The Relationship of Phylogenetic Distribution and Integron Gene Presence with Multiple Antibiotic Resistance in Escherichia coli Isolates Obtained from Bovine Milk with Mastitis

Abstract

Multiple antibiotic resistant (MDR) bacteria are one of the most important current threats to public health. Integrons are important mobile genetic mechanisms involved in the spread of resistance. Escherichia coli includes both commensal and pathogenic clones found in  different phylogenetic groups. Revealing the distribution of phylogenetic groups and the presence of integrons in E. coli, which causes clinical mastitis (CM), may assist in the development of specific strategies for the treatment and control of this pathogen. In this study, it was aimed to examine the relationship between phylogenetic distribution and integron related gene presence with multi-antibiotic resistance of bacteria in E. coli isolates obtained from bovine milk with clinical mastitis. The material of the study consisted of 73 E. coli (17.3%) isolates obtained from 422 milk samples. After bacterial isolation was performed with  conventional biochemical tests; Identification, phylogeny and presence of integron genes were examined by Polymerase Chain Reaction (PCR). The resistance patterns of the isolates against nine antibiotics belonging to nine antimicrobial families were analyzed using the Kirby-Bauer disc diffusion method. Isolates resistant to at least three drugs from various antimicrobial drug classes were defined as multi-drug resistant (MDR). Chi-square (χ2) test was used to calculate the relationship between multidrug resistance of isolates, their  phylogenetic distribution and presence of integron genes (p<0.05). According to the antibiotic test results, the highest resistance was found against ampicillin (79.4%) and amoxicillin clavulanate (61.6%), while the highest sensitivity was against ciprofloxacin (86.3%) and cefoperazone (84.9%). 52.1% (38/73) of the isolates were MDR. The majority of isolated E. coli strains belonged to group A (37.0%), followed by group B1 (24.7%), group C (15.0%), group D (5.5%), group E (% 8.2%) and then group F (4.1%). It was determined that 31.5% of the isolates carried integron related genes. There was no significant relationship between MDR and phylogenetic distribution of the isolates. However, the relationship between MDR and the prevalence of integron related genes was significant (p=0.001). The high rate of multi-antibiotic resistance of isolates belonging to phylogroups A and B1, which are generally considered commensal, is a concern. The increase presence of MDR bacteria in animals can lead to the emergence of resistant bacteria that can be transferred to humans through direct contact or through the food chain, reducing the effectiveness of antibiotics used to treat human diseases.

Keywords: Escherichia coli; Multiple Antibiotic Resistance; Phylogenetic Distribution; Integron

Introduction

Bacterial pathogens that cause mastitis can be contagious or environmental. Contagious mastitis agents have the feature of being easily transmitted from animal to animal. Environmental pathogens such as Escherichia coli are important because they are frequently isolated from bovine mastitis (1, 2) and can cause toxic or fatal effects in affected cattle (3). While commensal strains of E. coli are bacteria of the intestinal microbiota of humans and animals, virulent strains are pathogens that cause intestinal or extra-intestinal diseases (1). The barrier between commensalism and virulence of E. coli largely depends on the balance between the host’s immune status and the bacteria’s ability to elicit virulence factors (4). Recently, the term mammary pathogenic E. coli has been proposed to classify  mastitis-associated pathogens (5), however, specific virulence factors that may reveal the relationship between these strains and mastitis have not been identified (6). Therefore, the determination of pathogenic E. coli strains is important in terms of determining the risk profile of the isolate.
Phylotyping of E. coli isolates is very useful as it gives an idea about the source and  pathogenicity of the isolate obtained. Clermont et al. (2013) analyzed four genes (arpA, chuA, yjaA, and TspE4.C2) and classified E. coli isolates into eight phylogroups (A, B1, B2, C, D, E, F, and clade I) (7). According to this, birds, reptiles, fish and some mammals carry phylogroup A. The B1 phylogroup is generally environmental. This type of E. coli has a low risk to human health. Humans predominantly carry phylogroup B2. This type of E. coli has a high risk to human health. Recently, phylogroup C has been identified, which includes a group of strains closely related to but distinct from phylogroup B1. Phylogroup C and phyogroup F, about which little is known are acknowledged to have a low risk to human health, unlike Clade 1 strains, which include extraintestinal pathogenic strains. Phylogroup D is associated with humans and omnivores (pigs) while the phylogroup E is linked to herbivores and cattle. These two phylotypes pose a moderate risk to human health (7, 8). Phylogenetic analyzes have shown that virulent extraintestinal E. coli strains are mostly associated with group B2, while commensal strains are mostly associated with A or group B1 phylogroups (9). The mastitis-causing E. coli has great intraspecific diversity and has been classified in different phylogenetic groups (mostly A, B1, B2 or D) (10). It has been reported that the majority of E. coli strains causing bovine mastitis belong to phylogroups A and B1 (9-13). Information on the C, E and F groups that cause CM is quite scarce (14, 15).

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