Various mobile genetic elements carrying optrA in Enterococcus faecium and Enterococcus faecalis isolates from swine within the same farm.

OBJECTIVES: In this study, the distribution of the oxazolidinone/phenicol resistance gene optrA and the mobile genetic elements involved in its dissemination were analysed among enterococcal isolates from a farrow-to-finish swine farm. METHODS: Enterococcus faecium and Enterococcus faecalis isolates were obtained from all pig production stages in the farm. The optrA-carrying E. faecium and E. faecalis isolates were subjected to PFGE and antimicrobial susceptibility testing. Complete sequences of the genetically unrelated optrA-carrying E. faecium and E. faecalis isolates were determined using Illumina HiSeq and MinION platforms. RESULTS: The optrA gene was present in 12.2% (23/188) of the E. faecium and E. faecalis isolates, most of which originated from nursery and finishing stages. The 23 optrA-positive Enterococcus isolates represented 15 PFGE types. WGS of representative isolates of the 15 PFGE types showed that optrA was carried by diverse genetic elements either located in the chromosomal DNA or on plasmids. A novel optrA-bearing genetic element was identified on two distinct multi-resistance plasmids from E. faecium. Two new hybrid plasmids carrying several resistance genes were found in two E. faecalis isolates. pC25-1-like plasmids and chromosomally integrated Tn6674 and Tn6823-like transposons were prevalent in the remaining Enterococcus isolates. CONCLUSIONS: The gene optrA was found in genetically unrelated E. faecium and E. faecalis isolates from the same farm. Analysis of the genetic contexts of optrA suggested that horizontal transfer including different plasmids and transposons played a key role in the dissemination of optrA in this farm.

Molecular Characteristics and Virulence Gene Analysis of Non-O157 Shiga Toxin-Producing Escherichia coli from Cattle in Xinjiang.

Non-O157 Shiga toxin (stx)-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Cattle are the principal reservoirs of STEC, although other animals can be carriers. Humans are mainly infected by consuming contaminated drinking water or food. This study aimed to evaluate the virulence potential of isolated bovine non-O157 STEC to humans in Xinjiang. During 2015-2017, 978 rectal swab samples collected from cattle of 5 farms were screened for the presence of Shiga toxin-encoding genes by polymerase chain reaction. Strains identified as STEC were isolated from rectal swab samples, and were characterized for stx subtype, virulence genes, O serogroup, phylogenetic group, and hemolytic phenotype. Among 125 non-O157 STEC isolates, the prevalence percentages of stx1 and stx2 were 22 and 21, respectively, and 57% of the isolates carried both Shiga toxins. The stx subtypes were mainly found in the combination of stx1a/stx2a (57%), stx2a (20%), stx1a (22%), stx1a/stx2a/stx2c (1%), and stx2a/stx2c (1%). The enterohemolysin (ehxA) gene was found in 94% of the isolates. No intimin (eae) was detected. Hemolysis was observed in 33% of the isolates. Two STEC serogroups O145 (17%) and O113 (2%) were found, which were reported to be associated with outbreaks of human disease. Phylotyping assays showed that most strains largely belong to groups A (91%) and B1 (7%). The results of this study can help improve our understanding of the epidemiological aspects of bovine STEC and devise strategies for protection against it.

Identification of the natural product paeonol derived from peony bark as an inhibitor of the Salmonella enterica serovar Typhimurium type III secretion system.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic pathogen in public health and food safety. The type III secretion system (T3SS) encoded by Salmonella pathogenicity island (SPI) is a sophisticated molecular machine that facilitates active invasion, intracellular replication, and host inflammation. Due to increasing antibiotic resistance, new therapeutic strategies that target the Salmonella T3SS have received considerable attention. In this study, paeonol was identified as an inhibitor of the S. Typhimurium T3SS. Paeonol significantly blocked the translocation of SipA into host cells and suppressed the expression of effector proteins without affecting bacterial growth in the effective concentration range. Additionally, S. Typhimurium-mediated cell injury and invasion levels were significantly reduced after treatment with paeonol, without cytotoxicity. Most importantly, the comprehensive protective effect of paeonol was confirmed in an S. Typhimurium mouse infection model. Preliminary mechanistic studies suggest that paeonol inhibits the expression of effector proteins by reducing the transcription level of the SPI-1 regulatory pathway gene hilA. This work provides proof that paeonol could be used as a potential drug to treat infections caused by Salmonella.

High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed.

Campylobacter is a major foodborne pathogen in humans and a significant cause of abortion in sheep. Although ruminants are increasingly recognized as important reservoirs for Campylobacter species, limited information is available about the molecular epidemiology and antimicrobial resistance (AMR) profiles of sheep Campylobacter Here, we describe a two-trial study that examined Campylobacter profiles in sheep and determined whether in-feed tetracycline (TET) influenced the distribution and AMR profiles of Campylobacter Each trial involved 80 commercial sheep naturally infected with Campylobacter: 40 of these sheep were medicated with tetracycline in feed, while the other 40 received feed without antibiotics. Fecal and bile samples were collected for the isolation of Campylobacter The bacterial isolates were analyzed for antimicrobial susceptibility and genotypes. The results revealed that 87.0% and 61.3% of the fecal and bile samples were positive for Campylobacter (Campylobacter jejuni and Campylobacter coli), with no significant differences between the medicated and nonmedicated groups. All but one of the tested Campylobacter isolates were resistant to tetracycline. Although fluoroquinolone (FQ) resistance remained low in C. jejuni (1.7%), 95.0% of the C. coli isolates were resistant to FQ. Genotyping revealed that C. jejuni sequence type 2862 (ST2862) and C. coli ST902 were the predominant genotypes in the sheep. Feed medication with tetracycline did not affect the overall prevalence, species distribution, and AMR profiles of Campylobacter, but it did increase the total Campylobacter counts in bile and gallbladder. These findings identify predominant Campylobacter clones, reveal the high prevalence of FQ-resistant C. coli, and provide new insights into the epidemiology of Campylobacter in sheep.IMPORTANCECampylobacter is a major cause of foodborne illness in humans, and antibiotic-resistant Campylobacter is considered a serious threat to public health in the United States and worldwide. As a foodborne pathogen, Campylobacter commonly exists in the intestinal tract of ruminant animals, such as sheep and cattle. Results from this study reveal the predominant genotypes and high prevalence of tetracycline (TET) and fluoroquinolone (FQ) resistance in sheep Campylobacter The finding on fluoroquinolone resistance in sheep Campylobacter is unexpected, as this class of antibiotics is not used for sheep in the United States, and it may suggest the transmission of fluoroquinolone-resistant Campylobacter from cattle to sheep. Additionally, the results demonstrate that in-feed medication with tetracycline increases Campylobacter counts in gallbladders, suggesting that the antibiotic promotes Campylobacter colonization of the gallbladder. These findings provide new information on Campylobacter epidemiology in sheep, which may be useful for curbing the spread of antibiotic-resistant Campylobacter in animal reservoirs.

Physiochemical and functional properties of chum salmon (Oncorhynchus keta) skin gelatin extracted at different temperatures.

BACKGROUND: Aquatic source gelatins are gaining more attention due to the advantages in safety and religion acceptability compared with mammalian sources. For understanding the effects of extracting temperature on gelatins from chum salmon (Oncorhynchus keta) skins (GCSS), gelatins were extracted at temperatures from 40 to 90°C and the physiochemical properties of GCSS were investigated. RESULTS: GCSS yield increased while imino acids content declined as the increase of temperature. GCSS40, 50 and 60 showed strong ß-, α1- and α2-chains but the three faded in GCSS70, 80 and 90, with the presence of low molecular weight fragments. Amides A, I and III were shifted to higher wavenumber in GCSS70, 80 and 90 compared with that of GCSS40, 50 and 60. X-ray diffraction showed lower intensity of peak at 7° in GCSS80 and 90 than in the other GCSS. Gel strength declined while a*, b* and ΔE* value increased as temperature increased. Foam expansion and stability of GCSS40, 50 and 60 were lower than those of GCSS70, 80 and 90. Emulsion activity and stability decreased as temperature increased. CONCLUSION: Extracting temperature greatly affected yield, molecular composition and functionalities of GCSS. A temperature lower than 50°C is recommended for GCSS extraction. © 2017 Society of Chemical Industry.

Molecular epidemiology, phenotypic and genomic characterization of antibiotic-resistant enterococcal isolates from diverse farm animals in Xinjiang, China.

Multidrug-resistant (MDR) bacteria in farm environments can be transferred to humans through the food chain and occupational exposure. Enterococcus infections caused by linezolid resistant enterococci (LRE) are becoming more challenging to treat as their resistance to antibiotics intensifies. Therefore, this study investigated the molecular epidemiology, phenotypic and genomic characterization of enterococci in seven species of farm animals (sheep, chicken, swine, camel, cattle, equine, pigeon) anal swab from Xinjiang, China by agar dilution method, polymerase chain reaction (PCR), whole-genome sequencing (WGS) and bioinformatics analysis. A total of 771 samples were collected, 599 (78 %) were contaminated with Enterococcus spp., among which Enterococcus faecalis (350/599) was dominant. Antimicrobial susceptibility testing showed that high resistance was observed in rifampicin (80 %), tetracycline (71 %), doxycycline (71 %), and erythromycin (69 %). The results of PCR showed the highest prevalent antibiotic resistance genes (ARGs) were aac(6')-aph(2″) (85 %), followed by tet(M) (73 %), erm(B) (62 %), and aph(3')-IIIa (61 %). Besides, 29 optrA-carrying E. faecalis isolates belonging to 13 STs (including 3 new alleles) were detected, with ST714 (31 %, 9/29) being the dominant ST type. The phylogenetic tree showed that optrA-carrying E. faecalis prevalent in the intensive swine farm is mainly caused by clonal transmission. Notably, optrA gene in Enterococcus spp. isolate from camel was first characterized here. WGS of E. faecalis F109 isolate from camel confirmed the colocalization of optrA with other five ARGs in the same plasmid (pAFL-109F). The optrA-harboring genetic context is IS1216E-fexA-optrA-erm(A)-IS1216E. This study highlights the prevalence of MDR Enterococcus (≥88 %) and four ARGs (≥75 %) in swine (intensive farming), cattle (commercial farming), and chickens (backyard farming) are high and also highlights that optrA-carrying E. faecalis of farm animals incur a transmission risk to humans through environment, food consumption and others. Therefore, antibiotic-resistant bacteria (ARB) monitoring and effective control measures should be strengthened and implemented in diverse animals.

Characterization of small plasmids carrying florfenicol resistance gene floR in Actinobacillus pleuropneumoniae and Pasteurella multocida isolates from swine in China.

Actinobacillus pleuropneumoniae and Pasteurella multocida are two important bacterial pathogens in swine industry. In the present study, resistance profiles of nine commonly used antibiotics of A. pleuropneumoniae and P. multocida isolates of swine origin from different regions of China were investigated by determination of minimum inhibitory concentrations (MICs). In addition, genetic relationship of the florfenicol-resistant A. pleuropneumoniae and P. multocida isolates was determined by pulsed-field gel electrophoresis (PFGE). The genetic basis of florfenicol resistance in these isolates were explored by floR detection and whole genome sequencing. High resistance rates (>25%) of florfenicol, tetracycline and trimethoprim- sulfamethoxazole were observed for both bacteria. No ceftiofur- and tiamulin- resistant isolates were detected. Furthermore, all the 17 florfenicol-resistant isolates (nine for A. pleuropneumoniae and eight for P. multocida) were positive for floR gene. The presence of similar PFGE types in these isolates suggested that clonal expansion of some floR-producing strains occurred in the pig farms from same regions. WGS and PCR screening showed that three plasmids, named pFA11, pMAF5, and pMAF6, were the cargos of the floR genes in the 17 isolates. Plasmid pFA11 exhibited novel structure and carried several resistance genes, including floR, sul2, aacC2d, strA, strB, and bla ROB - 1. Plasmids pMAF5 and pMAF6 were presented in A. pleuropneumoniae and P. multocida isolates from different regions, suggesting horizontal transfer of the two plasmids are important for the floR dissemination in these Pasteurellaceae pathogens. Further studies of florfenicol resistance and its transfer vectors in Pasteurellaceae bacteria of veterinary origin are warranted.

Alternaria Mycotoxins Analysis and Exposure Investigation in Ruminant Feeds.

Alternaria mycotoxins are a class of important, agriculture-related hazardous materials, and their contamination in ruminant feeds and products might bring severe toxic effects to animals and even human beings. To control these hazardous compounds, a reliable and sensitive LC-MS/MS (liquid chromatography-tandem mass spectrometry) method was established for simultaneous determination of six target Alternaria mycotoxins in ruminant feeds, including ALT (Altenuene), AME (Alternariol Monomethyl Ether), AOH (Alternariol), ATX-Ι (Altertoxins I), TeA (Tenuazonic Acid), and TEN (Tentoxin). This developed analytical method was used for the determination of the presence of these substances in cattle and sheep feeds in Xinjiang Province, China. The results revealed that Alternaria mycotoxins are ubiquitously detected in feed samples. Especially, AME, AOH, TeA, and TEN are the most frequently found mycotoxins with a positive rate over 40% and a concentration range of 4~551 µg/kg. The proposed method could be applied for exposure investigation of Alternaria mycotoxins in ruminant feeds and for the reduction in the health risk to animals and even consumers.

Data mining of natural hazard biomarkers and metabolites with integrated metabolomic tools.

Data mining was one of the most important challenges in natural product analysis and biomarker discovery. In this work, we proposed an integrated data analysis protocol for natural products annotation and identification in data-dependent acquisition. Firstly, natural products and structure-related compounds could be identified by comparing mass spectrum behavior with commercial standard. Secondly, diagnostic fragmentation filtering (DFF) function in MZmine (http://mzmine.github.io/) was investigated for screening specific conjugation compounds with the same neutral loss. Thirdly, we present feature-based molecular networking (FBMN) in GNPS (https://gnps.ucsd.edu/) as a chromatographic feature detection and alignment tool. In addition, FBMN could enable natural products analysis based on molecular networks. This proposed integrated protocol should facilitate metabolomic data mining and biomarker discovery.

Differences in meat quality between Angus cattle and Xinjiang brown cattle in association with gut microbiota and its lipid metabolism.

Gut microbiota plays important roles in mediating fat metabolic events in humans and animals. However, the differences of meat quality traits related to the lipid metabolism (MQT-LM) in association with gut microbiota involving in lipid metabolism have not been well explored between Angus cattle (AG) and Xinjiang brown cattle (BC). Ten heads of 18-month-old uncastrated male AG and BC (5 in each group) raised under the identical conditions were selected to test MQT-LM, i.e., the backfat thickness (BFT), the intramuscular fat (IMF) content, the intramuscular adipocyte areas (IAA), the eye muscle area (EMA), the muscle fiber sectional area (MFSA) and the muscle shear force after sacrifice. The gut microbiota composition and structure with its metabolic function were analyzed by means of metagenomics and metabolomics with rectal feces. The correlation of MQT-LM with the gut microbiota and its metabolites was analyzed. In comparison with AG, BC had significant lower EMA, IMF content and IAA but higher BFT and MFSA. Chao1 and ACE indexes of α-diversity were lower. ß-diversity between AG and BC were significantly different. The relative abundance of Bacteroidetes, Prevotella and Blautia and Prevotella copri, Blautia wexlerae, and Ruminococcus gnavus was lower. The lipid metabolism related metabolites, i.e., succinate, oxoglutaric acid, L-aspartic acid and L-glutamic acid were lower, while GABA, L-asparagine and fumaric acid were higher. IMF was positively correlated with Prevotella copri, Blautia wexlerae and Ruminococcus gnavus, and the metabolites succinate, oxoglutaric acid, L-aspartic acid and L-glutamic acid, while negatively with GABA, L-asparagine and fumaric acid. BFT was negatively correlated with Blautia wexlerae and the metabolites succinate, L-aspartic acid and L-glutamic acid, while positively with GABA, L-asparagine and fumaric acid. Prevotella Copri, Blautia wexlerae, and Ruminococcus gnavus was all positively correlated with succinate, oxoglutaric acid, while negatively with L-asparagine and fumaric acid. In conclusion, Prevotella copri, Prevotella intermedia, Blautia wexlerae, and Ruminococcus gnavus may serve as the potential differentiated bacterial species in association with MQT-LM via their metabolites of oxoglutaric acid, succinate, fumaric acid, L-aspartic acid, L-asparagine, L-glutamic acid and GABA between BC and AG.

A survey of ß-lactamase and 16S rRNA methylase genes among fluoroquinolone-resistant Escherichia coli isolates and their horizontal transmission in Shandong, China.

The prevalence of ß-lactamase, 16S rRNA methylase genes, and plasmid-mediated fluoroquinolone-resistance (PMQR) determinants (qnrC and qnrD) was determined by polymerase chain reaction in fluoroquinolone-resistant Escherichia coli isolated from a chicken farm, a pig farm, and a hospital in Shandong, China in 2007. The bla(TEM) and bla(CTX-M) were the most prevalent ß-lactamase genes in isolates from chickens (88.4%, 175/198 and 81.3%, 161/198) and hospitalized patients (87.8%, 122/139 and 69.1%, 96/139). The bla(TEM) was the most prevalent ß-lactamase gene observed in isolates from pigs (98.5%, 135/137). The gene bla(CMY-2) was also predominant among isolates from chickens (20.2%, 40/198). The bla(LAP-1) gene was first detected in one strain from chickens and humans (pig farm workers) in China. Only one strain from hospitalized patients was found to possess bla(SHV). The rmtB was the most prevalent 16S rRNA methylase gene detected in isolates from chickens (19.7%, 39/198) and hospitalized patients (15.8%, 22/139). To our knowledge, this is the first report of the detection of the qnrD gene in E. coli from chickens and pigs in China. The qnrC and bla(KPC) genes were not detected in any of the isolates. Results of southern hybridization revealed that PMQR determinants, ß-lactamases, and 16S rRNA methylase genes were located on the same plasmid in E. coli strains derived from patients. Also, PMQR determinants and ß-lactamase genes were localized on the same plasmid in an E. coli strain of animal origin. Results of conjugation experiments revealed that all of these plasmid-based resistance genes can be transferred by conjugation through horizontal transmission.

Prevalence of antimicrobial resistance among Salmonella isolates from chicken in China.

We evaluated the antimicrobial resistance of Salmonella isolated in 2008 from a chicken hatchery, chicken farms, and chicken slaughterhouses in China. A total of 311 Salmonella isolates were collected from the three sources, and two serogroups of Salmonella were detected, of which 133 (42.8%) consisted of Salmonella indiana and 178 (57.2%) of Salmonella enteritidis. The lowest percentage of S. indiana isolates was found in the chicken hatchery (4.2%), followed by the chicken farms (54.9%) and the slaughterhouses (71.4%). More than 80% of the S. indiana isolates were highly resistant to ampicillin (97.7%), amoxicillin/clavulanic acid (87.9%), cephalothin (87.9%), ceftiofur (85.7%), chloramphenicol (84.9%), florfenicol (90.9%), tetracycline (97.7%), doxycycline (98.5%), kanamycin (90.2%), and gentamicin (92.5%). About 60% of the S. indiana isolates were resistant to enrofloxacin (65.4%), norfloxacin (78.9%), and ciprofloxacin (59.4%). Of the S. indiana isolates, 4.5% were susceptible to amikacin and 5.3% to colistin. Of the S. enteritidis isolates, 73% were resistant to ampicillin, 33.1% to amoxicillin/clavulanic acid, 66.3% to tetracycline, and 65.3% to doxycycline, whereas all of these isolates were susceptible to the other drugs used in the study. The S. indiana isolates showed resistance to 16 antimicrobial agents. Strains of Salmonella (n = 108) carrying the resistance genes floR, aac(6')-Ib-cr, and bla(TEM) were most prevalent among the 133 isolates of S. indiana, at a frequency of 81.2%. The use of pulsed-field gel electrophoresis to analyze the S. indiana isolates that showed similar antimicrobial resistance patterns and carried resistance genes revealed six genotypes of these organisms. Most of these isolates had the common pulsed-field gel electrophoresis patterns found in the chicken hatchery, chicken farms, and slaughterhouses, suggesting that many multidrug-resistant isolates of S. indiana prevailed in the three sources. Some of these isolates were not derived from a specific clone, but represented a variety of genotypes of S. indiana.

First Isolation and Molecular Characterization of bla CTX-M-121 -Producing Escherichia coli O157:H7 From Cattle in Xinjiang, China.

The bovine Escherichia coli O157:H7 is a major foodborne pathogen causing severe bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Cattle are recognized major reservoir and source of E. coli O157:H7. We investigated the antibiotic resistance, molecular profiles, and intrinsic relationship between 21 isolates of E. coli O157:H7 from cattle farms and slaughtering houses in Xinjiang. Using pulsed-field gel electrophoresis (PFGE) molecular typing, two types of PFGE were revealed through cluster analysis, including clusters I and II, with 66 and 100% similarity of PFGE spectra between 21 isolates. We also detected that 18 isolates (86%) carried at least one virulence gene, 16 isolates (76%) carried the eae gene, and 7 (33%) carried the stx1 + stx2 + eae + hly + tccp genes. Eighteen isolates were susceptible to antibiotics. Three isolates were resistant to antibiotics, and two were multidrug resistant. One of the two multidrug-resistant isolates detectably carried the bla CTX-M-121 gene. This is the first finding of the bla CTX-M-121 gene detected in E. coli O157:H7 isolated from cattle in Xinjiang. The bla CTX-M-121 gene is transferable between the bacterial strains via plasmid transmission. The results indicated that E. coli O157:H7 may have undergone clonal propagation in cattle population and cross-regional transmission in Xinjiang, China.

Characterization of NDM-1-Producing Carbapenemase in Proteus mirabilis among Broilers in China.

Carbapenem-resistant pathogens mediated by metallo-beta-lactamases (MBLs) have spread worldwide, where NDM-1 is a typical and key MBL. Here, we firstly discussed the distribution characterization of NDM-1, which produces multidrug-resistant Proteus mirabilis among broilers in China. From January to April 2019, 40 (18.1%, 40/221) blaNDM-1-carrying P. mirabilis strains were recovered from commercial broilers in slaughterhouse B in China. All the isolates were resistant to imipenem, meropenem and other ß-lactams. These isolates belong to five clusters identified via pulsed field gel electrophoresis (PFGE). Further studies on twenty representative strains revealed that seven blaNDM-1 genes were located on plasmids with sizes of 104.5-138.9 kb. Notably, only three strains (PB72, PB96 and PB109) were successfully transferred to Escherichia coli J53, while the other four isolates were located in nontransferable plasmids. The rest were harbored in chromosomes. Ulteriorly, based on whole genome sequencing (WGS), these twenty isolates showed four typical phylogenetic clades according to single nucleotide polymorphisms (SNPs) of a core genome and presented four main genomic backbone profiles, in which type II/III strains shared a similar genetic context. All of the above is evidence of blaNDM-1 transmission and evolution in P. mirabilis, suggesting that the prevalence may be more diverse in broiler farms. Accordingly, as intestinal and environmental symbiotic pathogens, blaNDM-1-positive P. mirabilis will pose greater threats to the environment and public health.

High Prevalence and Diversity Characteristics of blaNDM, mcr, and blaESBLs Harboring Multidrug-Resistant Escherichia coli From Chicken, Pig, and Cattle in China.

The objective of this study was to understand the diversity characteristics of ESBL-producing Escherichia coli (ESBL-EC) in chicken, pig, and cattle. A high prevalence of ESBL-EC (260/344) was observed in all food animals with prevalence rates of 78.6% (110/140) for chicken, 70.7% (58/82) for cattle, and 75.4% (92/122) for swine. However, the resistance rates presented significant differences in different animal origin ESBL-EC, where resistance to CTX, GEN, IMP, NEO, and OFL was the highest in chicken ESBL-EC, then in cattle, and the lowest in swine. Seriously, most ESBL-EC harbor multidrug resistance to antibiotics (MDR, ≥3 antibiotic categories), and the MDR rates of ESBL-EC were the highest in chicken (98.18%), followed by swine (93.48%), and the lowest in cow (58.62%), while the same trend also was observed in MDR of ≥5 antibiotic categories. This high prevalence and resistance can be partly interpreted by the high carriage rates of the ß-lactamases CTX-M (n = 89), OXA (n = 59), SHV (n = 7), and TEM (n = 259). A significant difference of ß-lactamase genes also presented in different animal species isolates, where the chicken origin ESBL-EC possessed higher carriage rates of almost all genes tested than cattle and swine. Notably, eight chicken origin ESBL-EC carried transferable plasmid-mediated blaNDM-1 or blaNDM-5, especially, of which four ESBL-EC also contained the colistin resistance gene mcr-1, as confirmed by genomic analysis. More interestingly, two deletion events with a 500-bp deletion in ΔISAba125 and a 180-bp deletion in dsbC were observed in three blaNDM-5 IncX3 plasmids, which, as far as we know, is the first discovery. This showed the instability and horizontal transfer of blaNDM genetic context, suggesting that blaNDM is evolving to "pack light" to facilitate rapid and stable horizontal transfer. Sequence types (STs) and PFGE showed diversity patterns. The most prevalent STs were ST48 (n = 5), ST189 (n = 5), ST206 (n = 4), ST6396 (n = 3), ST10 (n = 3), and ST155 (n = 3), where ST48 ESBL-EC originated from three food animal species. The STs of all blaNDM-positive ESBL-EC were attributed to three STs, namely, ST6396 (n = 2), ST206 (n = 2), and ST189 (n = 4), where ST189 was also the unique type for four mcr-1-carrying ESBL-EC. In conclusion, we suggest that the three animal species ESBL-EC show similar high prevalence, diversity in isolate lineages, and significant discrepancies in antibiotic resistance and resistance genes. This suggests that monitoring and anti-infection of different food animal origin ESBL-EC need different designs, which deserves more attention and further surveillance.

Prevalence and genomic investigation of Salmonella isolates recovered from animal food-chain in Xinjiang, China.

Salmonella is a major foodborne pathogen worldwide, causing serious cases of morbidity and mortality due to the consumption of contaminated foods. Animal-borne foods were considered the main source of transferring Salmonella to humans; however, route surveillance by genomic platforms along the food-chain is limited in China. Here, we proceeded to the application of whole genome sequencing in the epidemiological analysis of Salmonella isolated along the food-chain in Xinjiang, China. A total of 2408 samples were collected from farms, slaughterhouses, and markets, and subjected to the isolation of Salmonella strains. 314 (13.04%) of the samples were positive for Salmonella. Phenotypic antimicrobial resistance was conducted by the broth dilution method using 14 antimicrobial agents belonging to ten classes for all 314 isolates. A selection of representative 103 isolates was subjected to whole-genome sequencing for understanding the Salmonella diversity, including serovars, antimicrobial and virulence genes, plasmid types, multi-locus sequence types, and allelic types. We found that S. Agona was the dominant serovar and O:4(B) was the dominant serogroup. The dominant genotype was ST13 and each serovar has a unique MLST pattern. Plasmids prediction reported Col(MGD2)_1 and Col(Ye4449)_1 as the dominant plasmids, in addition to the detection of IncFII(S)_1 and IncFIB(S)_1 carried by all S. Enteritidis isolates. Importantly, virulence genes prediction showed the presence of cdtB gene encoding typhoid toxins, spv genes, and pef gene cluster encoding fimbriae in the genomes of S. Indiana and S. Enteritidis. Phenotypic antimicrobial resistance identified 92.04% of the sampled isolates as multi-drug resistance (MDR), with high resistance to tetracycline (78.03%; 245/314), amoxicillin/ clavulanic acid (75.80%; 238/314), and ampicillin (70.70%; 222/314). Together, we firstly reported the prevalence of MDR Salmonella isolates harboring critical virulence factors transmission via animal-borne food-chain in Xinjiang, hence route surveillance by whole-genome sequencing platform could facilitate recognition and project early warning for the emerging MDR clones along the food-chain.

Identification of mcr-1 and a novel chloramphenicol resistance gene catT on an integrative and conjugative element in an Actinobacillus strain of swine origin.

The aim of this study was to characterize a mcr-1-carrying integrative and conjugative element (ICE) in a novel Pasteurellaceae-like bacteria of swine origin. The mcr-1-positive GY-402 strain, recovered from a pig fecal sample, was subjected to whole genome sequencing with the combination of Illumina Hiseq and MinION platforms. Genome-based taxonomy revealed that strain GY-402 exhibited highest ANI value (84.89 %) to Actinobacillus succinogenes, which suggested that it represented a novel Actinobacillus species. Sequence analysis revealed that mcr-1 was clustered with eight other resistance genes in the MDR region of a novel ICE element, named ICEAsp1. Inverse PCR and mating assays showed that ICEAsp1 is active and transferrable. In addition, six circular forms mediated by four ISApl1 elements were detected with different inverse PCR sets, indicating that flexible composite transposons could be formed by pairwise combinations of multiple IS copies. Cloning experiment and phylogenetic analysis revealed that the novel Cat protein, designated CatT, belongs to type-A family and confers resistance to chloramphenicol. In conclusion, this is, to the best of our knowledge, the first report of mcr-1 gene on ICE structure and also in Pasteurellaceae bacteria. The diverse composite transposons mediated by multicopy IS elements may facilitate the dissemination of different resistance genes.

Antimicrobial resistance in Enterococcus faecium and Enterococcus faecalis isolates of swine origin from eighteen provinces in China.

Enterococcus faecium and E. faecalis are important human pathogens and also served as sentinel organisms for monitoring systems of antimicrobial resistance in both animals and humans. In this study, 106 E. faecium and 56 E. faecalis isolates were collected from 61 pig farms in 18 proveinces of China. Antimicrobial susceptibility was determined for 9 clinically important antibiotics and 3 antimicrobial growth promoters. The Enterococcus isolates showed high prevalence of resistance to medically important antibiotics, such as ampicillin (50.9% for E. faecium and 19.6% for E. faecalis), chloramphenicol (24.5% for E. faecium and 41.1% for E. faecalis), erythromycin (83.0% for E. faecium and 91.1% for E. faecalis), tetracycline (79.2% for E. faecium and 100% for E. faecalis), quinupristin/dalfopristin (26.4% for E. faecium) and ciprofloxacin (73.6% for E. faecium and 66.1% for E. faecalis). Resistance to tigecycline, linezolid and vancomycin was very rare. The resistance status of three representative in-feed antibiotics bacitracin, nosiheptide and enramycin was firstly investigated with Enterococcus as indicator bacteria. The Enterococcus isolates showed extremely high frequency of bacitracin resistance (96.7% for E. faecium and 87.8% for E. faecalis), while no nosiheptide and enramycin resistance was observed. Pulsed-field gel electrophoresis (PFGE) analysis showed that a majority of E. faecium and E. faecalis strains showed unrelated profiles, indicating high heterogeneity among the Enterococcus isolates. Our study provided basic data on the antimicrobial resistance of E. faecium and E. faecalis isolates.

First report of the multidrug resistance gene cfr and the phenicol resistance gene fexA in a Bacillus strain from swine feces.

A multidrug resistance gene, cfr, and a phenicol resistance gene, fexA, were detected in a Bacillus strain, BS-01, isolated from swine feces. The cfr gene was carried on a novel 16.5-kb plasmid, designated pBS-01. A complete Tn917 structure, which harbors the macrolide-lincosamide-streptogramin B resistance gene erm(B), was located downstream of the cfr gene. The fexA gene was discovered in the chromosomal DNA of the BS-01 strain and identified in a Tn558 variant.