application-intelligent-high-throughput-antimicrobial-sensitivity

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

Key Laboratory of Livestock and Poultry Multi-omics of MARA

The dissemination of drug-resistant Escherichia coli in poultry products is becoming a public concern, as it endangers food security and human health. It is very common for E. coli to exhibit drug resistance in the poultry industry in China due to the excessive use of antibiotics. However, few studies have examined the drug resistance endowed by integrons and integron-associated gene cassettes in different phylogenetic groups of E. coli isolated from broiler carcasses. In this study, 373 antibiotic-resistant E. coli strains were isolated from the surfaces or insides of broiler carcasses from a slaughterhouse in Shandong Province, China. According to phylogenetic assays of chuA, yjaA, and an anonymous DNA fragment, TSPE4-C2, these isolates belong to four phylogenetic groups (A, B1, B2, and D) and seven subgroups (A0, A1, B1, B21, B22, D1, and D2). Of the tested isolates, 95.71% (n=357) are multi-drug resistant, among which group B1 was predominant, accounting for 33.51% (n=125) of the tested isolates. A high percentage of the E. coli isolates were resistant to amoxicillin-clavulanic acid (99.20%, n=370), doxycycline (92.23%, n=344), sulfamethoxazole-trimethoprim (90.88%, n=339), ciprofloxacin, (64.61%, n=241), sulbactam-cefoperazone (51.21%, n=191), and amikacin (33.78%, n=126). Furthermore, among the 373 isolates, class 1 and 2 integrons were identified in 292 (78.28%) and 49 (13.14%) of the isolates, respectively, while no class 3 integrons were detected. The most prevalent gene cassette arrays were dfrA17-aadA5 and dfrA12-orfF-aadA2 in the variable region of class 1 integrons, while only one gene cassette array (dfrA1-sat2-aadA1) was detected in the variable region of class 2 integrons. Class 1 integrons were distributed in various physiological subtypes, whereas no predominant phylogenetic groups could be identified. The presence of class 2 integrons in the B21 subtype was significantly higher than in the other subtypes, and it coexisted with the class 1 integron. This study suggests that broiler products are potential sources of multi-drug resistant E. coli, and that resistance genes could be spread by lateral gene transfer.

Identification of integrons and phylogenetic groups of drug-resistant Escherichia coli from broiler carcasses in China.

The prevalence of antimicrobial resistant Klebsiella pneumoniae in poultry products has been a public concern, as it severely endangers food safety and human health. In this study, we investigated 90 antimicrobial resistant Klebsiella strains that were isolated from a commercial broiler slaughter plant in Shandong province of China. Nearly all (89/90) of the isolates were identified as infectious phylogenetic group KpI-type K. pneumoniae. Out of these 90 strains, 87 (96.7%) were multidrug-resistant isolates, and 87 (96.7%) were extended-spectrum beta-lactamase (ESBL)-producing isolates. An analysis of the prevalence of quinolone resistance genes showed that 7.8%, 77.8%, 26.7%, and 2.2% of the strains carried the qnrA, qnrB, qnrS, and qepA genes, respectively. An analysis of beta-lactam resistance genes showed that a high percentage of the strains contain the blaTEM (76.7%), blaSHV (88.9%), and blaCTX-M (75.6%) genes, among which three blaSHV subtypes (blaSHV-1, n=30; blaSHV-11, n=38; blaSHV-12, n=12) and three blaCTX-M subtypes (blaCTX-M-14, n=14; blaCTX-M-15, n=35; blaCTX-M-55, n=19) were found. A further investigation of mobile genetic elements involved in horizontal multidrug resistance gene transfer showed the presence of class 1 and 2 integrons in 77 (85.6%) and five (5.6%) isolates, respectively, while no class 3 integrons were detected. Four types of class 1 integrons containing specific gene cassette arrays (dfrA12-orfF-aadA2, dfrA17-aadA5, dfrA1-aadA1, and empty) were identified. Only one gene cassette array (dfrA1-sat2-aadA1) was detected in the class 2 integrons. Furthermore, four different types of insertion sequence common region 1 (ISCR1)-mediated downstream structures were successfully identified in 46 class 1 integron-positive isolates, among which ISCR1-sapA-like-qnrB2-qacEΔ1 was the most commonly observed structure. Chi-square tests revealed a significant association between ESBL genes, plasmid-mediated quinolone resistance (PMQR) genes, and class 1 integrons (p<0.01). Additional conjugation experiments confirmed this relationship (p<0.01) in transconjugants by finding that a high percentage of PMQR genes (74.0%) and class 1 integrons (73.7%) were co-transferred with ESBL genes. Finally, multilocus sequence typing (MLST) was performed, and it revealed that the isolates from chickens are widely distributed in humans, and that antimicrobial resistance is not only disseminated by clonal spreading, but largely by horizontal gene transfer. These results suggest that horizontal transfer of antimicrobial resistance genes by mobile genetic elements, such as integrons, plays a major role in the spread of antimicrobial resistance. Therefore, elucidating the structures of drug resistance integrons is of great importance to the commercial broiler slaughter plant in Shandong, China.

Characterization of antimicrobial resistance in Klebsiella species isolated from chicken broilers.

Salmonella spp. are one of the most important foodborne bacterial pathogens in human beings and animals. This study aimed to analyze the prevalence and characterization of Salmonella from broilers in Shandong, China. A total of 67 Salmonella were recovered from 600 rectal swabs collected from 3 large-scale intensive broiler farms (67/600, 11.2%) between May and October 2018. Among Salmonella isolates, the most common serovars were S. enteritidis and S. typhimurium. The highest occurrence of resistance observed was for polymyxin (100%), followed by ampicillin (68.7%). The multidrug-resistant Salmonella isolation rate was observed to be 53.7%. Four β-lactamase genes were detected among the isolates, and all the isolates carried bla (67/67, 100%), followed by bla (19/67, 28.4%), bla (17/67, 25.4%), and bla (7/67, 10.4%). Four plasmid-mediated quinolone resistance gene were detected among the isolates; the prevalent resistance genes was aac(6')-Ib-cr (18/67, 26.9%), followed by oqxB (9/67, 13.4%), qnrB (6/67, 9.0%), and qnrD (1/67, 1.5%). The prevalent rate of mcr-1 was 6.0% (4/67). Class 1 integrons were detected in 26.9% of these isolates and contained 7 groups of resistance gene cassettes. Multilocus sequence typing analysis revealed 7 sequence types, and ST11 was the most frequent sequence types. This study indicated that reduction of Salmonella and strict control on the use of antibiotics in more than 5,000 million broilers in Shandong are the vitally important measures to keep public health.

Characterization of integrons and antimicrobial resistance in Salmonella from broilers in Shandong, China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

 is an important zoonotic pathogen that poses a serious threat to the pig industry and human health. The massive use of macrolides has led to the emergence of resistance in , and  is suspected to be a reservoir of antimicrobial resistance genes. The mechanism to macrolide resistance in  is mainly due to  and . In this study, loop-mediated isothermal amplification (LAMP) methods were developed to detect  and  genes in  through turbidimetry detection. The sensitivity and specificity of the LAMP reactions were determined. All results of LAMP and polymerase chain reaction (PCR) assay were compared to determine whether LAMP method was accurate and reliable. The results showed that all 100 nonstreptococcus clinical isolates tested negative, indicating the high specificity of LAMP assays. The detection limit of LAMP assay was 1 fg per reaction, and 10-10-fold lower than those of conventional PCR methods. Evaluation of the performance of the LAMP assay in  clinical strains revealed a good consistency between LAMP and PCR assays. In conclusion, LAMP assays are specific, sensitive, and rapid methods to detect  and  in .

Development of Two Loop-Mediated Isothermal Amplification Assays for Rapid Detection of  and  Genes in .

Phage vB_CpeS-17DYC was isolated from wastewater from a poultry market using Clostridium perfringens strain DYC. The vB_CpeS-17DYC genome is 39,184 bp long, with 65 open reading frames and a GC content of 30.6%. It shared 93.95% nucleotide identity, with 70% query coverage, with  phage phiCP13O (GenBank accession number NC_019506.1). Virulence factor genes were not found in the vB_CpeS-17DYC genome.

Yuqing Liu

Key Laboratory of Livestock and Poultry Multi-omics of MARA

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance