Evaluation of Streptococcus species isolated from subclinical sheep mastitis by molecular methods and determination of virulence factors and antimicrobial resistance genes

Streptococcus (S.) species are important pathogens that cause mastitis in sheep. The study aimed to examine Streptococcus species in sheep milk with subclinical mastitis, assessing their prevalence, antimicrobial resistance, and virulence genes. A total of 200 milk samples were collected from sheep farms in İzmir’s five districts. Out of 32 (28.6%) Streptococcus isolates identified by phenotypic methods, 25 were genotypically identified as S. uberis, 5 as S. agalactiae, and 2 as S. dysgalactiae. Disk diffusion was used to determine the antimicrobial resistance of the isolates. PCR was employed to identify antimicrobial resistance and virulence genes in the isolates. The highest resistance was found for cloxacillin (100%), and the highest sensitivity was found for florfenicol (84%). The most common resistance gene combination was tetM+tetS (3/32) for S. uberis in 9.4%. A total of five virulence genes were detected. GapC+sua (56.2%) constituted the most common gene pattern. The highest virulence gene gapC was detected in 78.1% (25/32) of the isolates. The cylE gene was not detected (0%) in the isolates. Streptococcus species may play a role in mastitis in sheep, emphasising the need for meticulous hygienic milking practices.

https://doi.org/10.17221/42/2023-VETMEDwhich considers colony morphology, haemolysis, and serological specificity (Ruegg 2017;Kabelitz et al. 2021).Streptococcus (S.) uberis (Vezina et al. 2022), Streptococcus dysgalactiae (Guerreiro et al. 2013), and Streptococcus agalactiae are the most common species causing mammary gland infections.Among microorganisms responsible for sheep mastitis, streptococci rank second in importance after staphylococci (Kumar et al. 2013).Both S. uberis and S. agalactiae can induce chronic mastitis in cows (An et al. 2021).Although, S. uberis is primarily an environmental pathogen, cases of transmission have been observed.It typically exhibits gamma haemolysis (i.e., no haemolysis) on blood agar, though it can also display alphahaemolytic behaviour in some instances.Its identification can be confirmed through a variable CAMP phenotype test and degradation of esculin, sodium hippurate, and inulin (Kabelitz et al. 2021).S. dysgalactiae is a pathogen that can survive in both the host and the environment.While some strains are alpha-haemolytic, most are non-haemolytic.It is phenotypically CAMP-negative and does not degrade esculin (Wente and Kromker 2020;Kabelitz et al. 2021).Several potential virulence factors have been identified for S. uberis, including sua, which aids in mammary epithelial cell adhesion and invasion, and gapC, a surface dehydrogenase protein (Kerro Dego et al. 2021).As a major mastitis-causing pathogen, S. agalactiae possesses virulence factors like the CAMP factor cfb and the toxin cylE (Zastempowska et al. 2022).Furthermore, S. agalactiae is capable of transferring genetic material to other mastitis pathogens, such as S. uberis and S. dysgalactiae, through genes like napr and enobinding host plasminogen protein, thereby contributing to host infection and colonization.The adherence of plasminogen on the bacterial surface plays a pivotal role in the pathogenic mechanism of bacterial adhesion to host cells (Shen et al. 2021).However, antibiotic resistance is also a contributing factor to the failure of mastitis treatment.S. dysgalactiae isolates were found to carry blaZ, ermA, ermB, ermC, and lnuA genes, while S. agalactiae isolates were found to harbour blaZ, ermB, ermC, lnuA, tetK, tetL, and tetM genes (Ahmed et al. 2020).Streptococcal isolates from mastitis sheep milk confirmed via PCR were found to contain genes such as ermB, ermC, linB, and antibiotic resistance genes like tetM and tetO (Saed and Ibrahim 2020).Aminoglycoside resistance in streptococci is also mediated by genes like aad-6 and aphA-3, which lead to enzymatic inactivation of antibiotics.
The countries with the world's largest sheep populations include China, India, Australia, Nigeria, Iran, Ethiopia, and Türkiye (Sevinc et al. 2022).In Türkiye, the sheep population averages 46 million, and the country produced approximately 1 100 000 tons of sheep's milk in 2022.According to the Governor's office (MAF 2023), Izmir province and its districts have an average of 672 000 sheep, with around 320 000 animals being milked.Raw sheep milk production decreased by 6.7% in 2022 compared to the previous year (TUIK 2022).Although sheep milk production seems to be declining according to TUIK data, our region plays an important role in the country's sheep milk production.Therefore, investing in academic research on animal husbandry with the aim of enhancing and contributing to the nation's sheep milk production could yield positive outcomes for effective animal husbandry practices.
This study aimed to identify multiple antibioticresistant Streptococcus species in milk collected from sheep with subclinical mastitis in the Küçük Menderes Basin (including Bayındır, Beydağı, Kiraz, Ödemiş, and Tire) in Izmir province.The study aimed to assess the susceptibility of isolated Streptococcus species to antibiotics and identify the presence of virulence genes such as gapC, sua, cylE, cfb, eno, and napr.This would provide better insights into the potential pathogenicity of these bacteria and inform effective treatment strategies for mastitis.Additionally, the study revealed antibiotic resistance genes including lnuD, ermB, ermC, tetL, tetS, aad-6, and blaZ.

Sample collection
A total of 200 milk samples were collected from 100 sheep of various breeds and ages in selected dairy farms in the Küçük Menderes Basin between November and February 2022.These milk samples were collected from sheep suspected of having mastitis based on the anamnesis provided by veterinarians or technicians from farms in the districts of Kiraz, Tire, Ödemiş, Bayındır and Beydağı districts.Samples of sheep's milk were collected in equal quantities in each district.Teats were cleaned and disinfected with 75% ethanol before https://doi.org/10.17221/42/2023-VETMEDcollecting milk samples.The first foremilk was discarded and 200 milk samples were collected aseptically into sterile tubes containing 10-15 ml of milk.California mastitis test (CMT) was applied to the collected milk samples and the results were evaluated as -, +, ++ and +++.According to the CMT results, 88 samples were considered negative, and 112 samples were considered positive.The investigation was continued on 112 CMT-positive samples.The results of the CMT applied to milk were evaluated according to the method of Schalm et al. (1971).CMT-positive samples were kept under cold chain conditions and transported to the molecular laboratories of Aydin Adnan Menderes University, Faculty of Veterinary Medicine, Department of Microbiology for laboratory analysis.

DNA extraction from Streptococcus strains
The Streptococcus isolates identified through biochemical methods such as Gram staining, catalase test, and haemolysis patterns were suspended in 500 μl of sterile distilled water.Subsequently, a genomic DNA extraction kit (Hibrigen ® , Kocaeli, Türkiye) was employed as per the manufacturer's protocol to extract DNA from the prepared Streptococcus suspensions.The obtained Streptococcus DNAs were then stored in a deep freezer at -20 °C for further molecular studies such as PCR and sequencing.

Molecular identification of Streptococcus species
For PCR, the final concentration for each sample was 10X Taq enzyme buffer solution (1X), 50 mM MgCl 2 (2 mM), 10 mM dNTP (0.2 mM), 40 ρmol of each primer, and 1.5 U/µl of Taq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA).The reaction was performed in a volume of 25 µl with 3 µl of DNA added per sample.Primers and cycling conditions were used as defined (Kaczorek et al. 2017a).PCR products were visualised by electrophoresis of 10 µl of each sample on a 2% agarose gel at 80 V for 60 min, and the results were evaluated using a gel documentation system (Vilber Lourmat, Collégien, France).

Streptococcus species
The confirmation of Streptococcus isolates by Sanger sequencing used Universal primers 357F (5'-CTCCTACGGGGAGGCAGCAG-3') and 1 492R (5'-GTTACCTTGTTACGACTT-3').The previously described protocol for PCR analysis was applied to the isolates (Turner et al. 1999).PCR products were purified using ExoSAP-IT TM (GML ® ) PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA), followed by Sephadex (GML ® ) (Sigma-Aldrich, St. Louis, MO, USA) for sequence PCR product purification.Sequencing of purified PCR products was performed using the GenomeLab Dye Terminator Cycle Sequencing (DTCS) Quick Start Kit and the Sanger Sequence instrument (Beckman Coulter, Inc., Fullerton, CA, USA).Standard Nucleotide BLAST ® NCBI Genomic Reference Sequences were used to analyse the nucleotide sequences of the PCR products.

Detection of virulence genes of Streptococcus species
The multiplex PCR method was used to examine the presence of virulence-related genes in all strains that were phenotypically identified as S. uberis, S. dysgalactiae, and S. agalactiae.The preparation of the master mix involved adding 20 ng of DNA sample to a total volume of 50 µl, with 1 µM of each primer, 0.4 mM dNTP, 1.5 mM MgCl 2 , 1 × reaction https://doi.org/10.17221/42/2023-VETMED
The primer sequences, product sizes, and annealing temperatures special to virulence-related target genes were used in our study to detect S. uberis, S. agalactiae, and S. dysgalactiae (Kaczorek et al. 2017b).
In this study, upon examining the virulence genes of the identified Streptococcus isolates, it was found that the gapC+sua genes were positive in 18 (56.2%)S. uberis isolates, while only the gapC gene was positive in 7 (21.8%)S. uberis isolates.Eno and napr genes (6.2%) were positive in S. dysgalactiae (n = 2) isolates.The cylE gene was negative in all isolates.
In addition, all of the phenotypic isolation, 16S rRNA PCR, Sanger sequence, virulence genes, antimicrobial resistance genes and antibiogram results obtained in the study are given in the heatmap (Figure 4).

DISCUSSION
The incidence of clinical mastitis in sheep flocks sampled worldwide is relatively low (1.2-3%), but significant variation can exist within flocks (0-37%) (Murphy et al. 2018).Subclinical mastitis (SCM) lacks visual symptoms, but diagnosis is possible through bacterial culture and/or quantification of somatic cell count (SCC) in milk.The morbidity rate of SCM is much higher in sheep than in clinical mastitis (CM) (12-50%) (Murphy et al. 2018).S. uberis and S. dysgalactiae are opportunistic environmental animal pathogens, while S. agalactiae is a primary pathogen causing contagious disease (Chen et al. 2021).The incidence of clinical mastitis in sheep has generally been reported to be less than 5%, with the incidence of subclinical cases ranging from 16% to 35% (Guerreirot al. 2013).In another study, the incidence of Streptococcus strains in mastitis sheep milk was found to be 8% (Ceniti et al. 2017).We analysed 200 subclinical mastitis sheep milk samples and isolated Streptococcus spp. in 32 (28.6%).In addition, studies have reported the following species ratios 5.1% and 2.1% for S. uberis and S. dysgalactiae, respectively (Ceniti et al. 2017), and19% for S. agalactiae (Abd-Elfatah et al. 2023).Rosa et al. (2022) reported that the most common streptococcal species in sheep and goats with mastitis were S. uberis (89.5%) and S. dysgalactiae (3.5%).In our study, S. agalactiae (15.6%) and S. dysgalactiae (6.3%) were detected at low percentages, but this was consistent with these findings.In contrast, S. uberis was detected at a higher rate (78%).This raises the question of whether there is a similar risk in sheep, as has been suggested for goats, where mechanical milking has been associated with a higher risk of bacterial positivity and S. uberis infection (Novac and Andrei 2020).
There are few studies on the detection of virulence genes in Streptococcus infections in sheep.When considering the virulence genes for Streptococcus strains isolated from clinical cases of mastitis in dairy cattle, a study reported a wide frequency of the cfb gene (93%) and cylE gene (90.6%) in S. agalactiae (Kaczorek et al. 2017a).Shi et al. (2023) reported that the cfb gene encodes the haemolysis-promoting factor CAMP and that this gene is one of the causative factors of Streptococcus infection.However, in our study on subclinical sheep mastitis, these genes were detected only in S. dysgalactiae species.Kaczorek et al. (2017a) reported the presence of sua (96%) and https://doi.org/10.17221/42/2023-VETMEDgapC (98%) genes in S. uberis, and eno (76%) and napr (83%) genes in S. dysgalactiae.All our positive isolates showed at least one virulence gene, with the sua and gapC genes being the most frequently detected.These results align with existing research on virulence-associated genes.Furthermore, we found that the eno and napr genes were comparatively less prevalent than other genes, suggesting a potential role in S. dysgalactiae infection in dairy sheep.Notably, the cylE gene was not detected.
The characterisation of isolates is important to gather information on resistance and to optimise therapy.Pathogenic Streptococcus isolates causing mastitis in dairy cows can exhibit severe resistance to erythromycin and penicillin (Tian et al. 2019).El-Shafei et al. (2020) reported that S. dysgalactiae strains (87%) demonstrated higher resistance to tetracycline than S. agalactiae strains (37%) in cows and buffaloes.Additionally, both strains were susceptible to penicillin and amoxicillin.In contrast, we observed a high rate of penicillin resistance in S. uberis and S. dysgalactiae, while S. agalactiae displayed high sensitivity.S. agalactiae showed higher susceptibility to cloxacillin (75%) than S. dysgalactiae (43%).A study in dairy cows conducted in Switzerland between 2011 and 2013 reported high antimicrobial susceptibility to amoxicillin-clavulanic acid (99%; 100%) and ampicillin (92%; 94%) for S. uberis and S. dysgalactiae, respectively (Ruegsegger et al. 2014).However, we found lower sensitivity percentages for amoxicillin-clavulanic acid in the region.Moreover, our findings indicated that S. dysgalactiae did not show susceptibility to ampicillin, and S. uberis showed very low susceptibility to this antibiotic.Conversely, S. agalactiae exhibited high susceptibility to ampicillin.According to a study, S. agalactiae demonstrated resistance to tetracycline (46%) and erythromycin (15%), while S. dysgalactiae showed resistance to tetracycline (38.5%) and erythromycin (20%) (Kabelitz et al. 2021).
While our findings are consistent with this study, we observed higher percentages of resistance in our country.The tetL, tetS, and tetM genes have frequently been reported in bovine mastitis to be frequently encountered in Streptococcus isolates (Kaczorek et al. 2017a).In several countries, including France, Brazil, Canada, Portugal, Poland, Egypt, Argentina, and China, S. uberis, S. agalactiae, or S. dysgalactiae have been reported to harbour the gene tetM in bovine mastitis (Naranjo-Lucena and Slowey 2023).Also, it has been reported that the dominance of the tetM gene indicates that the mechanism of resistance is mainly mediated by the protection of the ribosomes and not by the efflux pump (Rosa et al. 2021).The dominance of the tetM gene suggests that resistance mechanisms primarily involve ribosome protection rather than efflux pumps (Rosa et al. 2021).We detected resistance to tetracycline in our isolates, with tetM being the most frequently detected resistance gene in S. uberis (62.5%), followed by tetS (50%), ermC (43.7%), and tetL (40.6%).Saed and Ibrahim (2020) reported the common presence of the ermB gene in Streptococcus isolates from mastitis in dairy cows, with a prevalence of 40% among erythromycin-resistant isolates.Erythromycinresistant Streptococcus isolates from sheep milk have also been reported to carry the ermB gene (Rosa et al. 2021).The ermC gene resistance was found in 11 S. uberis isolates and 3 S. agalactiae isolates.Resistance to the ermC gene was detected in multiple resistance groups.However, only one isolate of each strain demonstrated possessing or carrying the ermB gene, which is 3.1% of the samples.The aad-6 gene was detected in S. uberis and S. dysgalactiae species in dairy cows (Kaczorek et al. 2017a).Another study reported that aminoglycoside-resistant Streptococcus isolates are negative for aad-6 genes (Rosa et al. 2021).Our results indicate that only 6 S. uberis isolates carried the aad-6 gene, aligning with the inherent aminoglycoside resistance of Streptococcus strains.In Germany, the ermB gene was only detected in S. uberis, whereas both S. agalactiae and S. dysgalactiae species harboured the ermB and ermC genes in dairy cows.However, in our study, we found the ermB gene only in S. uberis isolates.The blaZ gene is frequently detected in France for S. uberis, lnuD for S. uberis, and S. dysgalactiae in Poland (Naranjo-Lucena and Slowey 2023).Detection of the blaZ gene has also been reported in sheep milk (Rosa et al. 2021).However, we detected the blaZ gene in S. uberis isolates, and the lnu gene in one S. agalactiae isolate (3.1%), which is consistent with its reported low prevalence (Haenni et al. 2018).
In conclusion, our findings suggest that Streptococcus isolates from sheep mastitis have high antimicrobial resistance and carry various virulence genes that may be harmful to sheep.Further studies are needed to explore the epidemiology of Streptococcus-derived herd infections and their ability to confer resistance to antimicrobials regarding new virulence and resistance.

Figure 2 .
Figure 1. Isolation percentages in districts for Streptococcus isolates

Figure 3 .Figure 4 .
Figure 3. Distribution of resistance genes and number of Streptococcus strains isolated from mastitis milk samples