Aguirre et al. Porcine Health Management (2020) 6:8 https://doi.org/10.1186/s40813-020-00146-2 RESEARCH Open Access Antimicrobial resistance profile and prevalence of extended-spectrum beta- lactamases (ESBL), AmpC beta-lactamases and colistin resistance (mcr) genes in Escherichia coli from swine between 1999 and 2018 Laia Aguirre1* , Anna Vidal1, Chiara Seminati1, Montse Tello1, Noelia Redondo1, Laila Darwich1,2 and Marga Martín1,2 Abstract The frequent usage of antibiotics in livestock has led to the spread of resistant bacteria within animals and their products, with a global warning in public health and veterinarians to monitor such resistances. This study aimed to determine antibiotic resistance patterns and genes in pig farms from Spain during the last twenty years. Susceptibility to six antibiotics commonly used in pig production was tested by qualitative (disk diffusion) and quantitative (minimum inhibitory concentration, MIC) methods in 200 strains of Escherichia coli which had been isolated between 1999 and 2018 from clinical cases of diarrhoea in neonatal and post-weaned piglets. Results showed resistance around 100% for amoxicillin and tetracycline since 1999, and a progressive increase in ceftiofur resistance throughout the studied period. For colistin, it was detected a resistance peak (17.5% of the strains) in the 2011–2014 period. Concerning gentamicin, 11 of 30 strains with intermediate susceptibility by the disk diffusion method were resistant by MIC. Besides, the most frequent antimicrobial resistance genes were the extended- spectrum beta-lactamase (ESBL) blaCTX-M (13.5% of strains, being CTX-M-14, CTX-M-1 and CTX-M-32 the most prevalent genomes, followed by CTX-M-27, CTX-M-9 and CTX-M-3), AmpC-type beta-lactamase (AmpC) blaCMY-2 (3%) and colistin resistance genes mcr-4 (13%), mcr-1 (7%) and in less proportion mcr-5 (3%). Interestingly, these mcr genes were already detected in strains isolated in 2000, more than a decade before their first description. However, poor concordance between the genotypic mcr profile and the phenotypical testing by MIC was found in this study. These results indicate that although being a current concern, resistance genes and therefore antimicrobial resistant phenotypes were already present in pig farms at the beginning of the century. Keywords: Antimicrobial resistance, Escherichia coli, Pig, Colistin-mcr genes, ESBL * Correspondence: laia.aguirre@uab.cat 1 Departament de Sanitat i d’Anatomia Animals, Universitat Autònoma de Barcelona, Edifici V, Travessera dels Turons, 08193 Bellaterra, Spain Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Aguirre et al. Porcine Health Management (2020) 6:8 Page 2 of 6 Background aerobically at 37 °C overnight. Faecal samples collected in Levy et al. (1976) observed the direct link between the 2017 and 2018 were plated in the same culture media and usage of antibiotics in livestock and the apparition of conditions in order to isolate E. coli strains. The qualitative resistances in human pathogens by plasmid transfer [1]. disk diffusion method on Mueller Hinton agar was used to Nowadays, antibiotics are still extensively used in the test susceptibility to amoxicillin, tetracycline, gentamicin, animal husbandry sector, especially in those farming enrofloxacin, ceftiofur and colistin according to the Clinical activities with the most intensive systems such as pork and Laboratory Standards Institute (CLSI) [3, 4] using the production. Pig industry is the most important livestock antibiotic disks and breakpoints shown in Table 1. sector in Spain. Recent studies show that the percentage Amoxicillin-clavulanic resistance was tested in strains re- of resistance to last-resort drugs such as colistin is con- sistant to ceftiofur to predict the production of AmpC siderably higher than in other European regions [2, 3]. beta-lactamase. At the same time, the minimum inhibitory Following these reports and considering the worldwide concentration (MIC) of colistin was determined for all increasing prevalence of antimicrobial resistance (AMR) strains by broth microdilution method in 96-wells micro- in food-producing animals, the aim of this study was to plates, using a dilution prepared from colistin sulphate of determine the present situation of AMR in pig farms from 21958 IU/mg (731,93 μg/mg) potency [4]. Additionally, for Spain and its evolution since 1999, as well as the prevalence strains with resistant, intermediate and sensitive but close of the most common resistance genes (colistin resistance to the breakpoint results to gentamicin, MIC values were plasmids (mcr), genes encoding for extended-spectrum beta- also determined by broth microdilution with a dilution lactamases (ESBL), AmpC-type beta-lactamases (AmpC) and from gentamicin sulphate. Both MIC microdilution proce- carbapenemases). dures were manually prepared and performed according to CLSI standards, using the reference strain E. coli ATCC Methods 25922 as control in each plate. CLSI breakpoints shown in One hundred and sixty pathogenic E. coli strains isolated Table 2 were used. from swine enteric clinical cases were randomly recovered The most common genes coding for ESBL (blaCTX-M, from the strain collection of the Infectious Diseases blaTEM, blaSHV), AmpCs (blaCMY-1, blaCMY-2), carbape- Laboratory of the Veterinary Faculty of Barcelona (Spain) nemases (blaOXA) and colistin resistance (mcr-1, mcr-2, between 1999 and 2014 (8–16 strains per year). Those mcr-3, mcr-4 and mcr-5) genes were analysed by PCR in cases came from farms of different counties of Spain with all strains as previously described [8]. Controls for all neonatal or post-weaning diarrhoea problems. In addition, genes were provided by Dr. Migura (IRTA-CReSA, forty different E. coli strains isolated throughout 2017 and Bellaterra, Spain). Amplified PCR products were Sanger 2018 were collected from ten farms in Catalonia (NE of sequenced for verification at the Genomic and Bioinfor- Spain). The primary objective was to determine the evolu- matics Service of the Universitat Autònoma de Barcelona tion of antimicrobial resistances in E. coli strains since (Bellaterra, Spain). Sequences were analysed by using 1999 against the most commonly used antibiotics in BioEdit software and blasted against the public database swine. A second objective was to study the presence of (National Center for Biotechnology Information, NCBI). resistance genes (ESBL, carbapenemase and mcr) by Statistics were applied for determining antimicrobial molecular methods in all the selected strains. resistance differences among the periods studied using For this purpose, strains from the collection were the Chi-Square Test (Fisher Exact P-values) and graphed recovered in brain heart infusion and then streaked using the program Excel. Antimicrobials with statistical on blood agar and MacConkey agar, both incubated differences were further analysed by T-Square. Finally, Table 1 Concentrations and breakpoints of the antibiotic disks used for the disk diffusion method Antibiotica Concentration Breakpoint (mm) Referenceb (μg/mg) S R Amoxicillin 25 ≥ 17 ≤ 13 CLSI M100; human [5] Amoxicillin - clavulanic 30 ≥ 18 ≤ 13 CLSI M100; human [5] Ceftiofur 30 ≥ 21 ≤ 17 CLSI VET08; cattle E. coli and swine Salmonella cholerasuis [6] Colistin 50 ≥ 15 < 15 CA-SFM; veterinary [7] Enrofloxacin 5 ≥ 23 ≤ 16 CLSI VET08; dog, cat and poultry [6] Gentamicin 10 ≥ 16 ≤ 12 CLSI VET08; dog, horse [6] Tetracycline 30 ≥ 15 ≤ 11 CLSI M100; human [5] a Antibiotic disks: BBL™ brand. bCLSI veterinary breakpoints were preferably used. If not available, CLSI human or CA-SFM veterinary breakpoints were used Aguirre et al. Porcine Health Management (2020) 6:8 Page 3 of 6 Table 2 Potency and breakpoints used for the microdilution 42.5% (85/200), a percentage that has remained practic- methods for colistin and gentamicin ally stable in these past twenty years (P > 0.05). Gentami- Antibiotica Potency Breakpoint (μg/mL) Referenceb cin resistance was detected in 25.5% (51/200) strains, (μg/mg) with no significant differences (P > 0.05) over the studied S R Colistin 731.93 ≤2 > 2 CLSI M100; human [5] years. Thirty strains (15%) presented intermediate Gentamicin 614 ≤2 ≥8 CLSI VET08; dog, horse [6] susceptibility to gentamicin. Among these, 36.67% (11/ a 30) had a resistant MIC when tested by microdilution. Antibiotic powder: Sigma®. bCLSI veterinary breakpoints were preferably used. If not available, CLSI human breakpoints were used Of the eight strains with a sensitive result to gentamicin by disk diffusion but close to the breakpoint (16 mm), associations between mcr genes and MIC values were five presented resistant MIC values. The qualitative assessed by Odds-ratio tests. results of resistance to colistin did not show significant changes throughout the period (P > 0.05), with only 4.67% Results of resistant strains. In contrast, the MIC procedure dem- The disk diffusion tests demonstrated that 110 of 200 onstrated significant differences among the studied years strains (55%) were multidrug-resistant (MDR), namely (P < 0.02). Whereas there was no colistin resistance in resistant to three or more antibiotics. Because of the low neither of the three first periods, an increase was observed number of samples per year, strains were grouped in 4- in P4 (P < 0.05), with 17.5% (7/40) of strains resistant to year periods from 1999 to 2014 (P1 = 1999, 2000, 2001 colistin. However, the percentage in 2017 and 2018 and 2002, P2 = 2003, 2004, 2005 and 2006, P3 = 2007, declined significantly (P < 0.02) to 7.5% (3/40) (Fig. 2). 2008, 2009 and 2010, P4 = 2011, 2012, 2013 and 2014) The frequencies of the AMR genes are shown in with 40 strains per period, and a last group of strains Table 3. Only the ESBL gene blaCTX-M, was detected, in from 2017 and 2018 (P5), of 40 samples. As shown in 13.5% (27/200) strains, which showed a slight a tendency Fig. 1, no significant changes in the MDR percentages to increase throughout the studied period (P < 0.05). Out were observed over time (P > 0.05). However, the of the 27 strains, the sequences of 18 strains could be percentage of MDR was significantly higher in P5 (65%) obtained (Table 3). The most prevalent was blaCTX-M-14, than in P1 and P2 (47.5%) (P < 0.02). with 33.33% (6/18), followed by blaCTX-M-1 with 27.78%, Amoxicillin resistance was observed in 98.5% (197/ blaCTX-M-32 with 16.67% (3/18) and blaCTX-M-3 and 200) of the strains. There were no significant differences blaCTX-M-9 both in 5.56% (1/18) of the strains. No in amoxicillin resistance among studied periods (P > significant trend patterns were described for any of the 0.05), as can be seen in Fig. 2. Similarly, 94.5% (189/200) other AMR genes. blaCMY-2 was detected in 3% (6/200) strains were resistant to tetracycline, with no significant of strains. The mcr-4 was the most frequent colistin re- changes between 1999 and 2018 (P > 0.05). Resistance to sistance gene, in 13% (26/200) strains. Both mcr-1 and ceftiofur was detected in 15.5% of the samples (31/200). mcr-5 were detected in 7% (14/200) and 3% (6/200) As can be observed in Fig. 2, resistance to this antibiotic strains respectively. Out of the 43 strains positive to at was significantly higher in P5 than in the earlier periods least one mcr gene, forty (93.02%) had a sensitive MIC (P < 0.02). Enrofloxacin showed a level of resistance of value (0.5–2 μg/mL), and seven (4.46%) of the 157 strains negative to mcr genes presented a resistant MIC. Therefore, no significant association was found between mcr-positivity and MIC results (P > 0.05). The MIC dis- tribution by mcr PCR results can be observed in Table 4. blaTEM, blaSHV, blaCMY-1, blaOXA, mcr-2 and mcr-3 genes were not detected in any of the studied strains. Discussion Fig. 1 Minimum, maximum and mean percentage of MDR during In this study, MDR E. coli strains were found in a high the studied years, grouped in five periods (P1 = 1999–2002, P2 = proportion of porcine diarrheic cases from Spanish 2003–2006, P3 = 2007–2010, P4 = 2011–2014, P5 = 2017 and 2018) farms since 1999. In addition, an increase of these MDR according to the results obtained by disk diffusion with the frequencies was observed in 2017 and 2018, being exception of colistin, for which the results of the MIC were considered. Strains with intermediate susceptibility to gentamicin by amoxicillin and tetracycline the antimicrobials with the disk diffusion but resistant MIC were considered resistant. The line highest levels of resistance (98.5 and 94.5% respectively). represents the interval of values in the defined period from the Similar resistance levels to these antibiotics have been minimum to the maximum percentage, and the mean (number of previously described in enterobacteria from pig farms of MDR strains in the period divided by the total of strains of the Catalonia in 2002 [9, 10]. According to the EMA [11], period) is represented by the triangle tetracycline was the most sold antibiotic family for Aguirre et al. Porcine Health Management (2020) 6:8 Page 4 of 6 Fig. 2 Percentages of resistance to each of the 6 antibiotics in the five defined periods (P1 = 1999–2002, P2 = 2003–2006, P3 = 2007–2010, P4 = 2011–2014, P5 = 2017 and 2018) according to the results obtained by disk diffusion with the exception of colistin, for which the results of the MIC were considered. Strains with intermediate susceptibility to gentamicin by disk diffusion but resistant MIC were considered resistant Table 3 Number of E. coli isolates positive to the different AMR genes and blaCTX-M genotype sequencing results by year of isolation Year (strains per year) mcr-1 mcr-4 mcr-5 blaCMY-2 blaCTX-M blaCTX-M genotype 2000 (10) 2 2001 (11) 2 3 2 1 2002 (9) 1 1 2 2003 (10) 5 1 2004 (9) 3 1 2005 (11) 3 2 2 CTX-M-14 (1) CTX-M-32 (1) 2006 (10) 1 2007 (10) 2 CTX-M-14 (1) 2008 (10) 2 3 2009 (8) 1 1 2 CTX-M-1 (1) CTX-M-14 (1) 2010 (12) 1 4 CTX-M-9 (1) CTX-M-32 (1) 2011 (16) 2 2 1 2 CTX-M-1 (2) 2012 (13) 2 2 2013 (6) 1 2014 (5) 3 2017 (34) 3 1 9 CTX-M-1 (2) CTX-M-14 (3) CTX-M-32 (1) CTX-M-3 (1) CTX-M-27 (2) 2018 (6) 1 2 Total (%) 14 (7) 26 (13) 6 (3) 6 (3) 27 (13.5) Aguirre et al. Porcine Health Management (2020) 6:8 Page 5 of 6 Table 4 Number of strains by MIC colistin values and mcr PCR use of colistin in swine from 2015 to 2018 in Spain re- results ported by the AEMPS [16]. Colistin MIC (μg/mL) mcr-1 mcr-4 mcr-5 mcr absencea In accordance with previous studies in humans and Resistant >8 1 2 0 4 pigs [17], blaCTX-M was the most frequent ESBL gene 4 0 0 0 3 in our study, with upward trend throughout the years. On the other hand, blaCMY-2 and the six blaCTX-M Sensitive 2 2 2 0 44 genotypes detected in this study (CTX-M-1, CTX-M-3, <2 11 20 6 83 CTX-M-9, CTX-M-14, CTX-M-27 and CTX-M-32) had mcr-1, mcr-2, mcr-3 mcr-4 and mcr-5 were tested a been already been reported in E. coli isolates from pigs [12, 18]. Since their first report by Liu et al. [19], mcr genes in animal production in Spain from 2010 to 2015 and is E. coli strains have been worldwide described in both still highly used in pigs and other species. Similarly, humans and livestock. It has been speculated that colis- penicillins proved to be the second most consumed anti- tin use in animal production could be the original source biotic family in the same report, which would explain of these genes [20]. Although mcr-4 was firstly described the high levels of resistance to amoxicillin in the studied in 2017 by Carattoli et al. [21], in the present study mcr- strains. 4 (13%,) was already detected in E. coli strains isolated in By contrast, resistance to ceftiofur was considerably 2001. Also, mcr-1 (7%,) was the second most prevalent lower (15.5%), as third and fourth generation cephalo- colistin resistance gene, with the first positive strains sporins are less used in livestock [11]. However, the detected in 2005. Finally, mcr-5, which was firstly de- ESBL increase described during the last decades [12] scribed in 2017 by Borowiak et al. [22], was identified in would account for the significant difference (P < 0.02) in four strains from 2001 and 2002, but it was not detected ceftiofur resistance between P5 and the earlier periods. afterwards. The existence of strains positive to a mcr On the other hand, enrofloxacin presented a level of re- gene but with a sensitive MIC value suggests that, sistance around 40% that has remained practically stable although they do not express it, strains with a “sensitive” in these past twenty years. Although this fluoroquinolone phenotype could contain a mcr gene and might end up is exclusively for veterinary use, previous studies have expressing it. Conversely, the seven strains found pheno- demonstrated that enrofloxacin treatments in pigs in- typically resistant to colistin and negative for the mcr creases Campylobacter coli resistance to other fluoroqui- genes analysed, might suggest the presence of other mcr nolones such as ciprofloxacin [13], broadly used in genes such as mcr-6 and mcr-8, originally described in humans. Out of the 28 strains resistant to ceftiofur, 16 pigs [23, 24] or other genes such as pmrA that can con- (56.14%) presented resistance to amoxicillin-clavulanic, fer intrinsic resistance to colistin in E. coli [25] and have which has been described as an indicator of the presence not been included in the study. Besides, colistin resist- of AmpC, although not highly specific [14]. ance could occur via chromosomal mutations, although Resistance to gentamicin in swine E. coli has been less this mechanism is often unstable [19]. reported than to other aminoglycosides like strepto- mycin [9, 15]. In this study, gentamicin resistance was detected in 25.5% (51/200) strains, with no significant Conclusions differences (P > 0.05) over the studied years. The low but E. coli strains containing blaCTX-M and mcr-4 and mcr-1 considerable percentage of intermediate strains (15%) genes have been the most frequently detected in pig farms, and the fact that 36.67% (11/30) of these presented a since at least 20 years ago in the case of mcr-4. However, resistant MIC suggests that disk diffusion results for the poor concordance between the genotypic mcr profile testing gentamicin susceptibility should be interpreted and the MIC phenotypical testing observed in this study with caution. deserves further investigation. Finally, it is concerning the Colistin results from the qualitative MIC method were high levels of AMR for tetracycline and amoxicillin and considered for the analysis, as the disk diffusion method the increase of cephalosporin and colistin resistance has been described as not reliable when testing poly- during these two decades. Thus, antimicrobial surveillance myxins susceptibility [5], and the results in the present should be maintained, and research strengthened in new study reinforce this fact. From 2013 to 2015, Spain was approaches to control of bacterial infections in pigs by the European country with the highest sales of poly- reducing antibiotic consumption. myxins for food-producing animals [11], which might explain the significant increase in colistin resistance in Abbreviations P4. The following significant decrease in colistin resist- AMR: Antimicrobial resistance; ESBL: Extended-spectrum beta-lactamases; ance in P5 might be related to the 97% reduction in the MDR: Multidrug-resistant; MIC: Minimal inhibitory concentration Aguirre et al. Porcine Health Management (2020) 6:8 Page 6 of 6 Authors’ contributions 12. Carattoli A. Animal reservoirs for extended spectrum β-lactamase producers. LA and MM initiated and planned the work. LA, AV and CS selected the Clin Microbiol Infect European Soc Clin Microbiol Infect Dis. 2008;14:117–23. samples, performed experiments and analysed data. MT and NR assisted for 13. Delsol AA, Sunderland J, Woodward MJ, Pumbwe L, Piddock LJV, Roe JM. experiments, media preparation and laboratory equipment. MM and LD Emergence of fluoroquinolone resistance in the native Campylobacter coli supervised the work. LA, MM and LD discussed the results and drafted the population of pigs exposed to enrofloxacin. J Antimicrob Chemother. manuscript. 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