ESBL-Producing Escherichia coli from Cows Suffering Mastitis in China Contain Clinical Class 1 Integrons with CTX-M Linked to IS CR1

Abstract

The prevalence of pathogenic multi-drug resistant (MDR) extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is rapidly increasing, becoming a global concern. In a veterinary context, ESBL-producing E. coli are mostly reported in poultry and pigs. Here, we report on the prevalence and characterize ESBL-producing E. coli isolated from diverse dairy farms in China. Overall, 36 (23.53%) out of 153 E. coli isolates from mastitic milk samples (n = 1252) were confirmed as ESBL-producers by double-disc synergy testing and PCR. Nucleotide analysis of PCR amplicons revealed that bla_CTX-M was the predominant ESBL gene detected in 28 (77.78%) isolates, with bla_CTX-M-15 being the major (78.57%) allele encoding for ESBLs. Also, 20 (55.56%) and 6 (16.67%) of the ESBL isolates were carrying bla_TEM and bla_SHV genes, respectively, in singlet or in combination. The majority of these isolates belonged to phylo-group A (69.44%) and D (16.67%). Strikingly, all these isolates were found to be MDR showing high resistance to cephalosporins including the fourth generation cefepime and common non β-lactams. Additionally, class 1 integrons (intI1) were found in 30 (83.33%) isolates. Analysis of the class 1 integrons variable regions indicated that they were carrying up to five different gene cassettes conferring resistance to various drugs with a predominant combination of dfrA17-aadA5 genes in tandem, conferring resistance to aminoglycosides and trimethoprim. However, no ESBL encoding genes were found in the cassettes. Interestingly, 22 (66.11%) of the ESBL isolates were also carrying insertion sequence common region 1 (ISCR1) which was found to be associated with most of the CTX-M genes. Altogether, the current study reports on the high prevalence of ESBL-positive E. coli, particularly CTX-M-15, carrying clinical class 1 integrons and ISCR1 elements are likely indicative of their rapid and wider dissemination, posing threats to veterinary and public health. To the best of our knowledge, this is the first comprehensive study to report on the alarming high occurrence of ESBL-producing E. coli from mastitic cows in China.

Keywords: CTX-M-15; E. coli; ESBLs; bovine mastitis; gene cassettes; integrons.

Figure 1

Map of China (mainland) showing 16 provinces from where samples were collected.

Figure 2

Distribution of ESBLs encoding genes and CTX-M subtypes among ESBL-producing E. coli (n = 36) isolated from bovine mastitis.

Figure 3

Detection of class 1 integrons variable regions in ESBL-producing E. coli. PCR product was separated on 1% agarose gel. Lane 1, I-3 (intI1+) isolate; Lane 2, I₁-8strain; Lane 3, G-2 isolate; Lane 4, H-5 (intI1-ve) isolate; Lane 5, I₂-3 E. coli; Lane 6, Hn₁-2 strain; Lane 7, positive control strain; Lane 8, 2K molecular marker (Transgen, Beijing, China).

Figure 4

Restriction fragment length polymorphism (RFLP) analysis of intI1 variable region amplicons using Hinf I enzyme. RFLP product was analyzed on 1.5% agarose gel. Lane 1, I-3 isolate; Lane 2, I-25 E. coli; Lane 3, Hn₁-2 isolate; Lane 4, I₂-3 isolate; Lane 5, G-2 strain; Lane 6, Hb₃-1isolate; Lane 7, Hn₁-7 E. coli strain; Lane 8, 2K molecular marker.