Characterization of Staphylococcus spp. isolates and β-lactam resistance in broiler chicken production

Ramon Loureiro Pimenta1 , Dayanne Araújo de Melo2* , Greiciane França Bronzato3 , Vinícius Rangel de Salles Souza4 , Thérèsse Camille Nascimento Holmström5 , Shana de Mattos de Oliveira Coelho6 , Irene da Silva Coelho7  & Miliane Moreira Soares de Souza8  1Veterinarian, MSc., Programa de Pós-graduação em Ciência, Tecnologia e Inovação em Agropecuária (PPGCTIA), Universidade Federal Rural do Rio de Janeiro (UFRRJ), Campus Seropédica, RJ, Brazil. 2Veterinarian, DSc., PPGCTIA, UFRRJ, Campus Seropédica, RJ, Brazil. 3Biologist, DSc., PPGCTIA, UFRRJ, Campus Seropédica, RJ, Brazil. 4Veterinarian, Autonomus, Rio de Janeiro, RJ, Brazil. 5Zootechnist, MSc., Programa de Pós-graduação em Ciências Veterinárias (PPGCV), Departamento de Microbiologia e Imunologia (DMI), Instituto de Veterinária (IV), UFRRJ, Campus Seropédica, RJ, Brazil. 6Biologist, DSc. Departamento de Microbiologia e Imunologia (DMI), IV, UFRRJ, Campus Seropédica, RJ, Brazil. 7Agronomist, DSc., DMI, IV, UFRRJ, Campus Seropédica, RJ, Brazil. 8Veterinarian, DSc., DMI, IV, UFRRJ, Campus Seropédica, RJ, Brazil.


Introduction
Animal production is a relevant activity of Brazilian agribusiness. Brazil stands out in poultry production as the second-largest producer of chicken meat globally, reaching 13.056 million tons, leaving behind only the US with a production of 18.596 million tons (Associação Brasileira de Proteína Animal, 2018). The use of antimicrobials in animal production has different purposes: to protect or improve animal health and to stimulate faster growth and maximize profits (Van Boeckel et al., 2015). The use of antimicrobials as growth promoters is of great concern as it increases the chances of developing resistant bacteria. Many countries have already banned this, especially in the European Union since 2006(O'Neill, 2015. In Brazil, the prohibition of β-lactams as additives was established in article 18 the Normative Instruction 26/2009 published by Ministry of Agriculture, Livestock and Supplies (Brasil, 2009).
Antimicrobial use is supported due to the importance of infectious diseases causing significant economic losses in poultry production. Both coagulase-positive, especially S. aureus, and coagulasenegative Staphylococcus spp. (CoNS) has been implicated as an etiological agent in many avian pathologies (Corrand et al., 2012). The clinical signs most associated with Staphylococcus spp. in birds are arthritis, synovitis, chondronecrosis and osteomyelitis, gangrenous dermatitis, pododermatitis, omphalitis, septicemia, blepharitis and mandibular osteomyelitis (Huynh et al., 2014).
Methicillin-resistant Staphylococcus spp. are important in both human and veterinary medicine once it is related to resistance to all β-lactam antimicrobials (Cohn & Middleton., 2010). Two mechanisms can mediate the resistance to penicillin: the blaZ gene that encodes the production of the enzyme β-lactamases, which degrades the β-lactam ring of β-lactam antimicrobials, and the mecA gene, that encodes the production of low-affinity penicillinbinding protein called PBP2a or PBP2', whereas this change prevents binding of penicillin, thereby maintaining regular cell wall production (Kuroda et al., 2001). The mecA gene is widely spread between both Staphylococcus coagulase-positive and negative species from animal and human origin (Huber et al., 2011). Nemeghaire et al. (2014) found a range of 12.5% Staphylococcus sciuri methicillin-resistant in broilers reinforcing that CoNS can be reservoirs and help in the dispersion of antimicrobial resistance genes (Schoenfelder et al., 2017).
So, besides the clinical importance of staphylococci antimicrobial resistance, it must be investigated the contribution of poultry production to the worsening of the global antimicrobial resistance framework related to the use of antimicrobials in sub-therapeutic doses and for long periods, establishing ideal conditions for the circulation and maintenance of resistance genes, which can be transmitted to adapted pathogens to the human intestinal microbiota (O'Neill, 2015).
In previous studies developed by the Veterinary Bacteriology Research Group at Federal Rural University of Rio de Janeiro, it was observed that the highly phenotypic resistance to β-lactams was not necessarily correlated to the detection of the mecA gene in strains isolated from animals (Mendonça et al., 2012;Soares et al., 2008). Melo et al. (2014) elucidated this puzzling question by detecting a mecA gene variant in Staphylococcus spp. from bovine in Brazil. From the sequencing and comparison of both variant and classical mecA genes, point mutations impair genotypic detection with the previously described primers. The detection of the mecA gene variant in strains isolated from different animal species should be expanded to enlarge the understanding of the resistance mediated by such genes in animal origin strains. This study aimed to screen Staphylococcus species and its phenogenotypic resistance to β-lactam antimicrobials in isolates from broiler chicken.

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Characterization of Staphylococcus spp. isolates and β-lactam resistance in broiler chicken production

Sampling and Staphylococcus species identification
A total of 120 samples were collected from two broiler chicken farms in the mountain region of Rio de Janeiro between April 2015 and March 2016. Farms were twice visited in this period and 30 cloacal and 30 tracheal swabs were collected at each time. Swabs were transported in Stuart's medium (Absorve®) at room temperature immediately to LABAC-VET UFRRJ. Samples were inoculated in Blood Agar (HiMedia®) and selective Mannitol Red Phenol Agar (HiMedia®) and incubated at 37 °C for 24h. The isolates were identified in a mass spectrometer (MALDI-TOF LT Microflex Bruker, Bruker) controlled by the FlexControl 3.3 program (Bruker). The spectra were analyzed by the MALDI Biotyper 2.0 (Bruker) program using the standardized configurations for bacterial identification. Scores must be equal or greater than 2 to achieve an acceptable identification. When scores were below 2, isolates were submitted to biochemical identification comprising catalase test, coagulase test, resistance to bacitracin with 0.04 IU disk (SENSIFAR-CEFAR). Coagulase-negative isolates were tested to novobiocin-resistance (5μg) (SENSIFAR-CEFAR), and the resistant ones were evaluated by fermentation test of maltose, sucrose, mannitol, mannose, trehalose, xylose, raffinose, and cellobiose (Koneman et al., 2012).

DNA extraction
A 1.5 mL aliquot of Brain and Heart infusion Broth with bacterial growth was transferred to microtubes and centrifuged for 5 minutes at 12.000 RPM, and the supernatant was discarded. The procedure was repeated three times. Cells were resuspended in 600 μl of extraction solution (200 mM Tris-HCl pH 8.0, 25 mM EDTA pH 8.0, 1% SDS, 25 mM NaCl) and incubated at 65 °C for 30 min. DNA was extracted with chloroform: isoamyl alcohol 25:24:1 and precipitated with two-volume ice-cold ethanol. The DNA pellet was washed with 70% ethanol and resuspended in 30 μl of TE buffer (10 mM Tris-HCL, pH 8.0, EDTA1 mM pH 8.0) and stored at -20 °C until use.

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Characterization of Staphylococcus spp. isolates and β-lactam resistance in broiler chicken production

Discussion
The most prevalent species identified in both broiler chicken farms was S. gallinarum (35.2%, 31/88), followed by S. simulans (17.0%, 15/88). Heba et al. (2014), when studying broilers in Egypt, found 5.0% of S. galinarum and 22.0% of S. simulans. Rueanghiran et al. (2017) found nine different species of CoNS including S. gallinarum, but they do not clarify which different species, when analyzed the diversity of Staphylococcus spp. in feces of broilers from Thailand.
For a long time, CoNS has been considered harmless and minor important species. However, the advances in molecular taxonomy and systematic allowed a better comprehension of the species dissemination and consequently its pathogenic role (Calazans-Silva et al., 2014). Besides that, None isolate tested positive for conventional mecA gene whereas the variant mecA was detected in 6.8% (6/88), of which a singular S. simulans from cloaca, and five S. sciuri isolates from trachea. One isolate of S. aureus (1.1%), from trachea, amplified using universal primer but was not possible to verify the presence of mec genes, however, this strain presented the blaZ gene. Table 2 displays the results of phenogenotypical β-lactam resistance analysis and the main characteristics of this of Staphylococcus isolates.  CONS are important reservoir of resistance genes, mainly located on mobile genetic elements, conferring resistance to all classes of antimicrobials which are able to be transfer among different bacterial species and spread further, being a challenge at human and veterinary medicine (Fišarová et al., 2019). Considering the present study, 23 isolates (26.0%) were methicillin-resistant CoNS at phenotypic analysis, presenting resistance to oxacillin, being 13 (56.5%) S. gallinarum. Rueanghiran et al. (2017) detected 52% of methicillin-resistant strains and also considered S. gallinarum as a potential source of resistance genes to other bacteria in the environment.
Despite the phenotypic resistance observed, methicillin-resistant expression is peculiar and heterogeneous, and because of this, detection of the mecA gene is considered the gold standard method for confirmation of methicillin-resistant isolates by Clinical and Laboratory Standards Institute (2018). However, it was not possible to establish any correlation between phenotypic resistance profiles and the mecA gene's presence. Otherwise, universal primer successfully amplified variant mecA, showing maximum correlation, confirming that this tool is excellent when screening for mec-positives isolates, presenting a high sensitivity and specificity, 93.1% and 98.1% respectively, in a recent study developed by Melo et al. (2020).
The use of universal primer, for conventional and mecA variant gene detection, improve methicillin-resistant Staphylococcus diagnosis, being crucial for its accurate and rapid identification as proposed by Melo et al. (2020).
Only one isolated presented the blaZ gene, that codified the enzyme β-lactamase, showing that the mechanism responsible for β-lactamic resistance, at the farms evaluated, was the expression of mecA gene. Besides that, the detection of variant mecA gene in broiler chicken samples reinforce the fact that this gene is widespread in the animal environment, mainly in CoNS species.
Despite the phenotypic resistance observed in S. gallinarum strains, none of resistance genes (mecA genes and blaZ) tested in this study was detected in this species. These findings corroborated with Melo et al. (2018), that observed the same pattern evaluating β-lactam resistance in CoNS isolates from bovine intramammary infection. These results could support the hypothesis that these isolates have higher degrees of variability or suffer mutations that prevent its detection requiring further studies to better understanding.

Conclusion
Evaluating the β-lactam resistance in samples from animals requires more specific analyses, including detecting the mecA variant, which is not allowed with the conventional mecA primers described. This study is the first report of mecA variant presence in samples from broiler chicken.