High Levels of Resistance to Cephalosporins Associated with the Presence of Extended-Spectrum and AmpC β-Lactamases in Escherichia coli from Broilers in Southern Brazil

Introduction

Extended-spectrum beta-lactamase (ESBL) producers are associated with infection and colonization in animals and humans worldwide. ¹ CTX-M-1, CTX-M-14, CTX-M-15, SHV-12, and TEM-52 are the most frequent ESBL types among Enterobacteriales obtained from animals and are also found consistently in humans. These genes confer resistance to cephalosporin and are often carried by plasmids, which may also carry genes that lead to resistance to other classes of antibiotics such as aminoglycosides. ² In Brazil, ESBL-producing Enterobacteriales are widely distributed in hospitals, but there has been no multicenter study establishing the real prevalence of ESBL. ³ CMY-2, an AmpC-type β-lactamase whose main substrate is ceftazidime (CAZ), is the β-lactamase most commonly identified in animals.^4–6

The bla_CMY-2 gene was originally described in the chromosome, but it has already been found in plasmids of extraintestinal Escherichia coli isolates from both animals and humans.^7–9 Studies performed at the Center for Disease Control and Prevention (CDC) have identified bla_CMY-2 plasmids in both zoonotic pathogens and commensal bacterial species from human and animal sources in several continents.^10,11 Only scattered data on ESBL and bla_CMY-2 in Brazil are available, so the objective of this study was to describe the presence of ESBLs and bla_CMY-2 in broiler chicken in southern Brazil.

Materials and Methods

Cloacal swabs were collected from broiler chickens over 5 days, between August and October 2015, in a slaughterhouse in the Rio Grande do Sul State of Brazil. A total of 340 broiler chickens from 17 batches (20 animals per batch) obtained from 12 distinct farms were included in this study. The swabs (1 per chicken) were cultured in 10 mL of tryptic soy broth (TSB-KASVI^®) containing ceftazidime (30 μg) and incubated for 24 hrs at 37°C. Ten microliters of the broth was subcultured on a MacConkey agar plate and incubated at 37°C for 24 hrs. In most cases, only one colony was selected from the MacConkey plate, but in cases where differences in colony phenotype were observed, more than one colony was selected.

A total of 343 isolates were recovered and confirmed as E. coli using conventional methods. Isolates that could not be identified using conventional methods were submitted to matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-TOF/MS; Brucker Daltonics, Germany). The animal bulletins of health indicated that all chickens had received bacitracin, narasin, and nicarbazin during the first period of life (2nd and the 18th day) and avilamycin and salinomycin during the second period (20th and 35th day); batches 10 and 11 also received doxycycline for 3 days in their second period of life. This study was approved by the Ethics Committee of Federal University of Rio Grande do Sul—project number 29445.

Results

Of the 343 E. coli isolates, 57 (16.6%) tested positive for at least 1 ESBL gene in PCR: 30 for bla_TEM-1, 18 for bla_CTX-M, 3 for bla_SHV, and 6 for both bla_TEM-1 and bla_CTX-M. After sequencing, 27 (7.9%) isolates were confirmed as ESBL producers: 23 bla_CTX-M-2, 1 bla_CTX-M-8, 2 bla_SHV-2a, and 1 bla_SHV-₁₈ (Table 2). The highest proportion of ESBL-positive isolates (14/27; 51.85%) was in the material recovered from farm “N” and the bla_CTX-M-2 gene was detected in each isolate. Conversely, farm “D” was the only farm from which no ESBL-positive isolates were recovered.

Overall, 24 of the 27 isolates with ESBL genes gave positive results in the phenotypic test for ESBL and cefotaxime proved to be the best substrate (21 of 24 positives). Ten of the 343 isolates were positive for the mcr-1 gene (data published elsewhere) ¹⁹ ; but none of the isolates was positive for the carbapenemase genes. Twenty-four of the 32 isolates were positive for the bla_CMY-2 gene and 8/24 harbored both the bla_CMY-2 and mcr-1 genes. bla_CMY-2-positive isolates were identified in all farms evaluated (Table 2).

All 27 ESBL-positive isolates and 24 bla_CMY-2-positive isolates were considered multidrug resistant because they were resistant to at least 3 antibiotic classes (penicillin, cephalosporins, sulfonamides, and quinolones). For the 32 isolates tested for bla_CMY-2, the MIC ranges of CAZ and CTX were 16-128 and 4-64 μg/mL, respectively (Table 2). The MIC50 and MIC90 of CAZ were 64 μg/mL and for CTX were 16 and 32 μg/mL, respectively.

Discussion

All the isolates grew in the presence of ceftazidime, although the animals had not received cephalosporin, according to animal bulletins of health presented by the farms. Although the isolates were obtained from a broth supplemented with ceftazidime, only 7.9% of them tested positive for ESBL genes, which is low when compared with data from Europe (www.efsa.europa.eu/en/press/news/180227). ¹

Cefotaxime was a better substrate than ceftazidime for phenotypic detection of ESBL, which is unsurprising as bla_CTX-M-2 was the most prevalent ESBL gene in our sample, and it is well known that CTX-M enzymes have a high affinity for cefotaxime. ²⁰ It is noteworthy that this report is, to the best of our knowledge, the first time that some of the other ESBL genes that were identified, such as bla_SHV-2a and bla_SHV-18, ²¹ have been described in Brazil. Extraintestinal E. coli isolates of animal origin have already been associated with human infections, as the plasmids that carry bla_SHV-2a have been found in the material of both human and animal origins.^2,22 The bla_SHV-2a gene is the main variant of SHV in food-producing animals, ²¹ and a Canadian study has already demonstrated the existence of cross-contamination between food-producing animals and retail meat. ²³

Indeed, the potential for horizontal transmission of these genes between Enterobacteriales of differing origin is a matter of high public health concern. Although bla_CMY-2 was described in Brazil more than 10 years ago, ²⁴ only a few studies have presented prevalence data for Brazil and most have reported a low prevalence.^25–27 We identified bla_CMY-2 in a high proportion of isolates from all farms evaluated. Given that the prevalence of ESBL genes was very low in the isolates evaluated in this study, it is reasonable to regard bla_CMY-2 as an important mechanism of cephalosporin resistance in E. coli from broilers in southern Brazil. The mcr-1 gene has been identified among isolates harboring both bla_CTX-M-2 and bla_CTX-M-8. ¹⁹ The mcr-1 gene confers resistance to bacitracin, which might explain its presence in the chicken microbiota. ²⁸

In this study, we observed high rates of cephalosporin-resistant E. coli among investigated farms. Various mechanisms of resistance were identified. We also observed isolates harboring both ESBL and mcr-1 genes, which illustrates the importance of using antibiotics prudently in food production, to reduce the environmental reservoir of resistance genes.