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.

Cinnamaldehyde targets SarA to enhance ß-lactam antibiotic activity against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a current global public health problem due to its increasing resistance to the most recent antibiotic therapies. One critical approach is to develop ways to revitalize existing antibiotics. Here, we show that the phytogenic compound cinnamaldehyde (CIN) and ß-lactam antibiotic combinations can functionally synergize and resensitize clinical MRSA isolates to ß-lactam therapy and inhibit MRSA biofilm formation. Mechanistic studies indicated that the CIN potentiation effect on ß-lactams was primarily the result of inhibition of the mecA expression by targeting the staphylococcal accessory regulator sarA. CIN alone or in combination with ß-lactams decreased sarA gene expression and increased SarA protein phosphorylation that impaired SarA binding to the mecA promoter element and downregulated virulence genes such as those encoding biofilm, α-hemolysin, and adhesin. Perturbation of SarA-mecA binding thus interfered with PBP2a biosynthesis and this decreased MRSA resistance to ß-lactams. Furthermore, CIN fully restored the anti-MRSA activities of ß-lactam antibiotics in vivo in murine models of bacteremia and biofilm infections. Together, our results indicated that CIN acts as a ß-lactam adjuvant and can be applied as an alternative therapy to combat multidrug-resistant MRSA infections.

Sleep Deprivation Induces Gut Damage via Ferroptosis.

Sleep deprivation (SD) has been associated with a plethora of severe pathophysiological syndromes, including gut damage, which recently has been elucidated as an outcome of the accumulation of reactive oxygen species (ROS). However, the spatiotemporal analysis conducted in this study has intriguingly shown that specific events cause harmful damage to the gut, particularly to goblet cells, before the accumulation of lethal ROS. Transcriptomic and metabolomic analyses have identified significant enrichment of metabolites related to ferroptosis in mice suffering from SD. Further analysis revealed that melatonin could rescue the ferroptotic damage in mice by suppressing lipid peroxidation associated with ALOX15 signaling. ALOX15 knockout protected the mice from the serious damage caused by SD-associated ferroptosis. These findings suggest that melatonin and ferroptosis could be targets to prevent devastating gut damage in animals exposed to SD. To sum up, this study is the first report that proposes a noncanonical modulation in SD-induced gut damage via ferroptosis with a clearly elucidated mechanism and highlights the active role of melatonin as a potential target to maximally sustain the state during SD.

Concentration-resistance relationship and PK/PD evaluation of danofloxacin against emergence of resistant Pasteurella multocida in an in vitro dynamic model.

AIMS: This study aimed to assess the pharmacokinetic/pharmacodynamic (PK/PD) targets of danofloxacin to minimize the risk of selecting resistant Pasteurella multocida mutants and to identify the mechanisms underlying their resistance in an in vitro dynamic model, attaining the optimum dosing regimen of danofloxacin to improve its clinical efficacy based on the mutant selection window (MSW) hypothesis. METHODS AND RESULTS: Danofloxacin at seven dosing regimens and 5 days of treatment were simulated to quantify the bactericidal kinetics and enrichment of resistant mutants upon continuous antibiotic exposure. The magnitudes of PK/PD targets associated with different efficacies were determined in the model. The 24 h area under the concentration-time curve (AUC) to minimum inhibitory concentration (MIC) ratios (AUC24h/MIC) of danofloxacin associated with bacteriostatic, bactericidal and eradication effects against P. multocida were 34, 52, and 64 h. This translates to average danofloxacin concentrations (Cav) over 24 h being 1.42, 2.17, and 2.67 times the MIC, respectively. An AUC/MIC-dependent antibacterial efficacy and AUC/mutant prevention concentration (MPC)-dependent enrichment of P. multocida mutants in which maximum losses in danofloxacin susceptibility occurred at a simulated AUC24h/MIC ratio of 72 h (i.e. Cav of three times the MIC). The overexpression of efflux pumps (acrAB-tolC) and their regulatory genes (marA, soxS, and ramA) was associated with reduced susceptibility in danofloxacin-exposed P. multocida. The AUC24h/MPC ratio of 19 h (i.e. Cav of 0.8 times the MPC) was determined to be the minimum mutant prevention target value for the selection of resistant P. multocida mutants. CONCLUSIONS: The emergence of P. multocida resistance to danofloxacin exhibited a concentration-dependent pattern and was consistent with the MSW hypothesis. The current clinical dosing regimen of danofloxacin (2.5 mg kg-1) may have a risk of treatment failure due to inducible fluoroquinolone resistance.

Mechanisms of gastrointestinal toxicity in neuromyelitis optica spectrum disorder patients treated with mycophenolate mofetil: insights from a mouse model and human study.

Mycophenolate mofetil (MMF) is commonly utilized for the treatment of neuromyelitis optica spectrum disorders (NMOSD). However, a subset of patients experience significant gastrointestinal (GI) adverse effects following MMF administration. The present study aims to elucidate the underlying mechanisms of MMF-induced GI toxicity in NMOSD. Utilizing a vancomycin-treated mouse model, we compiled a comprehensive data set to investigate the microbiome and metabolome in the GI tract to elucidate the mechanisms of MMF GI toxicity. Furthermore, we enrolled 17 female NMOSD patients receiving MMF, who were stratified into non-diarrhea NMOSD and diarrhea NMOSD (DNM) groups, in addition to 12 healthy controls. The gut microbiota of stool samples was analyzed using 16S rRNA gene sequencing. Vancomycin administration prevented weight loss and tissue injury caused by MMF, affecting colon metabolomes and microbiomes. Bacterial ß-glucuronidase from Bacteroidetes and Firmicutes was linked to intestinal tissue damage. The DNM group showed higher alpha diversity and increased levels of Firmicutes and Proteobacteria. The ß-glucuronidase produced by Firmicutes may be important in causing gastrointestinal side effects from MMF in NMOSD treatment, providing useful information for future research on MMF. IMPORTANCE: Neuromyelitis optica spectrum disorder (NMOSD) patients frequently endure severe consequences like paralysis and blindness. Mycophenolate mofetil (MMF) effectively addresses these issues, but its usage is hindered by gastrointestinal (GI) complications. Through uncovering the intricate interplay among MMF, gut microbiota, and metabolic pathways, this study identifies specific gut bacteria responsible for metabolizing MMF into a potentially harmful form, thus contributing to GI side effects. These findings not only deepen our comprehension of MMF toxicity but also propose potential strategies, such as inhibiting these bacteria, to mitigate these adverse effects. This insight holds broader implications for minimizing complications in NMOSD patients undergoing MMF therapy.

Daily occupational exposure in swine farm alters human skin microbiota and antibiotic resistome.

Antimicrobial resistance (AMR) is a major threat to global public health, and antibiotic resistance genes (ARGs) are widely distributed across humans, animals, and environment. Farming environments are emerging as a key research area for ARGs and antibiotic resistant bacteria (ARB). While the skin is an important reservoir of ARGs and ARB, transmission mechanisms between farming environments and human skin remain unclear. Previous studies confirmed that swine farm environmental exposures alter skin microbiome, but the timeline of these changes is ill defined. To improve understanding of these changes and to determine the specific time, we designed a cohort study of swine farm workers and students through collected skin and environmental samples to explore the impact of daily occupational exposure in swine farm on human skin microbiome. Results indicated that exposure to livestock-associated environments where microorganisms are richer than school environment can reshape the human skin microbiome and antibiotic resistome. Exposure of 5 h was sufficient to modify the microbiome and ARG structure in workers' skin by enriching microorganisms and ARGs. These changes were preserved once formed. Further analysis indicated that ARGs carried by host microorganisms may transfer between the environment with workers' skin and have the potential to expand to the general population using farm workers as an ARG vector. These results raised concerns about potential transmission of ARGs to the broader community. Therefore, it is necessary to take corresponding intervention measures in the production process to reduce the possibility of ARGs and ARB transmission.

Innovative Delivery System Combining CRISPR-Cas12f for Combatting Antimicrobial Resistance in Gram-Negative Bacteria.

Antimicrobial resistance poses a significant global challenge, demanding innovative approaches, such as the CRISPR-Cas-mediated resistance plasmid or gene-curing system, to effectively combat this urgent crisis. To enable successful curing of antimicrobial genes or plasmids through CRISPR-Cas technology, the development of an efficient broad-host-range delivery system is paramount. In this study, we have successfully designed and constructed a novel functional gene delivery plasmid, pQ-mini, utilizing the backbone of a broad-host-range Inc.Q plasmid. Moreover, we have integrated the CRISPR-Cas12f system into the pQ-mini plasmid to enable gene-curing in broad-host of bacteria. Our findings demonstrate that pQ-mini facilitates the highly efficient transfer of genetic elements to diverse bacteria, particularly in various species in the order of Enterobacterales, exhibiting a broader host range and superior conjugation efficiency compared to the commonly used pMB1-like plasmid. Notably, pQ-mini effectively delivers the CRISPR-Cas12f system to antimicrobial-resistant strains, resulting in remarkable curing efficiencies for plasmid-borne mcr-1 or blaKPC genes that are comparable to those achieved by the previously reported pCasCure system. In conclusion, our study successfully establishes and optimizes pQ-mini as a broad-host-range functional gene delivery vector. Furthermore, in combination with the CRISPR-Cas system, pQ-mini demonstrates its potential for broad-host delivery, highlighting its promising role as a novel antimicrobial tool against the growing threat of antimicrobial resistance.

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.

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.

Synergistic effects of ceftazidime/avibactam combined with meropenem in a murine model of infection with KPC-producing Klebsiella pneumoniae.

OBJECTIVES: The emergence and expansion of carbapenem-resistant Klebsiella pneumoniae infections is a concern due to the lack of 'first-line' antibiotic treatment options. The ceftazidime/avibactam is an important clinical treatment for carbapenem-resistant K. pneumoniae infections but there is an increasing number of cases of treatment failure and drug resistance. Therefore, a potential solution is combination therapies that result in synergistic activity against K. pneumoniae carbapenemase: producing K. pneumoniae (KPC-Kp) isolates and preventing the emergence of KPC mutants resistant to ceftazidime/avibactam are needed in lieu of novel antibiotics. METHODS: To evaluate their synergistic activity, antibiotic combinations were tested against 26 KPC-Kp strains. Antibiotic resistance profiles, molecular characteristics and virulence genes were investigated by susceptibility testing and whole-genome sequencing. Antibiotic synergy was evaluated by in vitro chequerboard experiments, time-killing curves and dose-response assays. The mouse thigh model was used to confirm antibiotic combination activities in vivo. Additionally, antibiotic combinations were evaluated for their ability to prevent the emergence of ceftazidime/avibactam resistant mutations of blaKPC. RESULTS: The combination of ceftazidime/avibactam plus meropenem showed remarkable synergistic activity against 26 strains and restored susceptibility to both the partnering antibiotics. The significant therapeutic effect of ceftazidime/avibactam combined with meropenem was also confirmed in the mouse model and bacterial loads in the thigh muscle of the combination groups were significantly reduced. Furthermore, ceftazidime/avibactam plus meropenem showed significant activity in preventing the occurrence of resistance mutations. CONCLUSIONS: Our results indicated that the combination of ceftazidime/avibactam plus meropenem offers viable therapeutic alternatives in treating serious infections due to KPC-Kp.

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.