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.

Pharmacokinetic/pharmacodynamic evaluation of gamithromycin against rabbit pasteurellosis.

BACKGROUND: Gamithromycin is an effective therapy for bovine and swine respiratory diseases but not utilized for rabbits. Given its potent activity against respiratory pathogens, we sought to determine the pharmacokinetic profiles, antimicrobial activity and target pharmacokinetic/pharmacodynamic (PK/PD) exposures associated with therapeutic effect of gamithromycin against Pasteurella multocida in rabbits. RESULTS: Gamithromycin showed favorable PK properties in rabbits, including high subcutaneous bioavailability (86.7 ± 10.7%) and low plasma protein binding (18.5-31.9%). PK analysis identified a mean plasma peak concentration (Cmax) of 1.64 ± 0.86 mg/L and terminal half-life (T1/2) of 31.5 ± 5.74 h after subcutaneous injection. For P. multocida, short post-antibiotic effects (PAE) (1.1-5.3 h) and post-antibiotic sub-inhibitory concentration effects (PA-SME) (6.6-9.1 h) were observed after exposure to gamithromycin at 1 to 4× minimal inhibitory concentration (MIC). Gamithromycin demonstrated concentration-dependent bactericidal activity and the PK/PD index area under the concentration-time curve over 24 h (AUC24h)/MIC correlated well with efficacy (R2 > 0.99). The plasma AUC24h/MIC ratios of gamithromycin associated with the bacteriostatic, bactericidal and bacterial eradication against P. multocida were 15.4, 24.9 and 27.8 h in rabbits, respectively. CONCLUSIONS: Subcutaneous administration of 6 mg/kg gamithromycin reached therapeutic concentrations in rabbit plasma against P. multocida. The PK/PD ratios determined herein in combination with ex vivo activity and favorable rabbit PK indicate that gamithromycin may be used for the treatment of rabbit pasteurellosis.

Qualitative and Quantitative Analytical Techniques of Nucleic Acid Modification Based on Mass Spectrometry for Biomarker Discovery.

Nucleic acid modifications play important roles in biological activities and disease occurrences, and have been considered as cancer biomarkers. Due to the relatively low amount of nucleic acid modifications in biological samples, it is necessary to develop sensitive and reliable qualitative and quantitative methods to reveal the content of any modifications. In this review, the key processes affecting the qualitative and quantitative analyses are discussed, such as sample digestion, nucleoside extraction, chemical labeling, chromatographic separation, mass spectrometry detection, and data processing. The improvement of the detection sensitivity and specificity of analytical methods based on mass spectrometry makes it possible to study low-abundance modifications and their biological functions. Some typical nucleic acid modifications and their potential as biomarkers are displayed, and efforts to improve diagnostic accuracy are discussed. Future perspectives are raised for this research field.

Targeted inhibition of gut bacterial ß-glucuronidases by octyl gallate alleviates mycophenolate mofetil-induced gastrointestinal toxicity.

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China.

OBJECTIVES: Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. METHODS: The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers' markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. RESULTS: Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6')-Ib-cr. CONCLUSION: These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

Increased Activity of ß-Lactam Antibiotics in Combination with Carvacrol against MRSA Bacteremia and Catheter-Associated Biofilm Infections.

ß-Lactam antibiotics are the mainstay for the treatment of staphylococcal infections, but their utility is greatly limited by the emergence and rapid dissemination of methicillin-resistant Staphylococcus aureus (MRSA). Herein, we evaluated the ability of the plant-derived monoterpene carvacrol to act as an antibiotic adjuvant, revitalizing the anti-MRSA activity of ß-lactam antibiotics. Increased susceptibility of MRSA to ß-lactam antibiotics and significant synergistic activities were observed with carvacrol-based combinations. Carvacrol significantly inhibited MRSA biofilms and reduced the production of exopolysaccharide, polysaccharide intercellular adhesin, and extracellular DNA and showed synergistic biofilm inhibition in combination with ß-lactams. Transcriptome analysis revealed profound downregulation in the expression of genes involved in two-component systems and S. aureus infection. Mechanistic studies indicate that carvacrol inhibits the expression of staphylococcal accessory regulator sarA and interferes with SarA-mecA promoter binding that decreases mecA-mediated ß-lactam resistance. Consistently, the in vivo experiment also supported that carvacrol restored MRSA sensitivity to ß-lactam antibiotic treatments in both murine models of bacteremia and biofilm-associated infection. Our results indicated that carvacrol has a potential role as a combinatorial partner with ß-lactam antibiotics to address MRSA infections.

Dynamic human exposure to airborne bacteria-associated antibiotic resistomes revealed by longitudinal personal monitoring data.

Airborne antibiotic-resistant bacteria (ARB) can critically impact human health. We performed resistome profiling of 283 personal airborne exposure samples from 15 participants spanning 890 days and 66 locations. We found a greater diversity and abundance of airborne bacteria community and antibiotic resistomes in spring than in winter, and temperature contributed largely to the difference. A total of 1123 bacterial genera were detected, with 16 genera dominating. Of which, 7/16 were annotated as major antibiotic resistance gene (ARG) hosts. The participants were exposed to a highly dynamic collection of ARGs, including 322 subtypes conferring resistance to 18 antibiotic classes dominated by multidrug, macrolide-lincosamide-streptogramin, ß-lactam, and fosfomycin. Unlike the overall community-level bacteria exposure, an extremely high abundance of specific ARG subtypes, including lunA and qacG, were found in some samples. Staphylococcus was the predominant genus in the bacterial community, serving as a primary bacterial host for the ARGs. The annotation of ARG-carrying contigs indicated that humans and companion animals were major reservoirs for ARG-carrying Staphylococcus. This study contextualized airborne antibiotic resistomes in the precision medicine framework through longitudinal personal monitoring, which can have broad implications for human health.

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.

Antibiotic Resistance Patterns and Molecular Characterization of Streptococcus suis Isolates from Swine and Humans in China.

Streptococcus suis is a zoonotic pathogen that causes disease in humans after exposure to infected pigs or pig-derived food products. In this study, we examined the serotype distribution, antimicrobial resistance phenotypes and genotypes, integrative and conjugative elements (ICEs), and associated genomic environments of S. suis isolates from humans and pigs in China from 2008 to 2019. We identified isolates of 13 serotypes, predominated by serotype 2 (40/96; 41.7%), serotype 3 (10/96; 10.4%), and serotype 1 (6/96; 6.3%). Whole-genome sequencing analysis revealed that these isolates possessed 36 different sequence types (STs), and ST242 and ST117 were the most prevalent. Phylogenetic analysis revealed possible animal and human clonal transmission, while antimicrobial susceptibility testing indicated high-level resistance to macrolides, tetracyclines, and aminoglycosides. These isolates carried 24 antibiotic resistance genes (ARGs) that conferred resistance to 7 antibiotic classes. The antibiotic resistance genotypes were directly correlated with the observed phenotypes. We also identified ICEs in 10 isolates, which were present in 4 different genetic environments and possessed differing ARG combinations. We also predicted and confirmed by PCR analysis the existence of a translocatable unit (TU) in which the oxazolidinone resistance gene optrA was flanked by IS1216E elements. One-half (5/10) of the ICE-carrying strains could be mobilized by conjugation. A comparison of the parental recipient with an ICE-carrying transconjugant in a mouse in vivo thigh infection model indicated that the ICE strain could not be eliminated with tetracycline treatment. S. suis therefore poses a significant challenge to global public health and requires continuous monitoring, especially for the presence of ICEs and associated ARGs that can be transferred via conjugation. IMPORTANCE S. suis is a serious zoonotic pathogen. In this study, we investigated the epidemiological and molecular characteristics of 96 S. suis isolates from 10 different provinces of China from 2008 to 2019. A subset of these isolates (10) carried ICEs that were able to be horizontally transferred among isolates of different S. suis serotypes. A mouse thigh infection model revealed that ICE-facilitated ARG transfer promoted resistance development. S. suis requires continuous monitoring, especially for the presence of ICEs and associated ARGs that can be transferred via conjugation.

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.

Prevalence and molecular characteristics of mcr-1-positive Escherichia coli isolated from duck farms and the surrounding environments in coastal China.

The emergence of colistin-resistance is considered a threat to public health and colistin-resistant bacteria have recently been reported in animal, environmental and human sources. Whereas, the epidemic and dissemination of colistin-resistant bacteria in duck farms have not been surveyed, especially the surrounding environmental contamination from duck farms. We investigated the prevalence and molecular characteristics of mcr-1-positive E. coli from duck farms in coastal China. 360 mcr-1-positive E. coli isolates were collected from 1112 samples from duck farms and surrounding environments. The prevalence of mcr-1-positive E. coli in Guangdong province was higher than other two provinces we examined. PFGE analysis indicated clonal spread of mcr-1-positive E. coli between duck farms and surrounding environments, including water and soil. MLST analysis demonstrated that ST10 was more common than ST1011, ST117, and ST48. Phylogenomic analysis also suggested mcr-1-positive E. coli collected from distinct cities were assigned to the same lineage and mcr-1 was primarily located on IncI2 and IncHI2 plasmids. Genomic environment analysis showed mobile gene elements ISApl1 most likely plays a key role in the horizontal transmission of mcr-1. WGS further revealed that mcr-1 was found associated with 27 different ARGs. Our findings emphasize the urgent need for effective colistin resistance surveillance in humans, animals and the environment.

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.

Phenotypic and Genotypic Characterization of Carbapenem-Resistant Enterobacteriaceae Recovered from a Single Hospital in China, 2013 to 2017.

Objective: Carbapenem-resistant Enterobacteriaceae (CRE) have become an increasingly common cause of healthcare-related infections and present a serious challenge to clinical treatment. This study examined the phenotypic, genotypic characterization, clinical, and microbiological data of CRE in the Huizhou Municipal Central Hospital. Methods: We conducted a phenotypic susceptibility evaluation and whole genome sequence analysis for 52 CRE strains isolated from 37 patients and 2 medical device-related samples during 2013-2017 to characterize risk factors, antimicrobial resistance profiles, dominant clones and hospital transmission. Results: Long-term hospitalization, treatment time with antibiotics and use of invasive devices were linked to the risk of CRE infection. The carbapenem resistance genes (CRGs) we found included blaNDM (82.7%), blaIMP (19.2%) and blaKPC (3.8%), Escherichia coli (44.2%) and Klebsiella pneumoniae (44.2%) were the dominant species we identified, and the type of CRG carried by isolates was highly correlated with species. The coexistence of CRGs with a variety of other antibiotic resistance genes leads to an increased prevalence of high resistance levels for CRE to ß-lactams and other antibiotic classes such as aminoglycosides and fluoroquinolones. These isolates were sensitive only to colistin and tigecycline. In addition to this, we observed significantly genomic diversity of CRE isolates in this hospital. Importantly, we found that long-term transmission of multiple CRE clones had occurred at this hospital between various wards. Conclusion: Evaluating and improving the current infection control strategies may be necessary, and reducing nosocomial transmission remains the primary control element for CRE infections in China.

H2S responsive PEGylated poly (lipoic acid) with ciprofloxacin for targeted therapy of Salmonella.

Targeted antibiotic delivery system would be an ideal solution for the treatment of enteropathogenic infections since it avoids the excessive usage of antibiotics clinically, which may lead to threat on public health and food safety. Salmonella spp. are Enteropathogens, but they are also robust H2S producers in the intestinal tracts of hosts. To this end, the PEGylated poly (α lipoic acid) (PEG-PALA) copolymer nanoparticles with hydrophilic exterior and hydrophobic interior were designated in this study to encapsulate the antibiotics and release them in response to H2S produced by Salmonella spp. The PEG-PALA nanoparticles demonstrated excellent stability in vitro and biocompatibility toward mammalian Caco-2 and 293 T cells. The release of ciprofloxacin from PEG-PALA nanoparticle was only 25.44 ± 0.57% and 26.98 ± 1.93% (w/w) in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) solutions without H2S stimulation. However, the release amounts of ciprofloxacin were up to 73.68 ± 1.63% (w/w) in the presence of 1 mM Na2S as H2S source. In the mouse infection model, PEG-PALA nanoparticles encapsulated with ciprofloxacin (PEG-PALA@CIP) reduced the Salmonella colonization in the heart, liver, spleen, lung, cecum, and faeces, prolonged ciprofloxacin persistence in the intestine while reducing its absorption into the blood. More importantly, these nanoparticles reduced 3.4-fold of Enterobacteriaceae levels and increased 1.5-fold of the Lactobacillaceae levels compared with the drug administered in the free form. Moreover, these nanoparticles resulted in only minimal signs of intestinal tract inflammation. The H2S-responsive antibiotic delivery systems reported in this study demonstrating a variety of advantages including protected the drug from deactivation by gastric and intestinal fluids, maintained a high concentration in the intestinal tract and maximally kept the gut microbiota homeostasis. As such, this targeted antibiotic delivery systems are for the encapsulation of antibiotics to target specific enteropathogens.

Classification and molecular characteristics of tet(X)-carrying plasmids in Acinetobacter species.

The rapid dissemination of plasmid-mediated tet(X) genes in Acinetobacter species has compromised the clinical effectiveness of tigecycline, one of the last-resort antibiotics. However, the classification strategy and homology group of tet(X)-positive Acinetobacter spp. plasmids remain largely unknown. In this study, we classified them by genome-based replicon typing, followed by analyses of structural characteristics, transferability and in vivo effect. A total of 34 plasmids distributed in at least nine Acinetobacter species were collected, including three tet(X3)-positive plasmids and one tet(X6)-positive plasmid from our genome sequencing results. Among them, there were 28 plasmids carrying Rep_3 superfamily replicase genes and classified into six homology groups, consisting of GR31 (82.1%), GR26 (3.6%), GR41 (3.6%), GR59 (3.6%), and novel groups GR60 (3.6%) and GR61 (3.6%). Our tet(X3)-positive plasmids pYH16040-1, pYH16056-1, and pYH12068-1 belonged to the dominant GR31 group, whereas the tet(X6)-positive plasmid pYH12068-2 was unclassified. Structurally, all tet(X)-positive GR31 plasmids shared similar plasmid replication (repB), stability (parA and parB) and accessory modules [tet(X) and sul2], and 97.6% of plasmid-mediated tet(X) genes in Acinetobacter species were adjacent to ISCR2. Conjugation and susceptibility testing revealed pYH16040-1, pYH16056-1, and pYH12068-2, carrying plasmid transfer modules, were able to mediate the mobilization of multiple antibiotic resistance. Under the treatment of tigecycline, the mortality rate of Galleria mellonella infected by pYH16040-1-mediated tet(X3)-positive Acinetobacter spp. isolate significantly increased when compared with its plasmid-cured strain (p < 0.0001). The spread of such plasmids is of great clinical concern, more effects are needed and will facilitate the future analysis of tet(X)-positive Acinetobacter spp. plasmids.

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.

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 New Delhi Metallo-ß-Lactamase-Producing Escherichia coli in Food-Producing Animals in China.

We conducted a molecular surveillance study for carbapenem-resistant Enterobacteriaceae (CRE) colonization in food-producing animals in China that included primarily swine and poultry for three consecutive years. A total of 2,771 samples from food-producing animals and their surrounding environments were collected from different regions in China from 2015 to 2017. Enrichment cultures supplemented with meropenem were used to isolate carbapenem non-susceptible isolates and these were subsequently identified by MALDI-TOF MS. Resistance phenotypes and genotypes were confirmed using antimicrobial susceptibility testing and molecular biological techniques. Genomic characteristics of the carbapenemase-producing isolates were investigated using whole genome sequencing (WGS) and bioinformatic analysis. In total, 88 NDM-positive Enterobacteriaceae were identified from 2,771 samples and 96.6% were Escherichia coli. The New Delhi metallo-ß-lactamase (NDM)-positive E. coli displayed a diversity of sequence types (ST), and ST48 and ST165 were the most prevalent. Three variants of bla NDM (bla NDM-1, bla NDM-4, and bla NDM-5) were detected and WGS indicated that bla NDM-5 predominated and was carried primarily on IncX3 plasmids. All these isolates were also multiply-drug resistant. These results revealed that food-producing animals in China are an important reservoir for NDM-positive E. coli and pose a potential threat to public health.