Core and variable antimicrobial resistance genes in the gut microbiomes of Chinese and European pigs

Monitoring the prevalence of antimicrobial resistance genes(ARGs)is vital for addressing the global crisis of antibiotic-resistant bacterial infections.Despite its importance,the characterization of ARGs and microbiome structures,as well as the identification of indicators for routine ARG monitoring in pig farms,are still lacking,particularly concerning variations in antimicrobial exposure in different countries or regions.Here,metagenomics and random forest machine learning were used to elucidate the ARG profiles,microbiome structures,and ARG contamination indicators in pig manure under different antimicrobial pressures between China and Europe.Results showed that Chinese pigs exposed to high-level antimicrobials exhibited higher total and plasmid-mediated ARG abundances compared to those in European pigs(P<0.05).ANT(6)-Ib,APH(3')-IIIa,and tet(40)were identified as shared core ARGs between the two pig populations.Furthermore,the core ARGs identified in pig populations were correlated with those found in human populations within the same geographical regions.Lactobacillus and Prevotella were identified as the dominant genera in the core microbiomes of Chinese and European pigs,respectively.Forty ARG markers and 43 biomarkers were able to differentiate between the Chinese and European pig manure samples with accuracies of 100%and 98.7%,respectively.Indicators for assessing ARG contamination in Chinese and European pigs also achieved high accuracy(r=0.72-0.88).Escherichia flexneri in both Chinese and European pig populations carried between 21 and 37 ARGs.The results of this study emphasize the importance of global collaboration in reducing antimicrobial resistance risk and provide validated indicators for evaluating the risk of ARG contamination in pig farms.

Genetic Diversity, Antimicrobial Resistance, and Virulence Genes of Aeromonas Isolates from Clinical Patients, Tap Water Systems, and Food

Objective This study aimed to evaluate the genetic diversity,virulence,and antimicrobial resistance of Aeromonas isolates from clinical patients,tap water systems,and food.Methods Ninety Aeromonas isolates were obtained from Ma’anshan,Anhui province,China,and subjected to multi-locus sequence typing(MLST)with six housekeeping genes.Their taxonomy was investigated using concatenated gyr B-cpn60 sequences,while their resistance to 12 antibiotics was evaluated.Ten putative virulence factors and several resistance genes were identified by PCR and sequencing.Results The 90 Aeromonas isolates were divided into 84 sequence types,80 of which were novel,indicating high genetic diversity.The Aeromonas isolates were classified into eight different species.PCR assays identified virulence genes in the isolates,with the enterotoxin and hemolysin genes act,aer A,alt,and ast found in 47(52.2%),13(14.4%),22(24.4%),and 12(13.3%)of the isolates,respectively.The majority of the isolates(≥90%)were susceptible to aztreonam,imipenem,cefepime,chloramphenicol,gentamicin,tetracycline,and ciprofloxacin.However,several resistance genes were detected in the isolates,as well as a new mcr-3 variant.Conclusions Sequence type,virulence properties,and antibiotic resistance vary in Aeromonas isolates from clinical patients,tap water systems,and food.

Seafood as a Reservoir of Gram-negative Bacteria Carrying Integrons and Antimicrobial Resistance Genes in Japan

PCR and DNA sequencing were used to screen and characterize integrons and resistance genes in Gram-negative bacteria isolated from seafood products in Japan.A total of 215 Gram-negative bacteria were isolated from local and imported seafood samples collected from retail markets in Hiroshima Prefecture.Class 1 integrons containing gene cassettes encoding resistance to trimethoprim

Comparative Study of the Genetic Diversity, Antimicrobial Resistance, and Pathogenicity of Aeromonas Isolates from Clinical Patients and Healthy Individuals

Objective This study was performed to compare the genetic diversity,virulence,and antimicrobial resistance of Aeromonas strains isolated from patients and healthy individuals.Methods A total of 38 clinical strains and 19 strains from healthy individuals were isolated from the samples collected in Ma’anshan City,Anhui Province.Their taxonomy was investigated using concatenated gyrB-cpn60 sequences,and their resistance to 12 antibiotics was evaluated.The pathogenicity of these strains was examined through beta-hemolysis,protease activity,and virulence gene assays.Results The 57 Aeromonas strains were divided into 55 sequence types.Of these types,21 were novel,suggesting that their genetic diversity was high.These Aeromonas isolates could be divided into 7 species,and the positive rates of beta-hemolysis and protease activity were 49.1%and 73.7%,respectively.The detection rate of clinical patients in terms of beta-hemolysis and protease activity was higher than that of healthy individuals.Among the four most common Aeromonas strains,A.dhakensis had the highest detection rate of virulence genes.The multidrug resistance rate of the clinical isolates was much higher than that of the strains isolated from healthy individuals.Conclusions The taxonomy,virulence properties,and antibiotic resistance of Aeromonas isolates from patients differ from those of the isolates from healthy individuals.

A New High-throughput Real-time PCR Assay for the Screening of Multiple Antimicrobial Resistance Genes in Broiler Fecal Samples from China

Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.

Response to Comment on "The role of wildlife (wild birds) in the global transmission of antimicrobial resistance genes"

DEAR EDITOR,Since our first identification of plasmid-mediated colistin resistance gene mcr-1 in 2015 （Liu et al., 2016）, it has been described in human clinics, domestic animals, foods, and the environment worldwide （Schwarz ＆ Johnson, 2016）. Although it is still rare, the emergence of mcr-I in wild animals is of great concern. We summarized two previous reports on mcr-1 in wild birds from Lithuania and Argentina to describe its emergence and characteristics in wildlife and highlight the potentially important role of wild animals, particularly birds, in its global transmission （Wang et al., 2017）. The first detection of mcr-1 in wildlife in Asia was identified in an extended-spectrum β- lactamase-producing Escherichia coil strain isolated from Eurasian coot （Fulica atra）,

Comment on ＂The role of wildlife (wild birds)in the global transmission of antimicrobial resistance genes＂

DEAR EDITOR, We read with interest the article by Wang and colleagues regarding the role of wildlife in the transmission of antimicrobial resistance （AMR） （Wang et al., 2017）. Although we appreciate the efforts in reviewing this important topic, we would like to comment on some statements that we believe are not up-to- date or properly cited.

Association between the Phenotypes and Genotypes of Antimicrobial Resistance in Haemophilus parasuis Isolates from Swine in Quang Binh and Thua Thien Hue Provinces, Vietnam

Haemophilus parasuis (H. parasuis) is one of the bacterial pathogens of great concern as it causes huge economic losses to the swine industry worldwide. One of the reasons why the control of H. parasuis has failed is the increase in antimicrobial resistance (AMR). The country of Vietnam has the second-largest pig production in Asia. However, there is still a lack of data about the AMR prevalence of H. parasuis in Vietnam.The purpose of this study is to investigate the prevalence of AMR and analyze the association between AMR and AMR genes (ARGs). The H. parasuis strains used in this research were isolated from swine in the Quang Binh and Thua Thien Hue Provinces, Central Vietnam, as reported in our previous study. All of the strains were tested for AMR against 25 antibacterial agents using the broth microdilution method and for the presence of ARGs using the polymerase chain reaction (PCR) method. The tested strains were shown to have a high frequency of resistance to trimethoprim/sulfamethoxazole (94.6%), followed by resistance to colistin, chloramphenicol, gentamicin, penicillin, lincomycin, and amoxicillin. The most prevalent ARGs in these strains were blaTEM-1 (94.6%), int (76.8%), gyrA (58.9%), and rmtD (50.0%). Cefuroxime, chloramphenicol, and tobramycin resistances were strongly correlated with the presence of the ARGs blaROB-1 (odds ratio (OR) = 26.3, 95% confidence interval (CI) 2.7–255.7, p = 0.002), catl (OR = 25.1, 95% CI 2.4–258.9, p = 0.004), and strB (OR = 23.5, 95% CI 2.6–212.6, p = 0.001), respectively.This study reveals for the first time the current situation of H. parasuis AMR in Central Vietnam, which is helpful for the clinical control of this disease, as well as for the development of policies and clinical practice guidelines to reduce AMR in swine production in Central Vietnam.

Exploring the abundance and influencing factors of antimicrobial resistance genes in manure plasmidome from swine farms

Plasmids play a critical role in the dissemination of antimicrobial resistance genes(ARGs),however,a systematical understanding of ARGs originated from plasmids in swine production is currently lacking.Herein,quantitative polymerase chain reaction was applied to determine the prevalence of ten ARGs and the class1 integron gene intI1 of plasmid source in swinemanure from 44 farms in Sichuan,Hubei and Hebei provinces,China.All assayed ARGs were observed in plasmid DNA samples,and the average absolute abundance of aac(6’)-Ib-cr,blaNDM,blaCTX-M,optrA,ermB,floR,mcr-1,qnrS,tetM,sul1 and intI1 were 7.09,2.90,4.67,6.62,7.55,7.14,4.08,4.85,7.16,7.11 and 8.07 of 10 log copies/gram,respectively.IntI1 showed a high correlation(r>0.8,P<0.01)with the abundance of aac(6’)-Ib-cr and sul1 in swine manure.Moreover,the farm scale(i.e.,herd population)and geographical location were not found to be critical factors influencing the absolute abundance of ARGs of plasmid DNA in swine farms.However,the concentrations of florfenicol,Cu,Zn,Fe,total phosphorus(TP)and total potassium(TK)demonstrated a significant correlation with the abundance of several ARGs.Particularly,Cu and Zn had high correlations with optrA and blaCTX-M,respectively.Our results demonstrated that antibiotics,heavy metals and environmental nutrients are likely jointly contributing to the long-term persistence of ARGs in swine production.This study provides insights into the abundance and influencing factors of ARGs from swine manure,which is of significance for assessing and reducing the public health risks in livestock production.

A review of the resistome within the digestive tract of livestock

Antimicrobials have been widely used to prevent and treat infectious diseases and promote growth in food-production animals.However,the occurrence of antimicrobial resistance poses a huge threat to public and animal health,especial y in less developed countries where food-producing animals often intermingle with humans.To limit the spread of antimicrobial resistance from food-production animals to humans and the environment,it is essential to have a comprehensive knowledge of the role of the resistome in antimicrobial resistance(AMR),The resistome refers to the col ection of al antimicrobial resistance genes associated with microbiota in a given environment.The dense microbiota in the digestive tract is known to harbour one of the most diverse resistomes in nature.Studies of the resistome in the digestive tract of humans and animals are increasing exponential y as a result of advancements in next-generation sequencing and the expansion of bioinformatic resources/tools to identify and describe the resistome.In this review,we outline the various tools/bioinformatic pipelines currently available to characterize and understand the nature of the intestinal resistome of swine,poultry,and ruminants.We then propose future research directions including analysis of resistome using long-read sequencing,investigation in the role of mobile genetic elements in the expression,function and transmission of AMR.This review outlines the current knowledge and approaches to studying the resistome in food-producing animals and sheds light on future strategies to reduce antimicrobial usage and control the spread of AMR both within and from livestock production systems.

Genetic factors related to the widespread dissemination of ST11 extensively drug-resistant carbapenemase-producing Klebsiella pneumoniae strains within hospital

Background:Carbapenemase-producing Klebsiella pneumoniae(CP-Kp)poses distinct clinical challenges due to extensively drug resistant(XDR)phenotype,and sequence type(ST)11 is the most dominant blaKPC-2-bearing CP-Kp clone in China.The purpose of this current retrospective study was to explore the genetic factors associated with the success of XDR CP-Kp ST11 strains circulated in the intensive care unit(ICU)of a Chinese tertiary hospital.Methods:Six ST11 XDR CP-Kp strains were identified between May and December 2014 and validated by minimum inhibitory concentration examination,polymerase chain reaction,and pyrosequencing.The six ST11 XDR CP-Kp,as well as three multi-drug resistant(MDR)and four susceptible strains,were sequenced using single-molecule real-time method.Comprehensively structural and functional analysis based on comparative genomics was performed to identify genomic characteristics of the XDR ST11 CP-Kp strains.Results:We found that ST11 XDR blaKPC-2-bearing CP-Kp strains isolated from inpatients spread in the ICU of the hospital.Functionally,genes associated with information storage and processing of the ST11 XDR CP-Kp strains were more abundant than those of MDR and susceptible strains,especially genes correlative with mobile genetic elements(MGEs)such as transposons and prophages.Structurally,eleven large-scale genetic regions taken for the unique genome in these ST11 XDR CP-Kp strains were identified as MGEs including transposons,integrons,prophages,genomic islands,and integrative and conjugative elements.Three of them were located on plasmids and eight on chromosomes;five of them were with antimicrobial resistance genes and eight with adaptation associated genes.Notably,a new blaKPC-2-bearingΔΔTn1721-blaKPC-2 transposon,probably transposed and truncated fromΔTn1721-blaKPC-2 by IS903D and ISKpn8,was identified in all six ST11 XDR CP-Kp strains.Conclusion:Our findings suggested that together with clonal spread,MGEs identified uniquely in the ST11 XDR CP-Kp strains might contribute to their formidable adaptability,which facilitated their widespread dissemination in hospital.

Global Genomic Variations of Pathogenic Bacteria <i>Salmonella</i>Reading

US Centers for Disease Control and Prevention (CDC) estimates food-borne pathogenic Salmonella bacteria cause about 1.35 million infections, 26,500 hospitalizations, and 420 deaths in the U.S. every year. Outbreaks of multidrug-resistant Salmonella Reading linked mainly to raw turkey products and alfalfa sprouts have accounted for human illness including mortality. Therefore, we downloaded and compared genome sequences of 897 Salmonella Reading isolated from 27 resources in 11 countries at 21 year-points from the National Center for Biotechnology Information database. Isolates were clustered into 10 clades which consisted of 3 major and 7 minor or single node clades. Although 3 major clades included both Europe and American isolates, one clade consisted of 605 North American isolates out of 614 isolates. Evolutionary distance is more related to the continent than the source of isolation. The host source, continent (North America and Europe) and phylogenetic clade were related to the prevalence of isolates encoding Antimicrobial Resistance Genes (ARGs). Prevalence of prophages was greater in bovine and swine isolates than poultry and human isolates and the least prevalence was found in human isolates. Between continents, the prevalence of phage was greater in North Ame- rican isolates than European. The diversity of virulence factors in swine isolates differed from poultry isolate while no difference was found among continents. In conclusion, evolutionary distance is related to isolation host source rather than the continent, and genome features were distinguished by host and cluster. Our genomic analysis implies that Salmonella Reading evolved independently to environments within its lineages.

Genomic characterization of multidrug-resistant extraintestinal pathogenic Escherichia coli isolated from grain culture soils

Multidrug-resistant(MDR)Escherichia coli,mainly extraintestinal pathogenic E.coli(ExPEC),has been widely reported in infections worldwide.In agricultural soils,manure is a hotspot for the dissemination of antimicrobial resistance genes(ARGs)and pathogenic bacteria;however,MDR bacteria have also been reported in soils with no history of manure use.In addition,cross-resistance and co-resistance have been described as responsible for the metal-driven selection of bacteria resistant to antimicrobials.Therefore,the aim of this study was to analyze three MDR E.coli isolates obtained from Brazilian grain culture soil samples with no history of manure use by whole-genome sequencing.The MDR E.coli isolates were recovered from soils from corn and coffee fields,and presented resistance toβ-lactams,quinolones,aminoglycosides,tetracyclines,sulphonamides,and dihydrofolate reductase inhibitor.Resistome analysis showed ARGs to several antimicrobials(i.e.,β-lactams,tetracyclines,aminoglycosides,sulphonamides,trimethoprim,phenicols,fosfomycin,and macrolides)as well as several metal resistance genes and antibacterial biocide resistance genes.In addition,known mutations in quinolone-resistance-determining regions of GyrA(Ser83Leu and Asp87Asn),ParE(Ser458Thr),and ParC(Ser80Ile)were also detected.Virulome analysis showed the presence of virulence genes(lpfA,mcmA,gad,mchF,iroN,cma,and iss)associated with ExPEC.Multidrug-resistant ExPEC isolates were assigned to phylogenetic group B1.The presence of MDR B1-ExPEC in soil samples shows the ability of these isolates to survive in soils.This study reports for the first time some sequence types(i.e.,ST345,ST448,and ST1146)of MDR E.coli in Brazilian soils.Therefore,these findings contribute to the monitoring of antimicrobial resistance and surveillance studies based on whole-genome sequencing worldwide.