). est, elt and F18 were also found to co-locate with ARGs on a single IncFIB/IncFII plasmid. InPEC isolates from different pathotypes also possessed different profiles of virulence genes and antimicrobial resistance genes. Population structure analysis demonstrated that InPEC isolates from different pathotypes were highly heterogeneous whereas those of the same pathotype were extremely similar. Plasmid analysis revealed that K88 and/or est/elt were found on pGX18-2-like/pGX203-2-like and pGX203-1-like IncFII plasmids, while F18 and elt/est, as well as diverse ARGs were found to co-locate on IncFII/IncFIB plasmids with a non-typical backbone. Moreover, these key virulence genes were flanked by or adjacent to IS elements. Our findings indicated that both clonal expansion and horizontal spread of epidemic IncFII plasmids contributed to the prevalence of InPEC and the specific virulence genes (F4, F18, elt and est) in the tested swine farms.

Antimicrobial resistance and population genomics of emerging multidrug-resistant Salmonella 4,[5],12:i:- in Guangdong, China.

Salmonella 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium, has emerged as a global cause of multidrug-resistant salmonellosis and has become endemic in many developing and developed countries, especially in China. Here, we have sequenced 352 clinical isolates in Guangdong, China, during 2009-2019 and performed a large-scale collection of Salmonella 4,[5],12:i:- with whole genome sequencing (WGS) data across the globe, to better understand the population structure, antimicrobial resistance (AMR) genomic characterization, and transmission routes of Salmonella 4,[5],12:i:- across Guangdong. Salmonella 4,[5],12:i:- strains showed broad genetic diversity; Guangdong isolates were found to be widely distributed among the global lineages. Of note, we identified the formation of a novel Guangdong clade (Bayesian analysis of population structure lineage 1 [BAPS1]) genetically diversified from the global isolates and likely emerged around 1990s. BAPS1 exhibits unique genomic features, including large pan-genome, decreased ciprofloxacin susceptibility due to mutation in gyrA and carriage of plasmid-mediated quinolone resistance (PMQR) genes, and the multidrug-resistant IncHI2 plasmid. Furthermore, high genetic similarity was found between strains collected from Guangdong, Europe, and North America, indicating the association with multiple introductions from overseas. These results suggested that global dissemination and local clonal expansion simultaneously occurred in Guangdong, China, and horizontally acquired resistance to first-line and last-line antimicrobials at local level, underlying emergences of extensive drug and pan-drug resistance. Our findings have increased the knowledge of global and local epidemics of Salmonella 4,[5],12:i:- in Guangdong, China, and provided a comprehensive baseline data set essential for future molecular surveillance.IMPORTANCESalmonella 4,[5],12:i:- has been regarded as the predominant pandemic serotype causing diarrheal diseases globally, while multidrug resistance (MDR) constitutes great public health concerns. This study provided a detailed and comprehensive genome-scale analysis of this important Salmonella serovar in the past decade in Guangdong, China. Our results revealed the complexity of two distinct transmission modes, namely global transmission and local expansion, circulating in Guangdong over a decade. Using phylogeography models, the origin of Salmonella 4,[5],12:i:- was predicted from two aspects, year and country, that is, Salmonella 4,[5],12:i:- emerged in 1983, and was introduced from the UK, and subsequently differentiated into the local endemic lineage circa 1991. Additionally, based on the pan-genome analysis, it was found that the gene accumulation rate in local endemic BAPS 1 lineage was higher than in other lineages, and the horizontal transmission of MDR IncHI2 plasmid associated with high resistance played a major role, which showed the potential threat to public health.

Tobramycin-resistant small colony variant mutant of Salmonella enterica serovar Typhimurium shows collateral sensitivity to nitrofurantoin.

The increasing antibiotic resistance poses a significant global health challenge, threatening our ability to combat infectious diseases. The phenomenon of collateral sensitivity, whereby resistance to one antibiotic is accompanied by increased sensitivity to another, offers potential avenues for novel therapeutic interventions against infections unresponsive to classical treatments. In this study, we elucidate the emergence of tobramycin (TOB)-resistant small colony variants (SCVs) due to mutations in the hemL gene, which render S. Typhimurium more susceptible to nitrofurantoin (NIT). Mechanistic studies demonstrate that the collateral sensitivity in TOB-resistant S. Typhimurium SCVs primarily stems from disruptions in haem biosynthesis. This leads to dysfunction in the electron transport chain (ETC) and redox imbalance, ultimately inducing lethal accumulation of reactive oxygen species (ROS). Additionally, the upregulation of nfsA/B expressions facilitates the conversion of NIT prodrug into its active form, promoting ROS-mediated bacterial killing and contributing to this collateral sensitivity pattern. Importantly, alternative NIT therapy demonstrates a significant reduction of bacterial load by more than 2.24-log10 cfu/g in the murine thigh infection and colitis models. Our findings corroborate the collateral sensitivity of S. Typhimurium to nitrofurans as a consequence of evolving resistance to aminoglycosides. This provides a promising approach for treating infections due to aminoglycoside-resistant strains.

Synergistic antibacterial effects of closantel and its enantiomers in combination with colistin against multidrug resistant gram-negative bacteria.

Drug combinations and repurposing have recently provided promising alternatives to cope with the increasingly severe issue of antibiotic resistance and depletion of natural drug molecular repertoires that undermine traditional antibacterial strategies. Closantel, an effective adjuvant, reverses antibiotic resistance in gram-negative bacteria. Herein, the combined antibacterial enantioselectivity of closantel is presented through separate enantiomer studies. Despite yielding unexpected differences, two closantel enantiomers (R, S) increased colistin activity against gram-negative bacteria both in vitro and in vivo. The fractional inhibitory concentration indices of R-closantel and S-closantel combined with colistin against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli ranged from 0.0087 to 0.5004 and from 0.0117 to 0.5312, respectively. This difference was further demonstrated using growth inhibition assays and time-killing curves. Mechanistically, a higher intracellular concentration of R-CLO is more effective in enhancing the antimicrobial activity of combination. A mouse cutaneous infection model confirmed the synergistic stereoselectivity of closantel. This discovery provides novel insights for developing precision medication and containment of increasing antibiotic resistance.

Distribution of antibiotic resistance genes and their pathogen hosts in duck farm environments in south-east coastal China.

Livestock farms are major reservoirs of antibiotic resistance genes (ARGs) that are discharged into the environment. However, the abundance, diversity, and transmission of ARGs in duck farms and its impact on surrounding environments remain to be further explored. Therefore, the characteristics of ARGs and their bacterial hosts from duck farms and surrounding environment were investigated by using metagenomic sequencing. Eighteen ARG types which consist of 823 subtypes were identified and the majority conferred resistance to multidrug, tetracyclines, aminoglycosides, chloramphenicols, MLS, and sulfonamides. The floR gene was the most abundant subtype, followed by sul1, tetM, sul2, and tetL. ARG abundance in fecal sample was significantly higher than soil and water sample. Our results also lead to a hypothesis that Shandong province have been the most contaminated by ARGs from duck farm compared with other four provinces. PcoA results showed that the composition of ARG subtypes in water and soil samples was similar, but there were significant differences between water and feces samples. However, the composition of ARG subtypes were similar between samples from five provinces. Bacterial hosts of ARG subtypes were taxonomically assigned to eight phyla that were dominated by the Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. In addition, some human bacterial pathogens could be enriched in duck feces, including Enterococcus faecium, Acinetobacter baumannii, and Staphylococcus aureus, and even serve as the carrier of ARGs. The combined results indicate that a comprehensive overview of the diversity and abundance of ARGs, and strong association between ARGs and bacterial community shift proposed, and benefit effective measures to improve safety of antibiotics use in livestock and poultry farming. KEY POINTS: • ARG distribution was widespread in the duck farms and surroundings environment • ARG abundance on the duck farms was significantly higher than in soil and water • Human bacterial pathogens may serve as the vectors for ARGs.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2 K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2 K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2 K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2 K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, bla CTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.

Exposure to salinomycin dysregulates interplay between mitophagy and oxidative response to damage the porcine jejunal cells.

Salinomycin (SAL) has caused widespread pollution as a feed additive and growth promoter in livestock such as pigs, exerting a negative impact on public health. The toxicity mechanism of SAL has been widely studied in chickens, but the underlying mechanisms of SAL-induced toxicity to pigs and the ecosystem remain undefined. In this study, we explored the potential damage of SAL in IPEC-J2 cells to identify the effects of excessive SAL on the interplay between mitophagy and oxidative stress. The results showed that a concentration-dependent response was observed for SAL in altering cellular morphology and inducing cell death in IPEC-J2 cells, including the induction of cell cycle arrest and lactic dehydrogenase (LDH) release. Meanwhile, we found that excessive SAL led to oxidative damage by activating the Nrf2/Keap1/HO-1 pathway, accompanied by reactive oxygen species (ROS) elevation and the reduction of antioxidant enzyme activity. We also found that PINK1/Parkin-dependent mitophagy was activated by SAL exposure, particularly with mitochondrial membrane potential reduction. Interestingly, SAL-induced oxidative damages were prevented after the autophagy inhibitor 3-methyladenine (3-MA) treatment, and mitophagy was alleviated following ROS scavenger (N-acetylcysteine, NAC) treatment. Overall, our findings showed that SAL stimulated oxidative stress and mitophagy in IPEC-J2 cells resulting in cellular injury, and there was a strong connection between SAL-induced oxidative stress and mitophagy. Targeting ROS/PINK1/Parkin-dependent mitophagy and oxidative stress could be a novel protective mechanism in SAL-induced cell damage.

Occurrence of extended- spectrum ß-lactamase harboring K. pneumoniae in various sources: a one health perspective.

This study was designed to investigate the occurrence and dissemination of extended-spectrum ß-lactamase (ESBL) harboring Klebsiella pneumoniae in various ecological niches under the one health approach. A total of 793 samples were collected from animals, humans, and the environment. The findings of the study revealed the occurrence of K. pneumoniae as follows: animals (11.6%), humans (8.4%), and associated environments (7.0%), respectively. A high occurrence rate of ESBL genes was found in animals compared to human and environmental isolates. A total of 18 distinct sequence types (STs) and 12 clonal complexes of K. pneumoniae were observed. Overall, six STs of K. pneumoniae were identified in commercial chickens, and three were found in rural poultry. The majority of K. pneumoniae STs found in this study were positive for blaSHV, while the positivity of other ESBL-encoding genes combinations was different in different STs. The high occurrence rate of ESBL-harboring K. pneumoniae found in animals as compared to other sources is alarming and has the potential to be disseminated to the associated environment and community.

Natural flavonoids disrupt bacterial iron homeostasis to potentiate colistin efficacy.

In the face of the alarming rise in global antimicrobial resistance, only a handful of novel antibiotics have been developed in recent decades, necessitating innovations in therapeutic strategies to fill the void of antibiotic discovery. Here, we established a screening platform mimicking the host milieu to select antibiotic adjuvants and found three catechol-type flavonoids-7,8-dihydroxyflavone, myricetin, and luteolin-prominently potentiating the efficacy of colistin. Further mechanistic analysis demonstrated that these flavonoids are able to disrupt bacterial iron homeostasis through converting ferric iron to ferrous form. The excessive intracellular ferrous iron modulated the membrane charge of bacteria via interfering the two-component system pmrA/pmrB, thereby promoting the colistin binding and subsequent membrane damage. The potentiation of these flavonoids was further confirmed in an in vivo infection model. Collectively, the current study provided three flavonoids as colistin adjuvant to replenish our arsenals for combating bacterial infections and shed the light on the bacterial iron signaling as a promising target for antibacterial therapies.

Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria.

OBJECTIVES: Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action. METHODS: Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time-kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli. RESULTS: In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time-kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity. CONCLUSIONS: Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

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.

ARSCP: An antimicrobial residue surveillance cloud platform for animal-derived foods.

Antibiotics have been widely used for improving human and animal health and well-being for many decades. However, the enormous antibiotic usage in agriculture especially for livestock leads to considerable quantities of antibiotic residues in associated food products and can reach potentially hazardous levels for consumers. Therefore, timely detection and systematical surveillance on residual antibiotics in food materials are of significance to minimize the negative impact caused by such unwanted antibiotic leftovers. To this end, we constructed a cloud-platform-based system (ARSCP) for comprehensive surveillance of antibiotic residues in food materials. With the system, we collected 126,560 samples from 68 chicken farms across China and detected the antibiotic residues using a rapid detection colorimetric commercial (Explorer 2.0) kit and UPLC-MS/MS. Only 108 (0.085 %) of the samples contained residual antibiotics exceeding the MRLs and all data were subjected to ARSCP system to provide a landscape of antibiotic residues in China. As a proof-of-concept, we provided an overview of residual antibiotics based on data from China, but the system is generally applicable to track and monitor the antibiotic residues globally when the data from other countries are incorporated. We used the combined Explorer 2.0 and MS data to construct ARSCP, an antimicrobial residue surveillance cloud platform for raw chicken samples. ARSCP can be used for rapid detection and real-time monitoring of antibiotic residues in animal food and provides both data management and risk warning functions. This system provides a solution to improve the management of facilities that must monitor antibiotic MRLs in food animal products that can reduce the pollution of antibiotics to the environment.

Acquisition of a novel conjugative multidrug-resistant hypervirulent plasmid leads to hypervirulence in clinical carbapenem-resistant Klebsiella pneumoniae strains.

The co-occurrence of plasmid-mediated multidrug resistance and hypervirulence in epidemic carbapenem-resistant Klebsiella pneumoniae has emerged as a global public health issue. In this study, an ST23 carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) strain VH1-2 was identified from cucumber in China and harbored a novel hybrid plasmid pVH1-2-VIR. The plasmid pVH1-2-VIR carrying both virulence and multidrug-resistance (MDR) genes was likely generated through the recombination of a virulence plasmid and an IncFIIK conjugative MDR plasmid in clinical ST23 18622 isolated from a sputum sample. The plasmid pVH1-2-VIR exhibited the capacity for transfer to the clinical ST11 carbapenem-resistant K. pneumoniae (CRKP) strain via conjugation assay. Acquisition of pVH1-2-VIR plasmid directly converted a CRKP into CR-HvKP strain characterized by hypermucoviscosity, heightened virulence for Galleria mellonella larvae, and increased colonization ability in the mouse intestine. The emergence of such a hybrid plasmid may expedite the spread of CR-HvKP strains, posing a significant risk to human health.

A Trade-Off for Maintenance of Multidrug-Resistant IncHI2 Plasmids in Salmonella enterica Serovar Typhimurium through Adaptive Evolution.

Understanding the fitness costs associated with plasmid carriage is a key to better understanding the mechanisms of plasmid maintenance in bacteria. In the current work, we performed multiple serial passages (63 days, 627.8 generations) to identify the compensatory mechanisms that Salmonella enterica serovar Typhimurium ATCC 14028 utilized to maintain the multidrug-resistant (MDR) IncHI2 plasmid pJXP9 in the presence and absence of antibiotic selection. The plasmid pJXP9 was maintained for hundreds of generations even without drug exposure. Endpoint evolved (the endpoint of evolution) S. Typhimurium bearing evolved plasmids displayed decreased growth lag times and a competitive advantage over ancestral pJXP9 plasmid-carrying ATCC 14028 strains. Genomic and transcriptomic analyses revealed that the fitness costs of carrying pJXP9 were derived from both specific plasmid genes and particularly the MDR regions and conjugation transfer region I and conflicts resulting from chromosome-plasmid gene interactions. Correspondingly, plasmid deletions of these regions could compensate for the fitness cost that was due to the plasmid carriage. The deletion extent and range of large fragments on the evolved plasmids, as well as the trajectory of deletion mutation, were related to the antibiotic treatment conditions. Furthermore, it is also adaptive evolution that chromosomal gene mutations and altered mRNA expression correlated with changed physiological functions of the bacterium, such as decreased flagellar motility, increased oxidative stress, and fumaric acid synthesis but increased Cu resistance in a given niche. Our findings indicated that plasmid maintenance evolves via a plasmid-bacterium adaptative evolutionary process that is a trade-off between vertical and horizontal transmission costs along with associated alterations in host bacterial physiology. IMPORTANCE The current idea that compensatory evolution processes can account for the "plasmid paradox" phenomenon associated with the maintenance of large costly plasmids in host bacteria has attracted much attention. Although many compensatory mutations have been discovered through various plasmid-host bacterial evolution experiments, the basis of the compensatory mechanisms and the nature of the bacteria themselves to address the fitness costs remain unclear. In addition, the genetic backgrounds of plasmids and strains involved in previous research were limited and clinical drug resistance such as the poorly understood compensatory evolution among clinically dominant multidrug-resistant plasmids or clones was rarely considered. The IncHI2 plasmid is widely distributed in Salmonella Typhimurium and plays an important role in the emergence and rapid spread of its multidrug resistance. In this study, the predominant multidrug-resistant IncHI2 plasmid pJXP9 and the standard Salmonella Typhimurium ATCC 14028 bacteria were used for evolution experiments under laboratory conditions. Our findings indicated that plasmid maintenance through experimental evolution of plasmid-host bacteria is a trade-off between increasing plasmid vertical transmission and impairing its horizontal transmission and bacterial physiological phenotypes, in which compensatory mutations and altered chromosomal expression profiles collectively contribute to alleviating plasmid-borne fitness cost. These results provided potential insights into understanding the relationship of coexistence between plasmids encoding antibiotic resistance and their bacterial hosts and provided a clue to the adaptive forces that shaped the evolution of these plasmids within bacteria and to predicting the evolution trajectory of antibiotic resistance.

Molecular Epidemiology of Plasmid-Mediated Types 1 and 3 Fimbriae Associated with Biofilm Formation in Multidrug Resistant Escherichia coli from Diseased Food Animals in Guangdong, China.

Types 1 and 3 fimbriae in Enterobacteriaceae play versatile roles in bacterial physiology including attachment, invasion, cell motility as well as with biofilm formation and urinary tract infections. Herein, we investigated the prevalence and transmission of plasmid-mediated types 1 and 3 fimbriae from 1753 non-duplicate Enterobacteriaceae from diseased food Animals. We identified 123 (7.01%) strong biofilm producers and all was identified as E. coli. WGS analysis of 43 selected strong biofilm producers revealed that they harbored multiple ARGs, including ESBLs, PMQR and mcr-1. The gene clusters mrkABCDF and fimACDH encoding types 1 and 3 fimbriae, respectively, were identified among 43 (34.96%) and 7 (5.7%) of 123 strong biofilm isolates, respectively. These two operons were able to confer strong biofilm-forming ability to an E. coli weak-biofilm forming laboratory strain. Plasmid analysis revealed that mrk and fim operons were found to co-exist with ARGs and were primarily located on IncX1 and IncFII plasmids with similar backbones, respectively. mrkABCDF operons was present in all of 9457 Klebsiella pneumoniae using archived WGS data, and shared high homology to those on plasmids of 8 replicon types and chromosomes from 6 Enterobacteriaceae species from various origins and countries. In contrast, fimACDH operons was present in most of Enterobacter cloacae (62.15%), and shared high homology to those with only a small group of plasmids and Enterobacteriaceae species. This is the first comprehensive report of the prevalence, transmission and homology of plasmid-encoded type 1 and 3 fimbriae among the Enterobacteriaceae. Our findings indicated that plasmid-encoded mrkABCDF and fimACDH were major contributors to enhanced biofilm formation among E. coli and these two operons, in particular mrk could be as a potential anti-biofilm target. IMPORTANCE Biofilms allow bacteria to tolerate disinfectants and antimicrobials, as well as mammalian host defenses, and are therefore difficult to treat clinically. Most research concerning biofilm-related infections is typically focused on chromosomal biofilm-associated factors, including types 1 and 3 fimbriae of biofilm-forming Enterobacterium. However, the transmission and homology of the mobile types 1 and 3 fimbriae among Enterobacteriaceae is largely unknown. The findings revealed that the plasmid-encoded type 3 fimbriae encoded by mrkABCDF and type 1 fimbriae encoded by fimACDH were major contributors to enhancing biofilm formation among strong biofilm E. coli from diseased food producing animals. Additionally, mrk operon with high homology at an amino acid sequence was present both on plasmids of various replicon types and on chromosomes from diverse Enterobacteriaceae species from numerous origins and countries. These findings provide important information on the transmission of the mobile types 1 and 3 fimbriae among Enterobacteriaceae, indicating a potential antibiofilm target.

Epidemiology of bla CTX-M-Positive Salmonella Typhimurium From Diarrhoeal Outpatients in Guangdong, China, 2010-2017.

Salmonella enterica can lead to intestinal diarrhea, and the emergence and spread of cephalosporin-resistant Salmonella have brought great challenges to clinical treatment. Therefore, this study investigated the prevalence and transmission of bla CTX-M genes among S. Typhimurium from diarrhoeal outpatients in Guangdong, China, from 2010 to 2017. A total of 221 bla CTX-M-positive isolates were recovered from 1,263 S. Typhimurium isolates from the facal samples of diarrhoea patients in 45 general hospitals from 11 cities. The most popular CTX-M gene was bla CTX-M-55 (39.6%, 72/182) in the CTX-M-1 group, followed by bla CTX-M-14 (22.5%, 41/182) and bla CTX-M-65 (19.2%, 35/182) in the CTX-M-9 group. The isolates that carried bla CTX-M-9G had significantly higher resistance rates to multiple antibacterials compared with bla CTX-M-1G (p < 0.01). Meanwhile, PFGE analysis not only showed the clonal transmission of bla CTX-M-55/14/65-positve isolates of diarrhoeal outpatients' origins from different hospitals in Guangdong province, but also the characteristic of bla CTX-M-55/14/65-positve isolates' bacterial persistence. Multilocus sequence typing (MLST) analysis indicated that these S. Typhimurium isolates possessed ST34 and ST19. Furthermore, genomic Beast phylogenomic analysis provided the evidence of a close relationship of bla CTX-M-positive S. Typhimurium isolates between the outpatients and pork. Most bla CTX-M-55/14/65 genes were transmitted by non-typeable or IncI1/IncFII/IncHI2 plasmids with the size of ranging from ~80 to ~280 kb. Moreover, whole-genome sequencing (WGS) analysis further revealed that bla CTX-M-55/14/65 coexisted with other 25 types of ARGs, of which 11 ARGs were highly prevalent with the detection rates >50%, and it first reported the emergence of bla TEM-141 in S. Typhimurium. This study underscores the importance of surveillance for bla CTX-M-positive microbes in diarrhea patients.

Phylogenomic analysis of Salmonella Indiana ST17, an emerging MDR clonal group in China.

OBJECTIVES: To reconstruct the genomic epidemiology and evolution of MDR Salmonella Indiana in China. METHODS: A total of 108 Salmonella Indiana strains were collected from humans and livestock in China. All isolates were subjected to WGS and antimicrobial susceptibility testing. Phylogenetic relationships and evolutionary analyses were conducted using WGS data from this study and the NCBI database. RESULTS: Almost all 108 Salmonella Indiana strains displayed the MDR phenotype. Importantly, 84 isolates possessed concurrent resistance to ciprofloxacin and cefotaxime. WGS analysis revealed that class 1 integrons on the chromosome and IncHI2 plasmids were the key vectors responsible for multiple antibiotic resistance gene (ARG) [including ESBL and plasmid-mediated quinolone resistance (PMQR) genes] transmission among Salmonella Indiana. The 108 Salmonella Indiana dataset displayed a relatively large core genome and ST17 was the predominant ST. Moreover, the global ST17 Salmonella Indiana strains could be divided into five distinct lineages, each of which was significantly associated with a geographical distribution. Genomic analysis revealed multiple antimicrobial resistance determinants and QRDR mutations in Chinese lineages, which almost did not occur in other global lineages. Using molecular clock analysis, we hypothesized that ST17 isolates have existed since 1956 and underwent a major population expansion from the 1980s to the 2000s and the genetic diversity started to decrease around 2011, probably due to geographical barriers, antimicrobial selective pressure and MDR, favouring the establishment of this prevalent multiple antibiotic-resistant lineage and local epidemics. CONCLUSIONS: This study revealed that adaptation to antimicrobial pressure was possibly pivotal in the recent evolutionary trajectory for the clonal spread of ST17 Salmonella Indiana in China.

Comparison of the prevalence and molecular characteristics of fosA3 and fosA7 among Salmonella isolates from food animals in China.

OBJECTIVES: To investigate the prevalence and molecular characteristics of fosA3 and fosA7 among Salmonella isolates. METHODS: Five hundred and fifty-one Salmonella isolates collected from food animals in China during 2016-19 were screened for fos genes. The drug resistance, serovars, clonal relationships and genetic environments of fosA were compared between fosA7- and fosA3-positive Salmonella. RESULTS: A relatively high prevalence of fosA7 (9.26%) and fosA3 (6.53%) was identified. fosA3 was associated with high-level fosfomycin resistance (≥512 mg/L), while fosA7 conferred relatively low-level resistance that was independent of the presence of glucose-6-phosphate. Additionally, fosA7 could facilitate Salmonella survival under oxidative stress. Both fosA3 and fosA7 were found in diverse serovars and STs, but segregated into distinct groups. The fosA3-positive Salmonella Typhimurium/Salmonella Indiana strains showed close genetic relationships, while fosA7-positive Salmonella Meleagridis/Salmonella Agona/Salmonella Derby showed a relatively high degree of whole-genome sequence heterogeneity. fosA3 was located on conjugative IncHI2 plasmids or chromosomes, while fosA7 was strictly chromosomal. Furthermore, two strains carried large chromosomal fosA7 regions within genomic islands. The fosA3 and fosA7 contigs from our isolates and the NCBI could be segregated into four primary and distinct genomic backbones. IS26 and the antibiotic resistance genes (ARGs) blaCTX-M, blaTEM-1B and rmtB were frequently adjacent to fosA3, while fosA7-carrying contigs generally lacked mobile elements and ARGs. CONCLUSIONS: fosA3 and fosA7 were the primary factors contributing to reduced fosfomycin susceptibility, to different degrees, in these Salmonella isolates. The distinct distributions and molecular characteristics of fosA7 and fosA3 indicated that their origin and evolution in Salmonella were most likely distinct.