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Antimicrobial Resistance in Campylobacter jejuni Isolated from Brazilian Poultry Slaughterhouses

Keywords:
Antimicrobial resistance; Campylobacter jejuni; gyrA; mutation; poultry; tetO

INTRODUCTION

Campylobacteriosis is one of the most common foodborne diseases in the world. It is considered the most frequently reported foodborne illness in the European Union (EU) and one of the most important in the United States (US) (EFSA & ECDC, 2018; CDC, 2019a; WHO, 2019). Poultry is known to be the major reservoir and an important source for pathogen transmission to humans (Kaakoush et al., 2015Kaakoush NO, Castaño-Rodríguez N, Mitchell HM, Man SM. Global epidemiology of Campylobacter infection. Clinical Microbiological Reviews 2015;28(3):687-720.). Campylobacteriosis is most often associated with the consumption of raw and undercooked poultry or the cross-contamination of other foods by these items (CDC, 2019a). Although Brazil is a leading supplier of the world’s poultry meat (ABPA, 2018), Brazil’s official data does not report Campylobacter infections.

Resistance in foodborne pathogens presents the potential for their transmission to humans through the food chain (Wang et al., 2013Wang H, Ye K, Wei X, Cao J, Xu X, Zhou G. Occurrence, antimicrobial resistance and biofilm formation of Salmonella isolates from a chicken slaughter plant in China. Food Control 2013;33(2):378-384.). Campylobacteriosis is generally a self-limiting disease. However, in some patients, Campylobacter infection can result in a systemic disease requiring the use of antimicrobials (CDC, 2019b). Erythromycin is considered the first-line treatment, but fluoroquinolones are also frequently used due to their broad-spectrum activity against enteric pathogens (Engberg et al., 2001Engberg J, Aarestrup F, Taylor D, Gerner-Smidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerging Infectious Diseases 2001;7(1):24-34.). Recently, however, multidrug-resistant Campylobacter strains have been detected in poultry and several other sources around the world (Szczepanska et al., 2017Szczepanska B, Andrzejewska M, Spica D, Klawe JJ. Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from children and environmental sources in urban and suburban areas. BMC Microbiology 2017;17(80):1-9.; Du et al., 2018Du Y, Wang C, Ye Y, Liu Y, Wang A, Li Y, et al. Molecular identification of multidrug-resistant Campylobacter species from diarrheal patients and poultry meat in Shanghai, China. Frontiers in Microbiology 2018;9:1-8.; Montgomery et al., 2018Montgomery MP, Robertson S, Koski L, Salehi E, Stevenson LM, Silver R, et al. Multidrug-resistant Campylobacter jejuni outbreak linked to puppy exposure- United States, 2016-2018. Morbidity and Mortality Weekly Report 2018;67(37):1032-1035.).

In the EU, Campylobacter isolated from human and poultry sources have shown high to extremely high resistance to ciprofloxacin and tetracycline (EFSA & ECDC 2018), and both substances have been widely used in Brazilian poultry production in recent decades (Machinski Júnior et al., 2005Machinski Júnior M, Benini A, Netto DP, Nunes MP, Vedovello Filho D, Benatto A, et al. Medicamentos veterinários utilizados na avicultura de postura no Estado do Paraná. Curitiba: PAMvet / PR; 2005. p.1-24.). Ciprofloxacin resistance in Campylobacter strains is usually related to the Tre-86-Ile mutation in the quinolone resistance-determining region (QRDR) of the gyrA gene, which results in the replacement of the amino acid threonine by isoleucine (Frasao et al., 2015Frasao BS, Medeiros V, Barbosa AV, Aguiar WS, Santos FF, Abreu DLC, et al. Detection of fluoroquinolone resistance by mutation in gyrA gene of Campylobacter spp. isolates from broiler and laying (Gallus gallus domesticus) hens, from Rio de Janeiro State, Brazil. Ciência Rural 2015;45(11):2013-2018.a). Resistance to tetracycline is usually related to the presence of the tetO gene (Pratt & Korolik, 2005Pratt A, Korolik V. Tetracycline resistance of Australian Campylobacter jejuni and Campylobacter coli isolates. Journal of Antimicrobial Chemotherapy 2005;55:452-460.). Our aim was to assess the minimum inhibitory concentrations (MICs) for Campylobacter jejuni strains and determine their molecular resistance profiles to tetracycline and ciprofloxacin.

MATERIALS AND METHODS

Bacterial strains and growth conditions

A total of 54 C. jejuni strains were selected for this study (Table 1). Isolates were obtained from broiler carcass samples collected between 2011 and 2012 from different Brazilian poultry slaughterhouses according to criteria described by the International Organization for Standardization (ISO 10272-1:2017). The bacterial isolates were stored at −80 °C in ultra-high temperature-processed milk and were reactivated on blood base agar (Oxoid, Hampshire UK) supplemented with 5% defibrinated sheep blood. The plates were incubated within a jar at 42 ± 1 °C under microaerobic conditions.

Table 1
Campylobacter jejuni strains: identification, source of isolation, phenotypic resistance profiles and molecular resistance profile.

Phenotypic characterization of antimicrobial resistance

As described by the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2013b), a broth microdilution test was performed to determine the MIC for six clinically relevant antibiotics (Sigma-Aldrich): chloramphenicol (0.25-128 mg/L), ciprofloxacin (0.007-16 mg/L), erythromycin (0.064-128 mg/L), gentamicin (0.064-32 mg/L), nalidixic acid (1-256 mg/L), and tetracycline (0.064-64 mg/L). The strains were classified as susceptible or non-susceptible (including intermediate strains) according to the breakpoints described in the CLSI standards (CLSI, 2013a; El-Adawy et al., 2015El-Adawy H, Ahmed MFE, Hotzel H, Tomaso H, Tenhagen BA, Hartung J, et al. Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli recovered from organic turkey farms in Germany. Poultry Science 2015;64(11):2831-2837.). The strains were also classified as wild type or non-wild type (nWT) based on their epidemiological MIC cut-off (ECOFFs), which were determined according to the EUCAST guidelines available at the time of data analysis (January, 2019) (EUCAST, 2019). A C. jejuni reference strain (ATCC 33560) was selected to ensure the validity of the tests. Strains that were resistant to three or more classes of antimicrobials were classified as multidrug resistant (MDR) strains (Schwarz et al., 2010Schwarz S, Silley P, Simjee S, Woodford N, Van Duijkeren E, Johnson AP, et al. Editorial: assessing the antimicrobial susceptibility of bacteria obtained from animals. Journal of Antimicrobial Chemotherapy 2010;65:601-604.). The multiple antibiotic resistance (MAR) index was determined as previously described (Krumperman, 1983Krumperman PH. Multiple antibiotic resistance indexing of Escherichia coli to identify high-risk sources of fecal contamination of foodst. Applied and Environmental Microbiology 1983;46(1):165-170.).

Molecular characterization of antimicrobial resistance

Thermal extraction of DNA was performed as described (Sambrook & Russel, 2012Sambrook J, Russel DW. Molecular cloning: a laboratory manual. 4th ed. New York: Cold Spring Harbor Laboratory Press; 2012.). The strains with tetracycline MICs ≥2 mg/L were selected for PCR detection of the tetO gene. The primers were designed by Bacon et al. (2000). All PCR reactions (25 µL) contained 10X PCR buffer, 2.5 mM dNTPs, 10 pmol primer, 2 mM MgCl2, 5 U Taq DNA polymerase, and 2 µL template DNA. The cycling program consisted of 30 cycles of 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min. The amplified products (559 bp) were separated by electrophoresis in a 1% agarose gel stained with ethidium bromide, which was photographed under UV illumination. A total of 31 strains with ciprofloxacin MICs ≥4 mg/L were selected to characterize their molecular resistance. First, the QRDR in the gyrA gene was detected by PCR with primers designed by Price et al. (2005Price LB, Johnson E, Vailes R, Silbergeld E. Fluoroquinolone-resistant Campylobacter isolates from conventional and antibiotic-free chicken products. Environmental Health Perspectives 2005;113:557-60.). All PCR reactions (25 µL) contained 10X PCR buffer, 1 mM dNTPs, 10 pmol primer, 2 mM MgCl2, 1 U Taq DNA polymerase, and 5 µL template DNA. The cycling program consisted of 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The amplified products (454 bp) were separated by electrophoresis in a 1% agarose gel, stained with ethidium bromide, and photographed under UV illumination. All reactions were repeated three times. A PCR control containing the PCR mixture without the addition of the template DNA was included with all reactions.

The amplified products of gyrA were then sequenced in triplicate in an automated sequencer (ABI-PRISM 3500® Genetic Analyzer; Applied Biosystems) with 50 cm capillaries and polymer POP7 (Applied Biosystems). The sequences obtained in the chromatograms were processed using Chromas Lite (Technelysium) and Geneious (Biomatters) software. To confirm the presence of the mutation, the sequence of strain C. jejuni (L04566.1), obtained from GenBank, was used as a ciprofloxacin-sensitive strain standard.

Statistical analysis

The data were subjected to a descriptive statistical analysis using PASW Statistics software. The kappa index (Landis & Koch, 1977Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33(1):159-174.) was determined to evaluate the concordance between the classifications based on the CLSI breakpoints and ECOFF values.

RESULTS

The phenotypic antimicrobial resistance profiles and MIC results are described in Tables 1 and 2. Only one strain was susceptible to all substances and all strains were clinically susceptible to gentamicin and chloramphenicol, regardless of the breakpoint (CLSI or EUCAST) evaluated. Resistance for tetracycline and erythromycin was higher when EUCAST parameters were applied. 46.3% (25/54) of the strains were classified as non-susceptible and 51.8% (28/54) as nWT for tetracycline, and 42.6% (23/54) of the strains were classified as non-susceptible and 48.1% (26/54) as nWT for erythromycin. Resistance to ciprofloxacin was equal for both parameters, and 94.4% (51/54) of the strains were classified as non-susceptible or nWT. Regarding resistance for nalidixic acid, 94.4% (51/54) of the strains were non-susceptible according to the CLSI breakpoints and 83.3% (45/54) were nWT according to EUCAST breakpoint. CLSI also classifies the strains as “intermediate”, which were considered as non-susceptible in the present study (Borges et al., 2019Borges KA, Furian TQ, de Souza SN, Salle CTP, Moraes HL, Nascimento VP. Antimicrobial resistance and molecular characterization of Salmonella enterica serotypes isolated from poultry sources in Brazil. Brazilian Journal of Poultry Science 2019;21(1):1-8.) due to their uncertain therapeutic effect in vivo (CLSI, 2013b).

Table 2
Minimum inhibitory concentration (MIC) results: non susceptible strains (CLSI breakpoints) and non-wildtype (ECOFF values).

The molecular antimicrobial resistance profiles are described in Table 1. Only 42.8% (12/28) of tetracycline non-susceptible strains presented the gene tetO. All strains tested for the presence of mutations in the QRDR fragment of the gyrA gene showed a threonine to isoleucine (Thr-86-Ile) mutation at codon 86 and 16,1% (5/31) of them presented a second mutation at codon 149 (Val-149-Ile). The silent mutations His-85-His, Ser-119-Ser, Ala-120-Ala, and Val-161-Val were observed in all the analyzed strains, while 22.6% (7/31) and 12.9% (4/31) also contained the silent mutations Pro-186-Pro and Gly-110-Gly, respectively.

DISCUSSION

Antimicrobial resistance is a complex challenge and a major problem for global public health. Each year, about 25,000 patients in the EU and 23,000 in the US die from infections caused by multiresistant bacteria, with annual treatment costs of approximately 1.5 billion euros and 20 billion dollars, respectively (WHO, 2014). The Brazilian government does not have an integrated program for monitoring antimicrobial resistance in the primary human and production animal pathogens such as Salmonella spp. and C. jejuni, making the adoption of new measures to control and restrict the use of antimicrobials difficult (Borges et al., 2019Borges KA, Furian TQ, de Souza SN, Salle CTP, Moraes HL, Nascimento VP. Antimicrobial resistance and molecular characterization of Salmonella enterica serotypes isolated from poultry sources in Brazil. Brazilian Journal of Poultry Science 2019;21(1):1-8.). In addition, unlike European countries, Brazil has no specific legislation mandating the analysis of campylobacteriosis. Therefore, studies addressing antimicrobial resistance are essential for characterizing the circulating C. jejuni strains in the Brazilian poultry production chain.

Although similar, the results based on the ECOFF values showed a great number of nWT strains (non-susceptible). Considering that MIC determinations depend on breakpoints and that MIC results affect clini-cal decisions and official data reports (Kassim et al., 2016Kassim A, Omuse G, Premji Z, Revathi G. Comparison of Clinical Laboratory Standards Institute and European Committee on antimicrobial susceptibility testing guidelines for the interpretation of antibiotic susceptibility at a University teaching hospital in Nairobi, Kenya: A cross-sectional stud. Annals of Clinical Microbiology and Antimicrobials 2016;15(1):1-7.), changes in the breakpoint parameters can result in significant changes in the final MIC. The breakpoints are set by three international agencies: the U.S. Food and Drug Administration Center for Drug Evaluation and Research (USDA-CDER), the CLSI, and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (Humphries et al., 2019Humphries RM, Abbott AN, Hindler JA. Understanding and addressing CLSI breakpoint revisions: a primer for clinical laboratories. Journal of Clinical Microbiology 2019;57(6):1-15.). The guidelines proposed by CLSI are some of the most used worldwide and are based on the drugs’ properties and mechanisms of resistance (Kassim et al., 2016), whereas EUCAST bases its breakpoints on the drugs’ properties and ECOFFs (EUCAST, 2019). We compared the results for both interpretative criteria through kappa analysis. It showed perfect agreement for ciprofloxacin, gentamicin and chloramphenicol, almost perfect agreement for tetracycline (κ = 0.889) and erythromycin (κ = 0.888) and fair agreement for nalidixic acid (κ = 0.400). Comparisons among studies is challenging due to the wide variation in interpretative techniques and parameters. The agreement seen between the EUCAST and CLSI guidelines not only provides important information about antimicrobial susceptibility, it indicates that international data on C. jejuni resistance could be compared, thus allowing the establishment of more specific control measures for this species in the poultry production chain.

The use of chloramphenicol in production animals has been banned in Brazil since 2003 (MAPA, 2003) and the use of gentamicin in poultry production is restricted (Giacomelli et al., 2014Giacomelli M, Salata C, Martini M, Montesissa C, Piccirillo A. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli from poultry in Italy. Microbial Drug Resistance 2014;20(2):181-188.). These are probably the reasons for the absence of non-susceptible strains in our study.

Our results indicate that almost 50% of the strains were resistant to erythromycin, which is higher than the previously published data (Bolinger & Kathariou, 2017Bolinger H, Kathariou S. The current state of macrolide resistance in Campylobacter spp.: trends and impacts of resistance mechanisms. Applied and Environmental Microbiology 2017;83(12):1-9.; Szczepanska et al., 2017Szczepanska B, Andrzejewska M, Spica D, Klawe JJ. Prevalence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from children and environmental sources in urban and suburban areas. BMC Microbiology 2017;17(80):1-9.). These results are a public health concern, because this agent is the treatment of choice for Campylobacteriosis (Engberg et al., 2001Engberg J, Aarestrup F, Taylor D, Gerner-Smidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerging Infectious Diseases 2001;7(1):24-34.). These high rates may be associated with the wide use of this drug in animal production up until 2012, when erythromycin was banned as a food additive in Brazil (MAPA, 2012). Higher erythromycin resistance rates can also be related to the several mechanisms by which Campylobacter acquires resistance to these antimicrobial agents (Bolinger & Kathariou, 2017).

We also observed a high level of resistance to tetracycline. Tetracycline resistance in Campylobacter has been previously reported in strains isolated from animal products (Abdi-Hachesoo et al., 2014Abdi-Hachesoo B, Khoshbakht R, Sharifiyazdi H, Tabatabaei M, Hosseinzadeh S, Asasi K. Tetracycline resistance genes in Campylobacter jejuni and C. coli isolated from poultry carcasses. Jundishapur Journal of Microbiology 2014;7(9):1-5.; Giacomelli et al., 2014Giacomelli M, Salata C, Martini M, Montesissa C, Piccirillo A. Antimicrobial resistance of Campylobacter jejuni and Campylobacter coli from poultry in Italy. Microbial Drug Resistance 2014;20(2):181-188.; Hungaro et al., 2015Hungaro HM, Mendonça RCS, Rosa VO, Badaró ACL, Moreira MAS, Chaves JBP. Low contamination of Campylobacter spp. on chicken carcasses in Minas Gerais state, Brazil: Molecular characterization and antimicrobial resistance. Food Control 2015;51:15-22.; Sierra-Arguello et al., 2015Sierra-Arguello YM, Morgan RB, Perdoncini G, Lima LM, Gomes MJP, Nascimento VP. Resistance to β-lactam and tetracycline in Campylobacter spp. isolated from broiler slaughterhouses in southern Brazil. Pesquisa Veterinária Brasileira 2015;35(7):637-642.). Over the past decade, the tetracycline compound class has been used in farm animal production as a growth promoter and for the treatment of diseases. The high resistance levels suggest that the overuse of tetracycline may have selected resistant strains. The majority of tetracycline resistance determinants confer increased resistance to the other compounds from the same class, though it is also possible that the use of oxitetracycline and doxycycline has also caused tetracycline resistance (Fairchild et al., 2005Fairchild AS, Smith JL, Idris U, Lu J, Sanchez S, Purvis LB, et al. Effects of orally administered tetracycline on the intestinal community structure of chickens and on tet determinant carriage by commensal bacteria and Campylobacter jejuni. Applied and Environmental Microbiology 2005;71(10):5865-5872.). A high level of tetracycline resistance in Campylobacter is usually associated with the presence of the tetO gene. This gene encodes the TetO protein, which protects ribosomes from the inhibitory effects of tetracycline (Connel et al., 2003Connel SR, Tracz DM, Nierhaus KH, Taylor DE. Ribosomal protection proteins and their mechanism of tetracycline resistance. Antimicrobial Agents and Chemotherapy 2003;47(12):3675-81.). A total of 28 isolates had tetracycline MICs ≥2 mg/L, and 42.8% of them carried the gene. Reports from Brazil have shown lower frequencies of this gene than in other countries (Sierra-Arguello et al., 2015; Du et al., 2018Du Y, Wang C, Ye Y, Liu Y, Wang A, Li Y, et al. Molecular identification of multidrug-resistant Campylobacter species from diarrheal patients and poultry meat in Shanghai, China. Frontiers in Microbiology 2018;9:1-8.). This gene can be present in conjugative plasmids containing resistance genes for other antimicrobials that continue to undergo selective pressure. The tetO gene can also be found as a chromosomal element. In this case, the stability of the chromosomal location ensures the gene replicates from generation to generation, even in the absence of the drug (Avrain et al., 2004Avrain L, Vernozy-Rozand C, Kempf I. Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens. Journal of Applied Microbiology 2004;97(1):134-140.; Crespo et al., 2012Crespo MD, Olson JW, Altermann E, Siletzky RM, Kathariou S. Chromosomal tet(O)-harboring regions in Campylobacter coli isolates from turkeys and swine. Applied and Environmental Microbiology 2012;78(23):8488-8491.).

Fluoroquinolones are considered the second-line treatment against Campylobacter infection in humans (Engberg et al., 2001Engberg J, Aarestrup F, Taylor D, Gerner-Smidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerging Infectious Diseases 2001;7(1):24-34.). Campylobacter strains showed high resistance to fluoroquinolones, with the CLSI breakpoints and ECOFF values indicating that 90.7% and 81.5% of the strains, respectively, were resistant to both ciprofloxacin and nalidixic acid. These high fluoroquinolone resistance rates have been previously described in Brazilian poultry sources (Sierra-Arguello et al., 2016Sierra-Arguello YM, Perdoncini G, Morgan RB, Salle CTP, Moraes HLS, Gomes MP, et al. Fluoroquinolone and macrolide resistance in Campylobacter jejuni isolated from broiler slaughterhouses in southern Brazil. Avian Pathology 2016;45(1):66-72.) and are likely due to the large use of this antimicrobial class in poultry production (Iovine & Blaser, 2004Iovine NM, Blaser MJ. Antibiotics in animal feed and spread of resistant Campylobacter from poultry to humans. Emerging Infectious Diseases 2004;10(6):1158-1189.). Although ciprofloxacin is more commonly used in humans, it is structurally related to enrofloxacin (WHO, 2011), which has been widely used in poultry production (Garcia-Migura et al., 2014Garcia-Migura L, Hendriksen RS, Fraile L, Aarestrup FM. Antimicrobial resistance of zoonotic and commensal bacteria in Europe: The missing link between consumption and resistance in veterinary medicine. Veterinary Microbiology 2014;170(1-2):1-9.), and previous studies have demonstrated that resistance for ciprofloxacin and enrofloxacin is developed through the same mechanisms (Frasao et al., 2015Frasao BS, Medeiros V, Barbosa AV, Aguiar WS, Santos FF, Abreu DLC, et al. Detection of fluoroquinolone resistance by mutation in gyrA gene of Campylobacter spp. isolates from broiler and laying (Gallus gallus domesticus) hens, from Rio de Janeiro State, Brazil. Ciência Rural 2015;45(11):2013-2018.b). Campylobacter resistance to fluoroquinolones is usually related to a mutation in the QRDR region of the gyrA gene (Frasao et al., 2015b). This gene codes for the ‘A’ subunit of the enzyme DNA gyrase and confers a decreased susceptibility to fluoroquinolones (Wilson et al., 2000Wilson DL, Abner SR, Newman TC, Mansfield LS, Linz JE. Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay. Journal of Clinical Microbiology 2000;38(11):3971-3978.). All strains tested for the presence of mutations in the QRDR fragment of the gyrA gene showed a threonine to isoleucine (Thr-86-Ile) mutation at codon 86 (Table 1). This result confirms that this substitution is always related to high fluoroquinolone MICs. A second mutation at codon 149 (Val-149-Ile) was observed in 19.3% of the strains. As these amino acids belong to the same class, the replacement may not lead to any significant conformational modifications of the protein. Consequently, its function probably remains unmodified (Taylor, 1986Taylor WR. The classification of amino acid conservation. Journal of Theoretical Biology 1986;119(2):205-218.). Other mutations associated with an intermediate level of resistance to quinolones such as Asp-90-Asn and Ala-70-Thr (Iovine, 2013) were not encountered in this study. Mutation in QRDR of gene gyrA is the main resistance mechanism to fluoroquinolones. However, chromosomal efflux pumps, especially those codified by cmeA, cmeB and cmeC genes, are important factors to antimicrobial in Campylobacter species (Wieczorek & Osek, 2013Wieczorek K, Osek J Antimicrobial resistance mechanisms among Campylobacter. BioMed Research International 2013;1-12.; Nascimento et al., 2019Nascimento RJ, Frasão BS, Dias TS, Nascimento ER, Tavares LSB, Almeida VL, et al. Detection of efflux pump CmeABC in enrofloxacin resistant Campylobacter spp. strains isolated from broiler chickens (Gallus gallus domesticus) in the state of Rio de Janeiro, Brazil. Pesquisa Veterinária Brasileira 2019;39:722-733.). Previous studies have demonstrated that almost all strains of Campylobacter jejuni isolated in Brazil presented these three genes (Nascimento et al., 2019).

Since 2000, several Latin American countries are part of the Pan American Health Organization Network for Monitoring/Surveillance of Antibiotic Resistance. However, very few of them are conducting surveillance for Campylobacter species. In this context, data of Campylobacter resistance are mainly published by academic research groups (Fernández & Pérez-Pérez, 2016). Available data shows that antimicrobial resistance in Campylobacter jejuni varies among Latin American countries. The higher rates are described for fluorquinolones in several countries besides Brazil, including Ecuador, Argentina and Peru (Pollett et al., 2012Pollett S, Rocha C, Zerpa R, Patiño L, Valencia A, Camiña M, et al. Campylobacter antimicrobial resistance in Peru: a ten-year observational study. BMC Infectious Diseases 2012;12:1-7.; Zbrum et al., 2015; Fernández & Pérez-Pérez, 2016; Vinueza-Burgos et al., 2017Vinueza-Burgos C, Wautier M, Martiny D, Cisneros M, Van Damme I, De Zutter L. Prevalence, antimicrobial resistance and genetic diversity of Campylobacter coli and Campylobacter jejuni in Ecuadorian broilers at slaughter age. Poultry Science 2017;96:2366-2374.). Resistance to aminoglycosides is usually lower among these countries (Zbrum et al., 2015; Vinueza-Burgos et al., 2017; Toledo et al., 2018Toledo Z, Simaluiza RJ, Fernández H. Occurrence and antimicrobial resistance of Campylobacter jejuni and Campylobacter coli isolated from domestic animals from Southern Ecuador. Ciência Rural 2018;48:1-5.). Resistance rates for erythromycin and tetracycline is variable according to the country (Pollett et al., 2012; Zbrum et al., 2015; Vinueza-Burgos et al., 2017).

The individual maximum and minimum multiple-antibiotic resistance (MAR) indices for the isolates were 0.7 and 0.2, respectively, with an average index of 0.5, regardless of the interpretative criteria used. According to Proroga et al. (2011Proroga YTR, Capuano F, Carullo MR, Tela I, Capparelli R, Barco L, et al. Antibiotic susceptibility pattern and multiple antibiotic resistances (MAR) calculation of extended spectrum β- lactamase (ESBL) producing Escherichia coli and Klebsiella species in Pakistan. African Journal of Biotechnology 2011;10(33):6325-6331.), the MAR index is a good risk assessment tool and can be applied to differentiate low- (MAR < 0.2) and high-risk (MAR > 0.2) regions where antibiotics are overused. Our results (overall MAR = 0.5) may indicate high antibiotic usage and high selective pressure in the poultry chain, but the practical significance of this finding may be lost, because antibiotic use is widespread in developing countries, including Brazil (Davis & Brown, 2016Davis R, Brown PD. Multiple antibiotic resistance index, fitness and virulence potential in respiratory Pseudomonas aeruginosa from Jamaica. Journal of Medical Microbiology 2016;65:261-271.).

Based on the CLSI results, 13% (7/54) of the strains were multidrug-resistant (MDR), whereas 16.7% (9/54) of the strains were classified as MDR using the ECOFF values. Emerging resistance to the antimicrobial agents of choice for treating Campylobacter infections is becoming a serious threat to public health (Du et al., 2018Du Y, Wang C, Ye Y, Liu Y, Wang A, Li Y, et al. Molecular identification of multidrug-resistant Campylobacter species from diarrheal patients and poultry meat in Shanghai, China. Frontiers in Microbiology 2018;9:1-8.). The frequency of MDR strains found in this study is similar to that in previous reports from Brazil (Sierra-Arguello et al., 2015Sierra-Arguello YM, Morgan RB, Perdoncini G, Lima LM, Gomes MJP, Nascimento VP. Resistance to β-lactam and tetracycline in Campylobacter spp. isolated from broiler slaughterhouses in southern Brazil. Pesquisa Veterinária Brasileira 2015;35(7):637-642.). Given such results, Brazilian authorities should consider establishing an integrated surveillance network for antibiotic resistance in Campylobacter.

Considering that poultry is the major source of human Campylobacter spp. infection and that antimicrobial-resistant strains can be easily transmitted to humans via the food chain, our results show the need for the implementation of prudent antimicrobial-use policies in the Brazilian food production chain.

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Publication Dates

  • Publication in this collection
    28 Oct 2020
  • Date of issue
    2020

History

  • Received
    27 Feb 2020
  • Accepted
    08 May 2020
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