Abstract
In this study we characterized the genetic environment of blaCTX-M and blaCMY-2 genes carried by 46 Escherichia coli isolates obtained from 20 chicken carcasses produced by five different brands in Brazil, including exporters and antibiotic-free-certified producers, purchased between 2010 and 2014. Similar plasmids characterized according to size and incompatibility group (Inc) were identified in E. coli belonging to different MLST-ST collected, regardless of carcass brand or production system. Hybridization assays with transconjugant strains revealed that blaCMY-2 gene (n = 19) was located on 85 kb plasmids of IncB/O, IncI1, IncFIB, or nontypeable groups. blaCTX-M-8 (n = 9) was located on 90 kb IncI1 plasmids. blaCTX-M-2 (n = 14) was inserted in class 1 integrons and conjugated only by one isolate in a 125 kb IncP plasmid. blaCTX-M-15 (n = 1), rarely described in isolates from food-producing animals in South America, was characterized by whole genome sequencing of transconjugant; the gene was carried in a 49.3 kb IncX1 plasmid. Sequencing of bla gene-flanking regions indicated the association of these genes with previously described insertion sequences. These results suggest that conserved genetic environments are related to ESBL and pAmpC genes in the Brazilian chicken production chain.
Introduction
Extended-spectrum β-lactamases (ESBL) and plasmid-mediated AmpC enzymes act against β-lactam antimicrobial agents and are frequently identified in Enterobacteriaceae isolated from food sources. First reports of blaCTX-M and blaCMY-2 genes in Brazilian chicken meat occurred in 2008 and 2010, in Escherichia coli obtained from chicken carcasses exported to Europe.1,2 In the following years, E. coli carrying these genes have also been detected in chicken meat acquired in retail markets in different Brazilian cities,3–5 and in isolates obtained from broilers in farms.6–8
The epidemiology of CTX-M enzymes indicates a higher prevalence of CTX-M-15 in clinical E. coli samples from Europe, North America, and the Middle East, and its increasing spread to other continents. 9 Regarding samples obtained from chicken or chicken meat, occurrence of the different variants shows a more specific geographical distribution: CTX-M-1 in the United States 10 and many countries of Europe,11,12 CTX-M-2 and CTX-M-8 in Brazil, 13 CTX-M-9 group variants in Asia and the recent increasing of CTX-M-55 in China and South Korea.14–16 On the contrary, most of pAmpC-producing E. coli isolated from food or food-producing animals carry blaCMY-2, regardless of region of origin.11,12,17
In a previous study, using antimicrobial drugs as selective pressure, we recovered 39 E. coli isolates carrying blaCTX-M and/or blaCMY-2 genes from 15 of 16 chicken carcasses analyzed from different batches of four Brazilian brands. 3 Producers included exporters, local, conventional, and certified antibiotic free, which suggested widespread occurrence of these genes. 3 Such results motivated this study, with the aim of investigating the mobile genetic elements associated with the successful dissemination of these β-lactamase encoding genes in Brazilian chicken meat. In addition to the previous sampling collection, we included in this study additional isolates obtained in a second round of carcass sampling, to evaluate a 4-year interval trend.
Materials and Methods
Samples
This study included two collections of isolates. The first one comprised 39 E. coli isolates carrying blaCTX-M and/or blaCMY-2 obtained from four frozen carcasses (1, 2, 3, 4) of different batches from four brands (A, B, C, D) commercialized during 2010 and 2011 in Rio de Janeiro. These isolates were selected as representatives of different biotypes among 136 E. coli (considering antimicrobial resistance phenotype, phylogenetic group, bla gene, and selective pressure). Isolates were obtained with or without selective pressure and their genetic diversity was previously characterized by ERIC2-PCR. 3
In addition, to evaluate the persistence of these genes in the production chain, we performed a second round of sampling in 2013 and 2014. Based on their availability in the retail market of Rio de Janeiro, brands A, C, and D were sampled again, and two additional brands (E and F) were included. Carcasses from brands A, B, C, and E were produced by conventional processes, and carcasses from brands D and F were produced in systems certified as antibiotic free.
In the second sampling time two carcasses of each brand were analyzed. Isolates were obtained in MacConkey agar plates (Difco, Sparks, MD) supplemented with 1 μg/mL ceftriaxone or 8 μg/mL cefoxitin (Sigma Aldrich, St. Louis, MO), and were identified by MALDI-TOF (Bruker Biotyper 3.1; Bruker Daltonics, Billerica, MA). Presence of pAmpC and CTX-M encoding genes was investigated in 24 E. coli isolates by PCR.18,19
Strain typing
Forty-six E. coli isolates carrying blaCTX-M and/or pAmpC genes from both collections were subjected to Achtman 7 gene multilocus sequence typing (MLST) as described (https://enterobase.warwick.ac.uk/species/index/ecoli).
Conjugation assays
The transfer of blaCTX-M and pAmpC genes was investigated by mating-out assays, using an azide-resistant E. coli J53 as a receptor. Transconjugant strains were obtained under selection with 100 μg/mL azide and 1 μg/mL ceftriaxone (Sigma Aldrich), confirmed by PCR and distinguished from donor strains by ERIC2-PCR typing. 20 Antimicrobial susceptibility testing was performed with donor and transconjugant strains to confirm β-lactamase expression and investigate the co-carriage of additional resistance genes.
Plasmid characterization
Plasmid incompatibility group typing of transconjugant strains was assessed by PCR. 21 Size and incompatibility groups of plasmids harboring blaCTX-M and/or pAmpC genes were determined by S1-PFGE using plasmids of known sizes from E. coli strains V517 (NCTC 50193) (53.7, 7.2, 5.6, 3.9, 3.0, 2.7, and 2.1 kb) and 39R861 (NCTC 50192) (147, 63, 43.5, and 6.9 kb) as controls, followed by hybridization with specific Inc and bla probes, using the AlkPhos Direct Labeling and Detection System (GE Healthcare, Life Sciences, United Kingdom). In addition, hybridization using S1-PFGE gels was performed to investigate the location of nontransferred blaCTX-M-2 genes.
Genetic context of bla genes
Integron carriage by donor and transconjugant strains was investigated by multiplex-PCR for integrase-encoding genes intI1 and intI2. 22 The genetic environment of the bla genes detected was investigated by PCR, using primers designed in this study, based on sequences available on NCBI GenBank database (Table 1). We investigated the presence of ISCR1 upstream and orf3 downstream to blaCTX-M-2, ISEcp1 upstream and orf477 downstream to blaCTX-M-15, ISEcp1 upstream and blc downstream to blaCMY-2, and IS26 and IS10 upstream to blaCTX-M-8. Cycling parameters for all reactions were initial denaturation at 95°C for 7 min, followed by 30 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 45 sec, and a final extension step at 72°C for 5 min, except for ISEcp1 upstream to blaCMY-2 amplification, with annealing temperature 58°C. Amplicons were subjected to Sanger sequencing (Macrogen, Seoul, Korea).
Primers Designed for Genetic Environments Surrounding bla Genes Amplification
Accession number refers to the sequences used as references to primer design.
Sequencing of blaCTX-M-15-harboring plasmid
Analysis of the blaCTX-M-15-harboring plasmid was performed after whole genome sequencing of the blaCTX-M-15-harboring E. coli transconjugant strain (Illumina MiSeq sequencer; Illumina). Reads obtained from DNA sequencing were trimmed with Trimmomatic 0.36. De novo assembly was performed by Unicycler Assembler tool. A single plasmid contig was obtained after separation of the entire chromosomal DNA of the recipient strain using the bbduk tool from bbmap package and the genome of E. coli J53 (NCBI GenBank accession number: CP028702.1) as reference. Antimicrobial resistance genes and plasmid incompatibility family were identified by Resfinder 23 and PlasmidFinder 24 online tools, respectively. Further annotation of the plasmid sequence was made by Patric online annotation service.
Results
Seven E. coli isolates, obtained from brands A, D, and F, were added to the collection from the second round of sampling, harboring genes blaCTX-M-2 (n = 2), blaCTX-M-8 (n = 1), or blaCMY-2 (n = 4). Overall, the collection of isolates characterized in this study included 46 E. coli isolates: 14 carrying blaCTX-M-2, 8 carrying blaCTX-M-8, 21 carrying blaCMY-2, 1 carrying blaCTX-M-15, 1 carrying both blaCTX-M-2 and blaCMY-2, and 1 carrying both blaCTX-M-8 and blaCMY-2 genes. Brands and year of isolation of the respective strains are given in Tables 2 and 3.
Characteristics of Plasmids Associated with blaCTX-M in Escherichia coli Obtained from Different Brands of Brazilian Chicken Meat, and Genetic Diversity of the Strains Carrying Them
Brands A, B, and C are conventional, Brand D is certified as antibiotic free. ND, not determined new ST; CIP, ciprofloxacin; CLO, chloramphenicol; FOS, fosfomycin/trometamol; GEN, gentamicin; SXT, sulfamethoxazole/trimethoprim; TET, tetracycline.
Characteristics of Plasmids Associated with blaCMY-2 in Escherichia coli Obtained from Different Brands of Brazilian Chicken Meat, and Genetic Diversity of the Strains Carrying Them
Brands A, B, and C are conventional, Brands D and F are certified as antibiotic free.
Our results demonstrated that blaCTX-M-8, blaCTX-M-15, and blaCMY-2 were mainly located in conjugative plasmids (9/9, 1/1, and 19/23 strains, respectively). In contrast, we did not detect blaCTX-M-2 in plasmids (14/15), except for one isolate (Tables 2 and 3).
Regarding the genetic environment of the bla genes, in all cases, blaCTX-M-2 were associated with ISCR1, orf3, and intI1 (even when incorporated into a conjugative plasmid), blaCTX-M-8 was associated with IS26 and IS10, and blaCTX-M-15 was associated with ISEcp1 and orf477. blaCMY-2 was associated with ISEcp1 and the blc gene in 21 of 23 strains. Using primers designed based on conserved genetic sequences, we were unable to detect blc in two blaCMY-2 carrying strains (ST1158, brand B and ST23, brand D).
Among the 30 transconjugant strains, only two, one each carrying blaCMY-2 or blaCTX-M-2, expressed resistance to an antimicrobial agent (tetracycline) in addition to those related to the β-lactamase genes. This finding suggests that although many donor strains exhibited resistance to different classes of antimicrobials, the genetic determinants of these drugs are not located in the same plasmid as the bla gene.
The blaCTX-M-2-harboring plasmid identified in this study was 125 kb and belonged to the IncP incompatibility group. blaCTX-M-8 was associated with 90 kb IncI1 plasmids (9/9 strains) and blaCMY-2 with 85 kb IncB/O, IncI1, IncFIB and nontypeable plasmids (7, 6, 3, and 3/23 strains, respectively), regardless of chicken brand. Notwithstanding, similar plasmids occurred in E. coli belonging to different STs. Moreover, we could observe isolates belonging to a same ST (ST115, ST155, ST602, ST1125, and ST3258) carrying diverse bla genes in their respective plasmids (Tables 2 and 3).
Sequencing of a blaCTX-M-15-carrying transconjugant strain revealed that this gene was located on a 49.3 kb plasmid, here named pF609, of the IncX1 incompatibility group. blaTEM-1B and qnrS1 resistance genes, and the genes encoding the conjugation machinery and a toxin–antitoxin system RelE/StbE were found in this plasmid. Blastn alignment showed 93% coverage and 99% identity with 47.6 kb plasmids from Shigella flexneri 1a strain 0670, Shigella flexneri 4c strain 072, and E. coli strain HP2 (accession numbers CP020088.1, KJ201886.1, and MH121702.1, respectively). Our data suggest a blaCTX-M-15 insertion in this plasmid mediated by an ISEcp1 element (Fig. 1). Plasmid DNA sequence was deposited in GenBank under the accession number MK965545.
Annotation and alignment of pF609 and pSF07202 (accession number KJ201886.1) indicating blaCTX-M-15 insertion mediated by ISEcp. Gray regions between the plasmids indicate nucleotide identity by BLASTN. Arrows represent predicted ORFs or relevant genes. Blue arrows represent the sequence inserted in TnpA (green arrow). The figure was generated by Easyfig genome comparison visualizer. 25 This figure is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0 Color images are available online.
Discussion
Genes encoding CTX-M and CMY-2 enzymes have been frequently detected in exported and inland distributed chicken meat from Brazil.1,3–5,11,26 In this article, the genetic environment associated with the dissemination of these genes were conserved regardless of brand, production system, or year of isolation.
The higher incidence of blaCTX-M-2, blaCTX-M-8, and blaCMY-2 genes in Brazilian chicken meat in comparison with other ESBL and pAmpC variants has been well documented throughout the years.2,3,26 Thus, this study is representative for the most relevant β-lactamase encoding genes carried by E. coli in this context. In contrast, blaCTX-M-15 was first detected in an isolate from Brazilian chicken meat in 2015, and additional reports of this occurrence are still uncommon.3,7,27 This led us to sequence the blaCTX-M-15 harboring plasmid.
Genetic environments surrounding blaCMY-2, blaCTX-M-2 and blaCTX-M-8 were similar to those classically described.28–30 This finding was expected based on the association of these genes with integrative or transposable elements. However, blaCTX-M-8 and blaCMY-2 genes were additionally located on conjugative plasmids with similar sizes and belonging to few Inc groups distributed regardless of brand, production system, or year of isolation. blaCTX-M-2, although detected in strains belonging to different STs, was in general nontransferable. Only one strain transferred blaCTX-M-2 by conjugation, and hybridization did not evidence the occurrence of this gene in plasmids from other blaCTX-M-2-harboring strains. This may suggest a chromosomal location for most isolates, as reported in other studies performed in Brazil.7,27 Understanding how these genetic elements disseminate and persist is challenging and demand further studies.
Co-transference of resistance genes to other classes of antimicrobial agents was uncommon in blaCTX-M- or blaCMY-2-harboring plasmids, thus indicating that cross-selection with non-β-lactam antimicrobial agents is not a factor determining the persistence of these plasmids in the respective strains. β-lactams are not approved as growth promoters in Brazil since 2009; however, these drugs may be used for prophylactic or therapeutic purposes based on veterinary prescription. 31 Additional studies will be necessary to quantitatively assign the role of extensive usage of β-lactams in farms producing these animals, and of other environmental factors, in the continuous occurrence of ESBL and pAmpC genes in E. coli from chicken meat. Noteworthy, many of the isolates included in this study were obtained from carcasses from animals produced in antibiotic-free system. Presence of antimicrobial resistance genes in isolates obtained from organic chicken meat or from farms with reduced usage of antimicrobial agents was also demonstrated in the Netherlands, Germany and France.27,32,33
pF609 was the first blaCTX-M-15-harboring plasmid isolated from a strain associated with chicken meat in Brazil. 3 Analysis of its sequence suggests the incorporation of blaCTX-M-15 into a plasmid previously described in Shigella (pSF07202, accession number KJ201886.1) and E. coli (pEQ2, accession number KF362122.2), mediated by ISEcp1. pF609 presents a functional conjugation machinery and one toxin–antitoxin system, which may contribute to its dissemination and persistence. More recently, Casella and colleagues published the draft genome of Ec39764, a blaCTX-M-15-harboring E. coli isolated in 2014. 27 Our analysis of pF609 and Ec39764 (accession number MUFZ01000000) indicates 98% of coverage and 100% identity among the blaCTX-M-15-harboring plasmids. STs of donor strains (ST1125 and ST355, respectively) are not closely related.
STs of β-lactamase-producing strains characterized in this study are unrelated to internationally successful clones. Still, one cannot exclude the possibility of transference of these genes to well-disseminated E. coli clones. Based on isolates obtained from a small number of carcasses, our data suggest a possible role of chicken meat contaminating strains as source of plasmids harboring clinically relevant β-lactamase-encoding genes. Additional studies are necessary to evaluate the impact of chicken meat consumption in the antimicrobial resistance profile of bacteria colonizing community-based individuals.
Footnotes
Acknowledgment
The authors thank Dr. Lee Riley for supporting MLST analysis.
Disclosure Statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Funding Information
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) that provided funds to R.R.B. (428693/2016-4) and research fellowships to G.B.K, R.C.P (311946/2016-0), and B.M.M. (311766/2015-3); by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) that provided fellowships to L.A.B.B and A.P.S.S.; and by Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) that provided grants to P.B.R, G.B.K., R.C.P. (E-26/203.282/2017) and B.M.M. (202.884/2017). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001.