Among the most important medical advances of the last century, antibiotic therapy is a vital resource in the fight against infectious bacterial diseases. However, its widespread use has led to the advent of antibiotic-resistant bacteria¹. Rapid spread of resistant strains, driven in part by human migration and the increasing industrialization of food and animal production, is a recogized worldwide health problem. One cause of strains developing resistance is the presence of antibiotic resistance genes (ARG), which can be acquired and transferred through mobile genetic elements (MGE) such as bacteriophages².

Lysogenic bacteriophages containing ARG have been identified, largely in aquatic environments^2,3. They have also been reported in fecal samples collected in hospitals from clinically healthy patients, suggesting that these phages may be universally present though undetected in health safety screening programs^4,5. Various studies of lysogenic bacteriophages have detected ARGs that have claimed the lives of thousands of people and generated millions of dollars in losses worldwide. These studies have also elucidated phages’ contribution to the spread of ARGs in the environment¹. The present study objective was to improve understanding of bacteriophages’ role in the spread of ARG by quantifying the prevalence of the bla _TEM , qnrA and qnrB genes in bacterial and phage DNA.

Water samples were collected from municipal slaughterhouses (SLH1, SLH2, SLH3 and SLH4) in the State of Mexico, Mexico, from September to December 2015. Samples were collected based on probabilistic criteria⁶, and following official guidelines⁷. A total of 48 samples were collected from the animal processing area of each slaughterhouse: 16 from potable water (PW); 16 from wastewater (WW); and 16 from sewers (SW). Each sample was collected with a swab, which was then rubbed along the edges of the lid surface⁷. Genotypic confirmation of E. coli isolation was done using an endpoint PCR to amplify the uidA gene (primers listed in Table 1), under established conditions⁸. Antibiotic susceptibility was quantified using fourteen antimicrobial substances in different concentration ranges following the disk diffusion method guidelines of CLSI⁹. Results interpretation was done according to CLSI guidelines¹⁰.

Phage isolation was done with mitomycin C, and phage lysis verified with the spot-test method and double layer test¹¹. Phage DNA was isolated with the phenol-chloroform method¹². Bacterial DNA removal and the presence of phage DNA were confirmed with an endpoint PCR test in a Multigene^TM Mini Personal thermal cycler (Labnet International Inc., Edison, NJ, USA) with uidA amplification as a negative control³.

Bacterial DNA extraction was done following a published protocol¹³, and phage and bacterial DNA concentration and purity quantified with a spectrometer (Quawell q500). Endpoint PCR was used to detect the qnrA, qnrB and bla _TEM genes, under previously reported conditions^14,15. Statistical analyses consituted an analysis of variance (ANOVA) and a linear correlation test run using StatCalc ver. 8.2.2 (Copyright © 2016 AcaStat Software). Significance was set at P<0.05.

Of the 48 collected samples, 37 (77 %) produced Escherichia coli isolates with varying resistance indices (Table 2). Of the 37 isolates, 13 (35.1 %) were from SLH1, while 8 each (21.6 %) were from SLH2, SLH3 and SLH4. The higher (P<0.05) number of isolates from SLH1 was probably a function of the larger number of animals pocessed there compared to the other slaughterhouses. A positive correlation (r= 1) was observed between the 19 (51.3 %) isolates from wastewater samples and the 18 (48.6 %) from sewage samples. This is to be expected since both sites exhibit similar conditions which are adequate for bacterial growth.

The number of isolates with ARG was higher in wastewater than in sewer water (P(0.05), and there were no isolates from the potable water samples (Table 3). Most probably due to the wide range of ARG variants and their different resistance mechanisms, the identified bacterial isolates which exhibited intermediate phenotypic resistance and susceptiblility also amplified bla _TEM , qnrA and qnrB.

A bacteriophage pool was obtained from each bacterial isolate for detection of bla _TEM , qnrA and qnrB; all the isolates presented temperate phages. The bla _TEM gene was the most prevalent in all the bacterial and phage isolates (Table 4). These results coincide with a previous study of water samples collected from wastewater treatment plants and slaughterhouses in which bla _TEM was highly prevalent (80 to 100 %) and had high gene copy densities (± 3.3 log₁₀)³. The bla _TEM gene is the most frequently reported worldwide^16,17, especially in Gram negative bacteria. This is assumed to be due to its broad dissemination via migratory waterfowl and the large number of β-lactamase enzymes synthesized by bacteria. In the bacterial isolates, prevalence of qnrA was 8.1 % and that of qnrB was 29.7 %, while in the phage isolates it was 2.7 and 10.8 %, respectively. In the phage isolates these prevalences contrast with previously reported prevalences in wastewater samples^3,18. High qnrA prevalences have also been reported in samples obtained from a health center⁴. Although the observed prevalence is not high, quinolone resistance has been increasing due to indiscriminate use in veterinary and human medicine¹⁸.

The amount of ARG identified in the bacterial DNA and phage DNA was positively correlated (r= 0.99) (Table 4). This correlation coincides with the widely reported presence, in multi-resistant bacteria, of phages capable of disseminating portions of ARG-containing bacterial genome throughout the environment. These phages can transduce ARG to commensal and pathogenic bacteria, providing them new adaptive (short-term) and evolutive (long-term) abilities, and contributing to generation of new resistant pathogenic strains which are potentially fatal.

The highest number of bacterial isolates exhibiting presence of the three evaluated ARG was recorded in SLH1, while in SLH2 all three genes were found in the phage isolates (Table 4). The contamination and unhygienic conditions in the sampled municipal slaughterouses provide many of the factors required for successful viral transduction: temperature, pH, concentration, and bacterial and phage physiological state.

Human lifestyles promote a steady increase in antibiotic resistance among microbes. Consciousness of the consequences of indiscriminate and unnecessary use of antimicrobials is slowly growing but numerous studies still report highly virulent and resistant bacteria in areas of human activity¹. The bacterial and phage isolates analyzed here exhibited the presence of bla _TEM , qnrA and qnrB in highly variable distributions; at least one of these genes was present in all the sampled slaughterhouses. Phages belonging to the MGE group play a vital role in the transfer of ARG between pathogenic-commensal and pathogenic-pathogenic bacteria⁵. Better understanding of the mechanisms of antimicrobial resistance is an important tool in the ongoing development of effective strategies to reduce this phenomenon.