Genetic environments and related transposable elements of novel cfr(C) variants in Campylobacter coli isolates of swine origin

Highlights

• Identified novel cfr(C) variants in C. coli isolates and confirmed they were functional.

• First report of cfr(C)-related transposable elements in Campylobacter.

• Some cfr(C) variants located on novel plasmids and were conjugative transferable.

• All cfr(C) gene were inserted in the downstream of kanamycin resistance gene aphA3.

Abstract

The cfr(C) is a cfr-like gene that confers cross-resistance to antibiotics targeting the 23S rRNA through methylation of nucleotide A2503. Here, we identified 7 C. coli isolates containing 4 novel cfr(C) variants from swine farm and slaughterhouses samples. Of the 7 cfr(C)-carrying isolates, one had a frame-shift mutation, while the other 6 had intact genes. However, one of the 6 intact genes did not show a PhLOPS_A phenotype in the original isolate, but was fully functional when cloned into C. jejuni NCTC 11168. Cloning of cfr(C) variants into C. jejuni NCTC 11168 and conjugative transfer of the two cfr(C)-containing plasmids further confirmed their role in conferring resistance to PhLOPS_A antimicrobials, and resulted in an 8-128-fold increase in their MICs. In all cfr(C)-carrying isolates, cfr(C) genes were located in the downstream of the kanamycin resistant gene aphA3. IS607* and IS1595-like were located immediately upstream of aphA3 gene and seemed to play a role in its recombination. A novel transposable element named ISCco7, which located immediately downstream of cfr(C) in two isolates, was probably associated with the integration of cfr(C). However, neither insertion sequence nor other transposable elements were identified near cfr(C) in the remaining five cfr(C)-positive isolates, indicating the mechanism underlying the integration of cfr(C) into plasmids or chromosomal DNA requires further investigation. These results reveal novel cfr(C) variants and their associated genetic environments in C. coli isolates and indicate the flexibility of C. coli in acquiring new antibiotic resistance genes.

Introduction

Florfenicol, a fluorinated synthetic analog of chloramphenicol, is widely used as an antimicrobial chemotherapeutic agent in animal husbandry, especially for the bovine and swine respiratory disease (Schwarz et al., 2004). Unfortunately, the extensive use of florfenicol potentially promotes the emergence of numerous florfenicol-resistant genes (FRGs), including the phenicol-specific exporters, such as fexA, fexB and floR (Cloeckaert et al., 2000; Kehrenberg and Schwarz, 2004; Liu et al., 2012), and multi-drug resistance (MDR) genes, including cfr/cfr-like, optrA and poxtA (Antonelli et al., 2018; Long et al., 2006; Wang et al., 2015a).

The cfr gene was firstly discovered in a bovine Staphylococcus sciuri isolate in 2000 (Schwarz et al., 2000). It is noteworthy because it provides cross-resistance to five chemically unrelated antimicrobial classes including phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A (known as the PhLOPS_A phenotype). Of particular concern is the resistance to the oxazolidinone class (e.g. linezolid), which is used as the last resort for treating MDR gram-positive bacterial infections in humans. To date, a number of cfr-like genes have been identified, including cfr(B) from Clostridium difficile (Deshpande et al., 2015), cfr(C) from Clostridium difficile and Campylobacter coli (Candela et al., 2017; Tang et al., 2017), cfr(D) from Enterococcus faecalis (Pang et al., 2020), and recently reported cfr(E) from Clostridium difficile (Stojkovic et al., 2019).

Campylobacter is a leading foodborne pathogen and exposed to both veterinary and human medicines (Shen et al., 2018). In recent years, Campylobacter had become increasingly resistant to clinically important antimicrobials and developed multiple mechanisms for antibiotic resistance, including mutations in target genes, such as 23S rRNA mutations to macrolides, gyrA mutations to fluoroquinolones, multidrug efflux pump CmeABC/RE-CmeABC extruding structurally diverse compounds and antimicrobials, and horizontally acquired antibiotic resistance genes, such as tet(O), erm(B), fexA, and fosX^CC (Liu et al., 2020; Shen et al., 2018; Tang et al., 2020; Wang et al., 2015b). Since 2013, the US CDC classified drug-resistant Campylobacter as a serious antibiotic resistance threat (CDC, 2013). In 2017, Tang et. al reported from USA the first identification of plasmid-mediated multi-drug resistance gene cfr(C) in C. coli isolates of feedlot cattle origin (Tang et al., 2017). The gene cfr(C), encodes a 379 aa protein that shows 55.1 % identity to the original Cfr from Staphylococcus sciuri (Schwarz et al., 2000). So far, cfr(C) has been found on plasmids of C. coli isolates from cattle in USA and in chromosomal DNA of C. coli isolates from swine in China (Liu et al., 2019; Tang et al., 2017; Zhao et al., 2019). However, no cfr(C)-related insertion sequence was identified from these cfr(C)-positive isolates. In this study, we collected Campylobacter isolates from a commercial swine farm and two slaughterhouses to investigate the prevalence and genetic environments of cfr(C) gene and analyze the potential transposable elements that may play a role in the dissemination of the cfr(C) gene.