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 swine manure 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.

Insights into the fates of plasmids and antimicrobial resistance genes during swine manure treatment and related factors based on plasmidome and metagenome analyses.

Swine manure treatment plants are important reservoirs of plasmid-harboring antibiotic resistance genes (ARGs) and physicochemical contaminants, but the changes in the abundances of plasmids and ARGs, and their interactions with the physicochemical properties of manure, are still unclear. Thus, in the present study, plasmidome and metagenome analyses were conducted for samples collected at different stages in the swine manure treatment process. The results indicated that anaerobic digestion and aerobic digestion were the most efficient stages for reducing the abundances of ARGs in swine manure. However, the plasmids associated with ARGs were not effectively removed in these stages. Through the whole treatment process, the IncL/M, IncQ1, IncHI2A, IncA/C, and IncN plasmid groups had strong correlations (r > 0.8, P < 0.01) with most ARG types, thereby indicating that these plasmids play important roles in the persistence of ARGs in this environment. Furthermore, the pH, total nitrogen, total phosphorus, and four heavy metals (Cu, Zn, As, and Fe) significantly affected the abundances of seven ARG subtypes (tetB(P), ant(6)-Ia, tet44, aph(3'')-Ib, mefB, tet(L), and tet(39)). In particular, florfenicol had the most positive correlations with ARGs. Our results indicated that nutrients, heavy metals, and antibiotics all contributed to the presence and persistence of plasmid-harboring ARGs. This study provides insights into the fate of plasmids and ARGs, and related factors during the swine manure treatment process, thereby facilitating the development of a new treatment technique for removing ARGs and reducing the public health risk associated with livestock production.

Exploring the profile of antimicrobial resistance genes harboring by bacteriophage in chicken feces.

Bacteriophage may play an important role in antimicrobial resistance genes (ARGs) transmission. However, the contribution of bacteriophage to the spread of ARGs in environment, especially in poultry farm environment, is rarely known. In this study, the prevalence of ARGs in bacteriophage DNA was investigated in chicken feces from 30 different poultry farms in China. Then the abundance of the aac(6')-Ib-cr, blaCTX-M, ermB, floR, mcr-1, sul1, tetM and intI1 genes was determined by qPCR in bacteriophage and compared with certain representative plasmid DNA samples. The results showed that 12 ARGs (aac(6')-Ib-cr, aph(3')-IIIa, blaCTX-M, ermB, ermF, floR, mcr-1, qnrS, sul1, sul2, vanA, tetM genes) and class 1 integron gene intI1 were detected in bacteriophage DNA fraction. The sul1, tetM and aac(6')-Ib-cr genes were most prevalent with high detection rates of 77%, 61% and 55%, respectively. To our best knowledge, this study firstly reported the presence of the mcr-1 gene in bacteriophage DNA derived from farms environments. We found that the gene copy (GC) numbers of the aac(6')-Ib-cr, ermB and sul1 genes were as high as 5.47, 5.22 and 5.54 log10 GC/g, respectively. Both the prevalence and abundance of ARGs in broiler fecal wastes were also generally higher than in laying hens. In addition, although the GC numbers of the aac(6')-Ib-cr, floR and tetM genes in plasmid DNA was higher than that in phage DNA fraction by 4.68, 3.59 and 3.9 orders of magnitude, respectively, the absolute abundances of the blaCTX-M and mcr-1 genes in phage DNA were close to or even higher than that in plasmid DNA at farm SIL2, SIL4 and SIB1. As potential vessels for ARGs, bacteriophage could not be ignored due to their unique extracellular persistence in environments. Overall, this is the first comprehensive survey about bacteriophage carried ARGs from farms in different regions in China.

The Yersinia high-pathogenicity island (HPI) carried by a new integrative and conjugative element (ICE) in a multidrug-resistant and hypervirulent Klebsiella pneumoniae strain SCsl1.

Multidrug-resistant and hypervirulent Klebsiella pneumoniae (hvKP) poses a significant risk to public health. To better understand the molecular characteristics of multidrug-resistant and hypervirulent K. pneumoniae of animal origin, fifteen K. pneumoniae strains from the liver, blood of sick pigs and chicken feces were collected. All K. pneumoniae isolates were subjected to antimicrobial susceptibility testing, string test, multi-locus sequence typing and whole genome sequencing. Seven K. pneumoniae isolates were found carrying the mcr-1.1 gene. Among them, a multidrug-resistant and hypervirulent K. pneumoniae strain SCsl1 isolated from the liver of a diseased pig was found to harbor 16 resistance genes (e.g., mcr-1.1) and 16 virulence genes including aerobactin. Moreover, a novel integrative and conjugative element, named ICEKpSL1, was identified in SCsl1, which contains a full Yersinia high-pathogenicity island (HPI). This element could be excised from the chromosome to form a circular intermediate, indicating potential transmission of the Yersinia pathogenicity island. The emergence of multidrug-resistance and hypervirulence in K. pneumoniae from animals warrants further surveillance.

Characterization of Plasmid-Mediated Quinolone Resistance in Gram-Negative Bacterial Strains from Animals and Humans in China.

The aim of this study was to determine the molecular epidemiology of plasmid-mediated quinolone resistance (PMQR) determinants in Escherichia coli, Salmonella enterica (Salmonella spp.), Klebsiella pneumoniae, and Proteus mirabilis. Four hundred seventy-two nonrepetitive isolates were collected from different sources in China and screened for the presence of PMQR genes (PMQRs). Then, 49 PMQR producers were selected to study the coexistence of PMQRs and other resistance genes using whole-genome sequencing (WGS). High rates of resistance to tetracycline (93.4%), nalidixic acid (81.5%), and norfloxacin (65.8%) were observed. The predominant PMQRs were aac(6')-Ib-cr (28.6%) and oqxAB (21.4%). The prevalence of PMQR determinants was significantly higher (p < 0.05) in E. coli from stockmen (55.9%, 19/34), pigs (51.1%, 70/137), and laying hens (43.1%, 28/65) than that from wild animals (21.7%, 5/23) and dairy cattle (20.1%, 5/24). WGS results showed that 89.8% of the PMQR-positive isolates co-harbored ß-lactamase genes, with blaCTX-M being the dominant ß-lactamase gene. In K. pneumoniae, the coexistence rate of oqxAB and qnrB with fosA, blaDHA-1, and blaSHV was significantly higher than that in P. mirabilis and E. coli. In contrast, the coexistence of qnrD and blaOXA-1 was more prominent (p < 0.001) in P. mirabilis than in the other two species. Particularly, oqxAB and mcr-1 had an obvious preference for E. coli than K. pneumonia and P. mirabilis (p < 0.001), which had not been reported in previous studies on the prevalence of PMQRs.

Characterization of Resistance Patterns and Detection of Apramycin Resistance Genes in Escherichia coli Isolated from Chicken Feces and Houseflies after Apramycin Administration.

The aim of this study was to evaluate the influence of apramycin administration on the development of antibiotic resistance in Escherichia coli (E. coli) strains isolated from chicken feces and houseflies under field conditions. Chickens in the medicated group (n = 25,000) were given successive prophylactic doses (0.5 mg/l) of apramycin in their drinking water from Days 1 to 5, while no antibiotics were added to the un-medicated groups drinking water (n = 25,000). Over 40 days, a total of 1170 E. coli strains were isolated from fecal samples obtained from medicated and un-medicated chickens and houseflies from the same chicken farm. Apramycin MIC90 values for E. coli strains obtained from the medicated group increased 32-128 times from Days 2 to 6 (256-1024 µg/ml) when compared to those on Day 0 (8 µg/ml). Strains isolated from un-medicated chickens and houseflies had consistently low MIC90 values (8-16 µg/ml) during the first week, but showed a dramatic increase from Days 8 to 10 (128-1024 µg/ml). The apramycin resistance gene aac(3)-IV was detected in E. coli strains from medicated (n = 71), un-medicated (n = 32), and housefly groups (n = 42). All strains positive for aac(3)-IV were classified into 12 pulsed-field gel electrophoresis (PFGE) types. PFGE types A, E, and G were the predominant types in both the medicated and housefly groups, suggesting houseflies play an important role in spreading E. coli-resistant strains. Taken together, our study revealed that apramycin administration could facilitate the occurrence of apramycin-resistant E. coli and the apramycin resistance gene acc(3)-IV. In turn, these strains could be transmitted by houseflies, thus increasing the potential risk of spreading multi-drug-resistant E. coli to the public.

Isolation of an IncP-1 plasmid harbouring mcr-1 from a chicken isolate of Citrobacter braakii in China.

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10-7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1-mcr-1-orf-ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids.

The Prevalence of Colistin Resistant Strains and Antibiotic Resistance Gene Profiles in Funan River, China.

Anthropogenic activities near urban rivers may have significantly increased the acquisition and dissemination of antibiotic resistance. In this study, we investigated the prevalence of colistin resistant strains in the Funan River in Chengdu, China. A total of 18 mcr-1-positive isolates (17 Escherichia coli and 1 Enterobacter cloacae) and 6 mcr-3-positive isolates (2 Aeromonas veronii and 4 Aeromonas hydrophila) were detected, while mcr-2, mcr-4 and mcr-5 genes were not detected in any isolates. To further explore the overall antibiotic resistance in the Funan River, water samples were assayed for the presence of 15 antibiotic resistance genes (ARGs) and class 1 integrons gene (intI1). Nine genes, sul1, sul2, intI1, aac(6')-Ib-cr, bla CTX-M, tetM, ermB, qnrS, and aph(3')-IIIa were found at high frequencies (70-100%) of the water samples. It is worth noting that mcr-1, bla KPC, bla NDM and vanA genes were also found in water samples, the genes that have been rarely reported in natural river systems. The absolute abundance of selected antibiotic resistance genes [sul1, aac(6')-Ib-cr, ermB, blaCTX-M, mcr-1, and tetM] ranged from 0 to 6.0 (log10 GC/mL) in water samples, as determined by quantitative polymerase chain reaction (qPCR). The sul1, aac(6')-Ib-cr, and ermB genes exhibited the highest absolute abundances, with 5.8, 5.8, and 6.0 log10 GC/mL, respectively. The absolute abundances of six antibiotic resistance genes were highest near a residential sewage outlet. The findings indicated that the discharge of resident sewage might contribute to the dissemination of antibiotic resistant genes in this urban river. The observed high levels of these genes reflect the serious degree of antibiotic resistant pollution in the Funan River, which might present a threat to public health.