Resistant enterococci isolated from raw sheep’s milk and cheeses from South region of Brazil

ABSTRACT: Enterococci have been used as sentinel organisms for monitoring antimicrobial resistance in food, humans, and other animals. In this sense, the present study evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in enterococci isolated from raw sheep’s milk and cheeses (colonial, feta-, and pecorino-type) from South region of Brazil. A total of 156 enterococci were isolated from milk (n=80) and cheese (n=76) samples, identified by MALDI-TOF. Enterococcus faecalis (50.6%; n=79) was the most frequent species isolated from both samples. According to in vitro susceptibility tests, enterococci strains were not susceptible to the most commonly antimicrobial agents used in human and veterinary medicine. The frequency of MDR strains in enterococci isolated from milk (53.7%) was higher than those from cheese (24.2%). The tet(M) gene was the most commonly detected among tetracycline not-susceptible strains. The present study provided the first evidence of antimicrobial not-susceptible enterococci in raw sheep’s milk and cheeses in South Brazil. Drug-resistant strains, particularly those that are MDR, constitute a One Health issue.


INTRODUCTION
Global milk production reached nearly 906 million tonnes in 2020, up 2% from 2019.The annual production of sheep's milk in the world is about 10.5 million tonnes and the major producers are Turkey, China, and Greek (FAO, 2021).In Brazil, the production and industrial processing of sheep's milk are still not very significant, corresponding to only 0.0019% of the total milk produced in the country, being the South and Southeast regions with the largest sheep raising for milk production flock (MERLIN JUNIOR et al., 2015).Despite low shares of production, this is an important economic activity for small and medium farmers (FERNANDES et al., 2017).
Enterococci is a part of the lactic acid bacteria (LAB) of importance in food.They often present on animal source foods, such as meats, fermented and cooked meats, as well as cheese and dairy products.They play an important role in modulating organoleptic properties as a starter culture, contributing to aroma and flavor development in cheeses, and as probiotics in humans and slaughter animals (DAPKEVICIUS et al., 2021).Despite the beneficial effects, enterococci can also be implicated in spoilage of foods and are commonly monitored as hygiene indicators in other types of food matrices.In the last decades, they are also related to health-associated infection (urinary tract infections, bacteremia, or endocarditis) in immunocompromised and hospitalized patients (LEBRETON et al., 2017).Enterococci are used as markers of animal fecal contamination of human foods and are employed as sentinel organisms for tracking trends in resistance to antimicrobial agents with activity against Gram-positive bacteria, in humans, retail meats and food animals (WHO, 2017).
The impact of the presence of antibioticresistant bacteria in food constitutes a One Health issue.One Health is an integrated, unifying approach that aims to sustainably balance and optimize the health of people, animals and ecosystems (WHO, 2022).Resistant microorganisms can reach humans directly through consumption of food or food products and/or contact with colonized animals or even as indirectly through environmental pollution containing human and/or animal excrement (ROBINSON et al., 2016).In this sense, this study detected and evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in Enterococcus spp.present in raw sheep's milk and cheeses from South Brazil.The data generated here also aims to contribute to the One Health antimicrobial resistance Network.

Isolation and identification of enterococci in raw milk and cheese samples of sheep
Seventeen samples comprising raw milk (n=9) and cheeses (Colonial, Feta-type, and Pecorinotype) (n=8) of sheep were collected from January 2017 to August 2019 in sheep's farming from Rio Grande do Sul and Santa Catarina states, Brazil (Table 1).
Cheeses were made from pasteurized milk with the addition of autochthonous starter cultures according to the regulations approved by Ministério da Agricultura, Pecuária e Abastecimento (MAPA), Brazil.Colonial cheeses were prepared by using a starter culture of Lactobacillus helveticus.Feta-and Pecorino-type cheeses were prepared using Streptococcus thermophilus, Lactobacillus bulgaricus, L. helveticus, and Lactobacillus casei as a starter culture.All samples were stored at 4 °C for transport.Due to the nature of the samples, ethical approval was not required for this study.
The enterococci isolation followed the methodology described by RIBOLDI et al. (2009) andPRICHULA et al. (2016).Samples were resuspended in 10 mL of saline 0.85 % and maintained under agitation (100 rpm) at 37 °C for 24 h.One mL was inoculated in 9 mL of selective medium Azide Dextrose Broth (Himedia, Mumbai, India) for 24 h at 37 °C, remaining under agitation (100 rpm).Aliquots of 1 mL were placed in 9 mL of sterile water, and the initial samples were further diluted 10-fold to obtain a final dilution factor of 1/1000.From each dilution, 100 μL were plated in triplicate on a Brain Heart Infusion Agar (Himedia, Mumbai, India) supplemented with 6.5 % NaCl, being incubated as described.The pure colonies were stored at -20 °C in a 10 % (w/v) solution of skim milk (Difco, Sparks, MD, USA) and 10 % (v/v) glycerol (Neon ComercialLtda, São Paulo, BR).Species were identified by matrix assisted laser desorption and ionization time-of-flight mass spectrometry method (MALDI-TOF) according to the protocol described by SAUGET et al. (2017).MALDI-TOF analysis was performed using a LT Bruker microflex mass spectrometer (Bruker Daltonik GmbH) and spectra were automatically identified using BrukerBioTyperTM 1.1 software.The identification by MALDI-TOF MS is based on the score value released by the equipment.A higher or similar 2.3 value indicated that the identifications of genus and species are reliable.The 2.0-2.29 values showed that the genus is reliable, and the species is probable, and the 1.7-1.99values indicated that the identification of genus is probable.
Colonies were suspended in peptone water until it reached 0.5 McFarland standard.The dilution was then streaked across a Mueller-Hinton Agar plate (Himedia, Mumbai, India), and the 12 antimicrobial disks were placed onto the surface of the agar.The plates were incubated for 24 h at 37 0 C. Zone diameters were recorded after a 24 h incubation period.Strains were classified as susceptible or not susceptible (intermediate susceptible or resistant) according to criteria from CLSI (2019) and BrCAST (2019) for linezolid.Enterococcus faecalis ATCC 29212 was used as a positive control.

Enterococci species isolated from raw milk and cheese of sheep
A total of 156 enterococci were isolated, 80 (51.3%) from raw milk and 76 (48.7%) from cheeses.
The most frequently isolated species was Enterococcus faecalis (50.6%; n=79), followed by E. faecium (20.5%; n=32), E. hirae (14.8%; n=23), E. durans (13.5%; n=21), and E. gallinarum (0.6%; n=1).According to the results shown in table 3, the most common species identified in milk samples were E. faecalis (58.8%; n = 47), E. hirae (28.8%; n = 23), and E. faecium (12.5%; n = 10) isolated from milk samples.As well as in other studies, E. faecalis was dominating species in sheep milk samples.DUCKOVÁ et al. ( 2009) identified 70% of E. faecalis in sheep´s milk samples in Slovakia.ARIZCUN et al. (1997) also found E. faecalis as a dominating species in ewe's milk in the Autonomous Region of Navarre in Spain.It is important to highlight that E. faecalis, E. hirae, and E. faecium occur in the gastrointestinal tract of sheep, and their presence in the raw sheep's milk might be associated with fecal contamination during milking and/or handling of animals (DAPKEVICIUS et al., 2021).
Out of 76 strains isolated from cheeses, 29 (38.2%) were collected from Colonial, 29 (38.2%) from Feta-type and 18 (23.7 %) from Pecorino-type.According to the results shown in table 3, differences in the species distributions were detected amongst cheese samples.Enterococcus durans (13.5%; n = 21) was found in Colonial and Feta-type cheeses; and E. faecium (75.9 %; n = 22) and E. gallinarum (3.5%; n = 1) were only isolated from Colonial cheese samples.These results are in line with VÝROSTKOVÁ et al. (2021), that reported different strains of enterococci in sheep cheeses samples.Enterococcus faecalis and E. faecium were also the most frequent species detected in artisanal cheeses produced with sheep, goat, buffalo, and cow pasteurized or raw milk (GELSOMINO et al., 2002;RIBOLDI et al., 2009;PRICHULA et al., 2013).Starter lactic acid bacteria, like enterococci, are naturally present in milk or beneficial contaminants originated from cheese production environments.The occurrence of E. faecalis in sheep's cheeses might be related to the development of aromatic and organoleptic features, since this species is able to produce acetaldehyde and diacetyl (ARIZCUN et al., 1997).Furthermore, some enterococci strains (such as E. durans, E. faecium, and E. faecalis) present in the raw milk and cheeses of sheep's are recognized to produce bacteriocins, with ability to killing or inhibits important foodborne pathogen, like Listeria monocytogenes (PRICHULA et al., 2021).

Antimicrobial resistance profile of enterococci isolated from raw milk and cheese of sheep's
To not overestimate the data referring to antimicrobial not susceptible profiles, strains isolated from the same sample with similar phenotypic and genotypic profiles, which could indicate clonal strains, were grouped.Thus, 156 strains were grouped into 74 strains (41 from milk, and 33 from cheese).Among the 74 strains, 65 (87.8%)strains were not susceptible to at least one antimicrobial agent evaluated, being this profile more frequently observed in strains collected from milk than those from cheese samples (Figure 1).All enterococci strains were susceptible to ampicillin, linezolid, and vancomycin.Single, double, and MDR profiles were observed in 21.6% (n = 16), 25.7% (n = 19), and 40.5% (n = 30) of the strains, respectively (Figure 1) (Table 4).The frequency of MDR strains isolated from milk (53.7%; n=22) were higher compared to cheese (24.2%; n=8).
The elevated frequency of tetracycline, rifampicin, erythromycin, and nitrofurantoin not susceptible strains in raw sheep milk, may be related to the fact that these drugs are widely used in veterinary medicine for the prophylaxis and treatment of infections (NOVAES et al., 2017).Tetracycline and streptomycin have been used to treat intramammary infections in sheep (LOLLAI et al., 2016;RÓŻAŃSKA et al., 2019).Another source of not susceptible strains in raw milk maybe been related to the milking equipment, since this genus is well known to survive desiccation, starvation, and disinfection, and are able to develop biofilms (ORTIGOSA et al., 2008;LEBRETON et al., 2017;PEREIRA et al., 2017).In addition, these resistance profiles observed might be also associated with environmental resistome (SURETTE & WRIGHT, 2017).
Frequency of tetracycline and erythromycin resistance genes between enterococci isolated from raw milks and cheese of sheep Among the 24 tetracycline not susceptible strains isolated from raw milks, 12 (50%) harbored the tet(M), 10 (41.7%) tet(M) and tet(L), and 1 (4.2%)only the tet(L).In contrast, all (n=4) tetracycline not susceptible strains collected from cheeses presented the tet(M), and none of them was positive for the tet(L).Erythromycin not susceptible strains (n=20) collected from milks, displayed two resistance genotypes: 6 (30%) carried the mrsC and 6 (30%) the ermB genes.Of the 21 strains not susceptible to erythromycin isolated from cheeses, 9 (42.9%)harbored the mrsC gene and 1 (4.8%) the ermB gene.These results are in agreement with other studies that evaluated these genes in enterococci isolated from milk and cheese samples (KANG et al., 2019;SILVETTI et al., 2019;VÝROSTKOVÁ et al., 2021).
Resistance to macrolides and tetracyclines in enterococci has been assumed to be related to the presence of a conjugative transposon.This mechanism is an important factor for spreading of tetracycline resistance between enterococci and other Gram-positive bacteria (RIZZOTTI et al., 2009).The occurrence of antimicrobial resistance genes in our samples, especially in cheeses, is a very important issue since these genes can be transmitted by conjugative transposons, facilitating horizontal genetic exchange and, therefore, promote the acquisition and spread of resistance genes.

CONCLUSION
The present study provided the first report of resistant enterococci in raw sheep's milk and cheeses in South Brazil.However, other points need to be explored to understand the origin, emergence, and dissemination of antimicrobial not susceptible and MDR enterococci in sheep samples.The dissemination of antimicrobial-resistant bacteria between human-animal-environment interface has been recognized as a One Health issue.

ACKNOWLEDGEMENTS
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001 and Conselho Nacional de Desenvolvimento Table 5 -Main phenotypic multidrug-resistant profiles observed among enterococci isolates from raw milk and cheeses-types of sheep´s in South Brazil.

Table 1 -
Information of samples collected from raw milk and cheese of sheep's in South Brazil.

Table 2 -
Primers used in the PCR reactions carried out for detection of resistance genes.

Table 3 -
Distribution of Enterococcus species among sheep's milk and cheeses-types samples collected in South Brazil from 2017 to 2019.

Table 4 -
Antibiotic resistance patterns among enterococci recovered from raw milk and cheeses-types of sheep in South Brazil.