Abstract
Aims:
The objectives of this study were to genotype a total of 48 Campylobacter jejuni and 39 Campylobacter coli strains isolated in Brazil from 1995 to 2016 by multilocus sequence typing (MLST) and to determine their resistance profile. The presence or points of mutation in the related resistance genes was verified.
Results:
By MLST, C. jejuni strains were typed into 36 STs and C. coli strains were typed into 27 STs. A total of 70.8% of C. jejuni and 35.9% of C. coli were resistant to at least one antimicrobial tested. The tet(O) gene was detected in 43.7% C. jejuni and in 12.8% C. coli. The ermB gene was not detected and one C. jejuni presented the mutation in the 23S rRNA gene. Besides, 58.3% C. jejuni presented the substitution T86I in the quinolone resistance-determining region of gyrA and 15.4% C. coli presented the substitution T38I. The cmeB gene was detected in 97.9% C. jejuni and in 97.4% C. coli.
Conclusion:
The presence of C. jejuni and C. coli resistant to some antimicrobial agents of clinical use is of public health concern. The presence of STs shared between Brazilian strains and isolates of different countries is of concern since it might suggest a possible spread of these shared types.
Introduction
C
The main risk factor for human campylobacteriosis is the consumption of contaminated food, such as poultry meat, raw milk, and water, as well as close contact with animals, including pets.4–8
Usually, campylobacteriosis is a self-limiting disease. However, antimicrobial treatment, preferably with macrolides or fluoroquinolones, is indicated in severe or prolonged cases or in immunocompromised patients. 9 The increased bacterial resistance to antibiotics is a matter of special concern, representing a significant public health problem. Campylobacter has developed resistance to several antimicrobial agents over the years, including macrolides, quinolones, and fluoroquinolones that have been used in human medicine.10,11 Specifically, resistance to erythromycin can be caused by the presence of the ermB gene and by a point of mutation in the domain V of the 23S rRNA gene.
Resistance to ciprofloxacin is mainly caused by mutations in the quinolone-resistance determining region (QRDR) of the gyrA gene and the presence of tet genes have conferred resistance to tetracyclines.12,13 The multidrug efflux system (CmeABC), which belongs to the Resistance Nodulation Division (RND), a family of transporters, is widely distributed in Campylobacter and confers intrinsic resistance to various antimicrobials, including fluoroquinolones and macrolides.14,15
Campylobacter spp. is a genetically diverse organism, and several typing methods have been used to successfully genotype strains of this genus.16,17 Multilocus sequence typing (MLST) is used to type microorganisms into arbitrary STs, which can be further grouped into clonal complexes (CCs) and has been successfully used for typing Campylobacter spp.16,18–21 This methodology analyzes the DNA sequence variations in a set of housekeeping genes and characterizes strains according to their sequencing profiles. Sequences differing at even a single nucleotide are assigned as different alleles and thus as different STs. The genotyping data are stored in an online database facilitating international comparisons.18,22
In Brazil, campylobacteriosis has been underdiagnosed and underreported; therefore, there is a paucity of studies about this pathogen in this country.23–30 Thus, due to the impact of Campylobacter on public health in the world concomitantly with the fact that Brazil is the largest exporter of chicken meat in the globe, studies that genotype and assess the antimicrobial resistance profiles of strains of this genus isolated from diverse origins in this country are of utmost importance.
The aims of this study were to genotype C. jejuni and C. coli strains isolated from clinical and nonclinical sources in Brazil by MLST and to assess its antimicrobial resistance profiles by the minimum inhibitory concentrations (MICs) against some important antimicrobials of clinical use and their molecular resistance mechanisms to better characterize strains of this important pathogen.
Material and Methods
Bacterial strains
A total of 48 C. jejuni and 39 C. coli strains were studied. The strains were isolated from humans (C. jejuni, n = 21; C. coli, n = 9), animals (C. jejuni, n = 16; C. coli, n = 11), food (C. jejuni, n = 9; C. coli, n = 9), and the environment (C. jejuni, n = 2; C. coli, n = 10) from some cities of Rio de Janeiro, Sao Paulo, Minas Gerais, and Rio Grande do Sul States in Brazil, between 1995 and 2016. Specifically, the strains isolated from monkeys were isolated from ones of the species, saimiri, rhesus, and cynomolgus, all from captive monkeys. Also, some strains were isolated from marmosets of wild life.
These strains were selected from the collections of the Campylobacter References Laboratories of the Oswaldo Cruz Institute of Rio de Janeiro (FIOCRUZ-RJ) and of the Adolfo Lutz Institute of Ribeirao Preto in Brazil (IAL-RP). They were systematically chosen to represent isolates from sporadic cases occurred during different years. Table 1 lists the strains studied.
Characteristics of the 48 Campylobacter jejuni and 39 Campylobacter coli Strains Studied Isolated from Clinical and Nonclinical Sources During a 20-Year Period in Brazil
STs in boldface indicate new STs.
CC, clonal complex; Chicken, nondiarrheal feces; Cip, ciprofloxacin; Dox, doxycycline; Ery, erythromycin; Human, diarrheal feces; MG, Minas Gerais; MIC, minimum inhibitory concentration; Monkey, nondiarrheal feces; ND, not detected; R, resistance; RJ, Rio de Janeiro; RS, Rio Grande do Sul; SP, São Paulo; ST, sequence typing; Tet, tetracycline; UA, unassigned to any CC.
It is important to mention that, the strains studied were chosen considering previous results of 111 C. jejuni and 63 C. coli strains typed by pulsed-field gel electrophoresis, sequencing of the small variable region of flaA gene and high-resolution melting analysis of CRISPR locus.26,27 Those methodologies provided information for selecting 48 C. jejuni and 39 C. coli strains that were the most genetically distinct as possible to be typed by MLST (Table 1).
Genus and species confirmation
The 48 C. jejuni and 39 C. coli strains were grown at 42°C overnight on BBL™ Columbia Agar Base (Becton Dickinson and Company, East Rutherford, NJ), supplemented with charcoal (Neon, Sao Paulo, SP, Brazil) and ferrous sulfate; sodium metabisulfite sodium pyruvate (FPB) (0.5% ferrous sulfate [Labsynth Ltda, Diadema, SP, Brazil], 0.5% sodium pyruvate [Vetec Ltda, Duque de Caxias, RJ, Brazil], and 0.5% sodium metabisulfite [Labsynth Ltda] diluted in sterile water) under microaerobic conditions (10% CO2, 5% O2, and 85% N2).
The growth of the strains was placed directly in Solution 1 (20% of sucrose, 50 mM of Tris HCl pH 8, 50 mM of ethylenediaminetetraacetic acid [EDTA]), and the genomic DNA of each strain was extracted as described by Campioni and Falcão. 31
The quantity of DNA was determined using a NanoDrop 1000 (Thermo Fisher Scientific, Walthan, MA), and its purity was estimated as described by Sambrook and Russel. 32 Specific regions of the 16S rRNA (857 bp), ceuE (462 bp) and mapA (589 bp) genes, were amplified by polymerase chain reaction (PCR) to molecularly confirm the genus and species C. coli and C. jejuni, respectively, as described by Denis et al. 33
Antimicrobial susceptibility testing
MICs were performed for the 48 C. jejuni and 39 C. coli strains listed in Table 1 using the agar dilution method as recommended by the Clinical Laboratory and Standards Institute M45Ed3. 34 The strains were grown as described in Genus and Species Confirmation section. The bacterial suspension was adjusted to match the 0.5 McFarland (Probac, Brazil) turbidity standard as recommended by the Clinical and Laboratory Standards Institute, 35 seeded in Mueller Hinton Broth supplemented with blood (bioMérieux, France) and then the Etest® (bioMérieux) of the antimicrobial agents, ciprofloxacin, doxycycline, tetracycline, and erythromycin were used. After inoculation, the plates were incubated at 42°C under microaerophilic atmosphere for 24 hours and then screened. The C. jejuni strain ATCC 33291 was included as quality control.
Resistance genes by PCR
The presence of tetracycline [tet(O)], erythromycin (ermB), and multidrug efflux pump (cmeB) resistance genes were searched in all the strains. The general PCR procedure was performed according to the method described in Falcão et al. 36 The primers used and the PCR conditions of each resistance genes searched were described previously in the following references: Obeng et al. 37 for tet(O) and cmeB, and Zhang et al. 38 for ermB. A template PCR without DNA was used as a negative control. The PCR products were analyzed by agarose gel electrophoresis and visualized by UV light after staining the gel with ethidium bromide (0.5 μg/mL). Representative positive amplicons generated by tet(O) and cmeB genes primers pairs were purified for sequencing with the PureLink Quick PCR Purification Kit (Life Technologies) according to the manufacturer's recommendations and it was sequenced using ABI 3500xL (Life Technologies). The DNA sequences were analyzed using the software ChromasPro and the BLAST program of the GenBank sequence database, and DNA of Cj 02 (C. jejuni) and CCAMP 394 (C. coli) strains were used as positive controls, for tet(O) and cmeB, respectively. For the ermB gene the Enterococcus faecalis strain, ATCC 700802/V583, was used as a positive control.
Detection of mutations in the 23S rRNA and QRDR of the gyrA
The point mutation in domain V of the 23S rRNA gene and mutations in QRDR of the gyrA gene were analyzed in all the strains resistant to erythromycin and ciprofloxacin, respectively. The primers used to sequence each gene searched were described previously by Zhang et al. 38 for domain V of the 23S rRNA gene and Lindmark et al. 39 for QRDR of the gyrA gene. The general PCR procedure was performed as described in the item 2.4. Afterward, the amplicons were purified for sequencing with the PureLink Quick PCR Purification Kit (Life Technologies) according to the manufacturer's recommendations. Automated DNA sequencing was performed with an ABI 3500xL (Life Technologies) and the DNA sequences were analyzed using the software ChromasPro.
Multilocus sequence typing
MLST was performed as described previously. 16 Briefly, fragments of seven housekeeping genes (aspartase A [aspA], glutamine synthetase [glnA], citrate synthase [gltA], serine hydroxymethyl transferase [glyA], phosphoglucomutase [pgm], transketolase [tkt], and ATP synthase alpha subunit [uncA]) were amplified by PCR, using the primers and reactions described by Dingle et al. 16 and Miller et al. 40 Sequencing was done using ABI 3500xL automated DNA analyzer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). The consensus sequence was queried against the Campylobacter database to give an allele number. The combination of the alleles of the seven housekeeping genes generated the ST. STs were assigned to CCs based on sharing four or more alleles with the defined ST founder. MLST alleles and CCs were assigned using the Campylobacter PubMLST database developed by Jolley and Maiden. 22 New alleles and STs were submitted to the database.
Results
Genus and species confirmation
All the 87 strains analyzed in this study were confirmed by PCR to belong to their species. The PCR performed for C. jejuni strains amplified DNA fragments of 857 and 589 bp that correspond to specific regions of the 16S rRNA and mapA genes for this species while for C. coli strains it was amplified DNA fragments of 857 and 462 bp that correspond to specific regions of the 16S rRNA and ceuE gene for C. coli.
Antimicrobial susceptibility
The antimicrobial resistance patterns of the 48 C. jejuni and 39 C. coli strains studied are summarized in Table 1. Among the 48 C. jejuni strains, 34 (70.8%) were resistant to at least one antimicrobial tested. The number of C. jejuni strains resistant to ciprofloxacin, doxycycline, tetracycline, and erythromycin was 28 (58.3%), 22 (45.8%), 22 (45.8%), and 3 (6.25%), respectively. Specifically, 15 C. jejuni strains isolated from humans (5), animals (7), and food (3) were resistant to ciprofloxacin, tetracycline, and doxycycline, simultaneously. Two C. jejuni strains isolated from humans were resistant to tetracycline, doxycycline, and erythromycin, simultaneously. One strain of this species isolated from food was resistant to all antimicrobial agents tested and this strain was considered multidrug resistant (Table 1).
Regarding the 39 C. coli strains, 14 strains (35.9%) were resistant to at least one antimicrobial agent. The number of C. coli strains resistant to ciprofloxacin, doxycycline, tetracycline, and erythromycin was 7 (18%), 8 (20.5%), 8 (20.5%), and 3 (7.7%), respectively. Five C. coli strains isolated from humans (1) and the environment (4) were resistant to tetracycline and doxycycline, simultaneously and 4 C. coli strains isolated from humans (3) and food (1) were resistant only to ciprofloxacin (Table 1). Two strains isolated from animal and food were resistant to ciprofloxacin, tetracycline, and doxycycline simultaneously; one strain isolated from human was resistant to erythromycin, tetracycline, and doxycycline simultaneously; one strain isolated from the environment was resistant to erythromycin and ciprofloxacin simultaneously. Finally, one strain isolated from food was resistant only to erythromycin (Table 1).
Resistance genes by PCR
The cmeB gene was detected in 47 (97.9%) C. jejuni strains and in 38 (97.4%) C. coli strains. Twenty-one (43.7%) C. jejuni strains and five (12.8%) C. coli strains carried the tet(O) gene. The ermB gene was not detected in any of the strains studied (Table 1).
Detection of mutations in the 23S rRNA and QRDR of the gyrA
One out of the three C. jejuni strains resistant to erythromycin presented the mutation A2075G in the domain V of the 23S rRNA gene. On the other way, no mutation points were found in the same gene for the two C. coli strains resistant to erythromycin. All the 28 C. jejuni strains resistant to ciprofloxacin showed the mutation T86I in the QRDR of the gyrA gene. Six out of seven C. coli strains resistant to ciprofloxacin presented a gyrA mutation that led to the substitution T38I. The strain CCAMP 761, also resistant to ciprofloxacin, did not show mutations in the studied region (Table 1).
Multilocus sequence typing
The MLST analysis showed 36 different STs among the 48 C. jejuni strains studied. Twelve STs (ST8741, ST8742, ST8743, ST8744, ST8745, ST8746, ST8747, ST8748, ST8749, ST8751, ST8752, and ST8753) have not been described before in the Campylobacter spp. MLST database until the writing of this article. The ST353 was the most prevalent with 7 strains isolated from humans (3) and food (4). The second most prevalent ST was the ST8741 with three strains being one strain isolated from human and two strains isolated from food. Thirty out of the 36 STs obtained in this study were represented by only one strain. Twenty-eight STs found belonged to a defined CC in the database and eight STs did not belong to any known CC at the time of this analysis. CC ST-353 and ST-354 were the most prevalent ones. Specifically CC ST-353 comprised 11 strains of STs 1522 (1 strain), ST8741 (3 strains), ST353 (5 strains), ST8743 (1 strain), and ST 8752 (1 strain). The CC ST-354 comprised five strains of ST3852, ST6257, ST8747, ST1723, and ST354 (Table 1).
Regarding C. coli, 27 different STs were obtained from the 39 C. coli studied. Only six STs (ST830, ST1096, ST1145, ST1166, ST1581, and ST7370) were previously described in the Campylobacter spp. MLST database, being the ST1581 the most common one found for four strains (10%) studied. Moreover, other 5 out of the 27 STs (ST7713, ST7716, ST7717, ST7718, and ST7719) found were classified as singletons, with no phylogenetic relationship among these strains and any other strain available in the database (Table 1). Twenty-one STs (77.8%) designated ST7628, ST7713, ST7714, ST7715, ST7716, ST7717, ST7718, ST7719, ST7720, ST7721, ST7722, ST7723, ST7724, ST7725, ST7727, ST8155, ST8156, ST8157, ST8158, ST8159, and ST8161 have not been described before and were exclusively composed by strains of this study. Among those 21 STs, 7 were represented by only 1 strain. The ST7628 and ST7720 were represented each one by two strains of this study, being one from human and one from food sources. The ST7724 was represented by two strains from animal sources and the ST8157 was represented by two strains isolated from environmental sources. Thirteen STs comprising a total of 19 strains studied belonged to the 828-CC, which is the main CC of C. coli species (Table 1).
In addition, all the strains studied were deposited in PubMLST database and the submissions ids are: BIGSdb_20180320002057_111341_33216 and BIGSdb_201803242019223_75819_31026.
Discussion
MLST has been an effective tool for genotyping Campylobacter strains from a wide range of sources and geographical locations. Moreover, this methodology has been allowing the study of the population structure and the evolution of this organism.16,40–46
This study assessed the genetic diversity, the antimicrobial resistance patterns, and the molecular resistance mechanisms of C. jejuni and C. coli strains isolated from humans, animals, food, and the environment over a 20-year period in Brazil.
Regarding the 48 C. jejuni strains studied, 34 strains (70.8%) were resistant to at least one antimicrobial tested and the number of strains resistant to ciprofloxacin, doxycycline, tetracycline, and erythromycin were 28 (58.3%), 22 (45.8%), 22 (45.8%), and 3 (6.25%), respectively (Table 1). It is interesting to mention that no difference regarding the resistance pattern was observed between the oldest and the newest C. jejuni strains. Some studies performed in other countries corroborated with the results obtained in the present one and showed a prevalent resistance to ciprofloxacin and tetracyclines.42,47–50 Duarte et al. 42 studied 89 C. jejuni strains isolated from humans feces, chicken feces, and chicken meat in Portugal and these authors observed that 82 (92%), 59 (66%), and 6 (6.7%) strains were resistant to ciprofloxacin, tetracycline, and erythromycin, respectively. According to Gblossi et al., 47 15 (38.5%) strains were resistant to ciprofloxacin and 7 (18%) strains were resistant to erythromycin among 39 C. jejuni strains isolated from chicken in Côte d'Ivoire. A study performed in Brazil showed that 94% and 2% of C. jejuni strains isolated from chicken (cloacal swab, carcasses, and chiller tank processing water) in Rio Grande do Sul State were resistant to ciprofloxacin and erythromycin, respectively. 48
Interestingly, a relatively low proportion of antimicrobial resistance (37.5%) was found in the 39 C. coli strains in this study (Table 1), which was different from data published by Duarte et al. in 201442 that observed a rate of resistance to ciprofloxacin and tetracycline remarkably high (90.7%), irrespective of the source of isolation, in 107 C. coli strains isolated in Portugal between 2009 and 2012. Likewise, a study performed in Italy showed high levels of resistance among the 145 Campylobacter spp. strains studied and the antibiotics resistance was significantly more frequent for 54 C. coli strains when compared with 91 C. jejuni strains studied. 10 A recent study with C. coli and C. jejuni published by Thomrongsuwannakij et al. in 2017 51 showed that isolates from commercial broiler production chains in Thailand presented high levels of resistance, which included isolates with resistance to three or more antimicrobial classes. Similarly in Spain, 96.9% of the Campylobacter spp. strains isolated from broilers were resistant to ciprofloxacin and 90.6% to tetracycline in 2012. 52 In a study performed by Wieczorek et al. 13 in Poland, 68% of the 47 C. coli strains were resistant to one or more antimicrobial agents. In Tanzania, 66 (95.6%) out of the 69 C. coli strains were resistant to one or more antimicrobial agents. 53 A more detailed analysis of the data obtained in this present work regarding the 39 C. coli strains studied allowed us to observe that 6 (60%) out of 10 strains isolated from humans and 5 (50%) out of 10 strains isolated from the environment were resistant to at least one antimicrobial agent. These data are interesting compared with a lower proportion of resistant strains from animals that was 1 (9%) in 11 strains and from food that was 3 (33.3%) in 9 strains. Nevertheless, no resistance pattern correlation was observed among the C. coli strains isolated in different years (Table 1).
The better correlations between the phenotypic and genotypic antimicrobial resistance were observed for tetracycline and ciprofloxacin-resistant strains. Specifically, 95% of the C. jejuni and 50% of the C. coli tetracycline-resistant strains presented the tet(O) gene. Regarding the mutation in the QRDR of the gyrA gene, all the 28 C. jejuni ciprofloxacin-resistant strains presented the mutation T86I and six out of seven C. coli ciprofloxacin-resistant strains had the substitution T38I. The C. coli-designated CCAMP 761 was the only ciprofloxacin-resistant strain studied that did not present any mutation in the QRDR. However, this strain presented the cmeB gene (Table 1).
Otherwise, for some strains of C. coli and C. jejuni studied, there was no correlation between the presence of the gene or mutation and the phenotypic resistance profile obtained by Etest (Table 1). No obvious explanation for these results was found, and further investigations are required. The presence of the cmeB gene in the majority of the C. jejuni and C. coli strains suggests that the resistance might be mediated by the multidrug efflux system (CmeABC). 14 The mutation in the domain V of the 23S rRNA gene was observed in just one of the three C. jejuni erythromycin-resistant strains and in none of the C. coli erythromycin-resistant strains. A possible explanation for this fact is the existence of other points of mutation that play a role in the mechanism of resistance to erythromycin, such as, amino acid substitution in the L4 and L22 ribosomal proteins. 53
Thus, those findings regarding the resistance profiles obtained for C. jejuni and C. coli studied are somewhat alarming once the bacterial resistance is a global concern in any degree.3,5,10,20
To date, information of more than 72,475 C. jejuni/coli strains isolated worldwide have been deposited in the MLST database and 9,831 STs were designated demonstrating the high genetic diversity of this bacterial genus. This is the first study that used MLST to type C. jejuni and C. coli strains isolated from diverse sources in Brazil. The diversity of the strains studied was established by identification of various STs and according to our results, it was not observed as a predominant ST among the C. jejuni and C. coli strains studied in this present work (Table 1). In contrast, some researchers in United Kingdom and China have shown that there were dominant genotypes among the human C. jejuni population, such as ST21, ST48, ST257, and ST353.54,55 However, only ST21 and ST353 were found in the present study comprising one and seven strains, respectively. It is interesting to mention that it was not observed any predominance of some STs when the oldest and the newest C. jejuni strains studied were compared. Although, the C. jejuni strains herein studied belonged to different CC, the fact that these strains were structured in clusters of related isolates showed a possible phylogenetic relationship between these strains (Table 1).
Two studies analyzed the genetic diversity of C. jejuni strains by MLST in South America. Collado et al. 56 studied 50 strains isolated from Chilean patients with gastroenteritis and observed that the STs, ST50, ST257, ST469, and ST475, were the most prevalent among those strains. A study performed in Ecuador 57 with 40 C. jejuni strains isolated from broilers showed STs, ST462, ST824, ST6244, ST7669, ST8308, ST8309, and ST8310, as the most prevalent. According to our results, only the ST475 was shared with these more prevalent STs found in the studies mentioned above.
Cody et al. 54 in a longitudinal 6-year study performed in United Kingdom showed that the six CC ST21, ST45, ST48, ST257, ST353, and ST443 were represented by 59.8% of all C. jejuni strains studied. Comparing these data with our results, only the CCs ST21, ST48, and ST353 were found in this study comprising 19 strains (39.6%) isolated from humans, animals, and food.
In the same way, the C. jejuni strains of our study shared other STs with isolates from different sources worldwide. For example, according to the Campylobacter spp. MLST database, ST51, ST1398, ST1723, and ST2304 were represented by strains isolated from chicken feces of this study and isolates from humans, animals, and food from countries, such as the United States, United Kingdom, Germany, Australia, and Greece. Likewise, four strains isolated from food and three strains isolated from humans in this study belonged to the ST353 that has also contained strains isolated from humans, animals, and food from Canada, United Kingdom, the United States, Germany, and Greece and has been reported as one of the most frequent STs.54–56
In addition, Duarte et al. 42 analyzed 89 C. jejuni strains isolated from humans, animals, and food and found 48 different STs, four of them, the ST51, ST52, ST354, and ST607, were also found in our study. The same was observed for strains isolated from food in the present work. The ST1359 and ST2282 were represented each by only one strain of this study isolated from food and also by strains isolated from China, Senegal, United Kingdom, Switzerland, Luxemburg, and Thailand from different sources, such as human feces and chicken meat, according to the PubMLST database.
A study carried by Llarena et al. 44 showed that the ST45 was the predominant one in 380 C. jejuni strains isolated from chickens in Finland. Ohishi et al. 45 studied 106 C. jejuni strains from humans in Japan, and ST19, ST50, and ST4526 were the most prevalent ones in 36% of the strains. However, according to our results, these STs mentioned above were not found in our study.
Regarding the C. coli strains studied, 13 STs belonged to the CC828, which suggest a possible phylogenetic relation among these strains. The CC828 accounts for ∼70% of the isolates submitted to PubMLST/campylobacter, with most of the other isolates sharing alleles with this CC and; therefore, being phylogenetically related (Table 1). Furthermore, CC828 is one of the most frequent CC identified in patients with diarrhea worldwide57,58 and isolates from agricultural and environmental sources are also often associated to CC828. 57 This is an important fact because researchers have reported the presence of a founder strain of the CC828 in human, swine, poultry, and cattle from different parts of the world that accounts for 74.6% of the C. coli human isolates reported into the PubMLST.57–63
The ST1581 was the most commonly founded ST in the C. coli strains studied in our work and was represented by four Brazilian isolates from human, animal, food, and the environment; in addition, the MLST database comprised one Danish isolate from human, one Dutch isolate from animal, and one isolate of an unknown country from food. In Ecuador, a study carried out in a slaughterhouse showed the ST5777 as the prevalent one among the C. coli strains studied. 64 In Switzerland, the two most frequently found STs were ST1049 and ST3345. 65 On the other hand, in a study by Thakur et al. the predominant ST was ST1413 among strains from the United States. 66 Thus, data reported by different researchers highlighted the overall weak clonal population and the genetic diversity of this specie.
In addition, the high number of new STs that have been detected in our study indicates that the Brazilian C. coli population is to some extent different from that seen in other countries that have strains deposited in the MLST database. In contrast to those reported by researchers around the world, the majority of the STs found in the present study were associated with different resistance patterns55,56,59 and no correlation regarding the MLST types was observed in the C. coli strains studied based on the year of isolation.
The present study revealed 36 unique STs among the 48 C. jejuni strains and 27 unique STs among the 39 C. coli strains studied, showing a high genetic diversity among those Brazilian strains. Our research group was the first to deposit sequences of C. jejuni and C. coli strains isolated in Brazil in the MLST database making it possible to compare those strains with more than 72,475 Campylobacter spp. strains deposited in this database. The results obtained allowed us to identify STs shared between the Brazilian strains studied and isolates from different countries worldwide which might suggest that a possible transmission might have occurred. Furthermore, MLST data are highly relevant to the extent that it may allow monitoring a possible spread of C. jejuni and C. coli strains from Brazil to other parts of the world.
In conclusion, the antimicrobial resistance observed in C. jejuni and C. coli strains isolated from different sources in Brazil is of public health concern mainly in terms of resistance to tetracyclines and quinolones, which have been the two antibiotic classes used to treat severe and chronic Campylobacter infections in humans. 9 Furthermore, the presence of STs shared between Brazilian strains and isolates from different countries is of concern once it might suggest that a possible transmission might have occurred between those countries. In addition, STs containing exclusively Brazilian isolates may contribute to monitoring the possible spread of those strains, which is important once Brazil is the largest chicken meat exporter, sending meat to more than 150 countries worldwide. Thus, our study contributed for a better characterization of C. jejuni and C. coli isolated in this country and provided information that may help monitoring a possible spread of isolates from Brazil to other parts of the world.
Ethics Statement
The authors declare that ethics approval was not required. The study was conducted using isolates belonging to culture collections of the Oswaldo Cruz Institute (FIOCRUZ-RJ) and Adolfo Lutz Institute (IAL-SP).
Footnotes
Disclosure Statement
No competing financial interests exist.
Funding Information
We thank the São Paulo Research Foundation (FAPESP) (Proc. 2014/13029-0 and Proc. 2016/24716-3), Brazil for financial support. During the course of this work, C.N.G. and M.R.F (Proc. CNPq 141495/2016-2) were supported by a scholarship from the National Council for Scientific and Technological Development (CNPq) and J.P.F. received a productive fellowship (CNPq 303475/2015-3 and CNPq 304399/2018-3) from the same financial agency and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Finance Code 001.