Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this study, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609, and enzymes of DNA metabolism were closely related to CRISPR-Cas. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the results of PCA that they may be important factors affecting the plasmid with CRISPR.

This research aimed to investigate the microbial composition and diversity of antimicrobial resistance genes (ARGs) found in Chinese broiler and layer family poultry farms. We focused on the differences in resistance phenotypes and genotypes of multidrug-resistant Enterobacteriaceae (MDRE) isolated from the two farming environments and the existence and transmissibility of colistin resistance gene mcr-1. Metagenomic analysis showed that Firmicutes and Bacteroides were the dominant bacteria in broiler and layer farms. Many aminoglycoside and tetracycline resistance genes were accumulated in these environments, and their absolute abundance was higher in broiler than in layer farms. A total of 526 MDRE were isolated with a similar distribution in both farms. The results of the K-B test showed that the resistance rate to seven antimicrobials including polymyxin B and meropenem in broiler poultry farms was significantly higher than that in layer poultry farms (P ≤ 0.05). PCR screening results revealed that the detection rates of mcr-1, aph(3’)Ia, aadA2, blaoxa–1, blaCTX–M, fosB, qnrD, sul1, tetA, and catA1 in broiler source MDRE were significantly higher than those in layers (P ≤0.05). A chimeric plasmid p20432-mcr which carried the novel integron In1866 was isolated from broiler source MDRE. The high frequency of conjugation (10–1 to 10–3) and a wide range of hosts made p20432-mcr likely to play an essential role in the high detection rate of mcr-1, aph(3’)-Ia, and aadA2 in broiler farms. These findings will help optimize disinfection and improve antimicrobial-resistant bacteria surveillance programs in poultry farms, especially broilers.

IntroductionThis research aimed to investigate the antibiotic resistance of Enterococcus faecalis from swine farms in Zhejiang Province and the prevalence and transmission mechanism of oxazolidone resistance gene optrA.MethodA total of 226 Enterococcus faecalis were isolated and their resistance to 14 antibiotics was detected by broth microdilution. The resistance genes were detected by PCR.ResultsThe antibiotic resistance rate of 226 isolates to nearly 57% (8/14) of commonly used antibiotics was higher than 50%. The resistance rate of tiamulin was highest (98.23%), that of tilmicosin, erythromycin, tetracycline and florfenicol was higher than 80%, and that of oxazolidone antibiotic linezolid was 38.49%. The overall antibiotics resistance in Hangzhou, Quzhou and Jinhua was more serious than that in the coastal cities of Ningbo and Wenzhou. The result of PCR showed that optrA was the main oxazolidinone and phenicols resistance gene, with a detection rate of 71.68%, and optrA often coexisted with fexA in the isolates. Through multi-locus sequence typing, conjugation transfer, and replicon typing experiments, it was found that the horizontal transmission mediated by RepA_N plasmid was the main mechanism of optrA resistance gene transmission in E. faecalis from Zhejiang Province. Two conjugative multi-resistance plasmids carrying optrA, RepA_N plasmid pHZ318-optrA from Hangzhou and Rep3 plasmid from Ningbo, were sequenced and analyzed. pHZ318-optrA contain two multidrug resistance regions (MDR), which contributed to the MDR profile of the strains. optrA and fexA resistance genes coexisted in IS1216E-fexA-optrA-ferr-erm(A)-IS1216E complex transposon, and there was a partial sequence of Tn554 transposon downstream. However, pNB304-optrA only contain optrA, fexA and an insertion sequence ISVlu1. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterococcus through inter-replicon gene transfer.DiscussionThis study can provide theoretical basis for ensuring the quality and safety of food of animal origin, and provide scientific guidance for slowing down the development of multi-antibiotic resistant Enterococcus.

Background: Existing studies on poultry (broilers and layers) suggest that strong screening of antibiotics leads to the wide spread of antibiotic resistance genes (ARGs). However, due to the different breeding cycle, antibiotic use and breeding environment characteristics of layers and broilers, they have different antibiotic resistance risks. Until now, there are few studies on the difference in risk of antibiotic resistance between broilers and layers, which is still a gap in our knowledge. Results: In the current study, high-throughput sequencing-based metagenomic revealed that firmicutes and bacteroidetes were the dominant bacteria in broiler and layer farms. Aph(3’’’)-III and ermF were the ARGs with the highest absolute abundance in broiler farms, which was higher than that of laying farms. TetW was the highest absolute abundance of ARG in laying hens, but it was still lower than broiler chickens. Three hundred fecal samples were used to isolate multidrug-resistant Enterobacteriaceae (MDRE). It was found that the detection rates of mcr-1, aph(3')-Ia, aadA2, blaOXA-1, blaCTX-M, fosB, qnrD, sul1, tetA and catA1 in broiler source MDRE were significantly higher than that in laying farms (P<0.05). Further studies showed that a chimeric plasmid p20432-mcr which carried the novel integron In1866 was isolated from broiler source MDRE. High frequence of conjugation (10-1 to 10-3) and wide range of hosts made p20432-mcr likely play an important role for the high detection rate of mcr-1, aph(3')-Ia and aadA2 in broiler farms. Conclusions: We comprehensively compared the prevalence of pathogen MDRE and their ARGs in the two environments and the mechanism of transmission. It is found that the mcr-1 positive plasmid p20432-mcr has a wide host range which may play an important part in the high detection rate of ARGs in broiler source MDRE. These findings will help to optimize disinfection and antibiotic resistant bacteria surveillance programs in poultry farms, especially in broilers.

Microorganisms have developed many strategies in the process of long-term defense against external attacks, one of which is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) bacterial immunological system. In this paper, the whole genome of 300 strains of Klebsiella was collected, the CRISPR-Cas system in the strains was statistically analyzed, and the types and structures of CRISPR system in Klebsiella were explored, as well as the correlation between CRISPR and mobile genetic elements (MGEs). Through principal component analysis (PCA), we found that Cas gene, plasmids, integron, IS1, IS609 and DNA-related enzymes were closely related to CRISPR. Compared the structural characteristics of plasmids, the DinG family helicases, Cas6, Csf2, and IS5 were observed near the CRISPR loci in plasmid, which is also confirmed by the the results of PCA that they may be important factors affecting the plasmid whith CRISPR.