Characterization of Staphylococci and Streptococci Isolated from Milk of Bovides with Mastitis in Egypt

The aim of this study was to characterize staphylococci and streptococci in milk from Egyptian bovides. In total, 50 milk samples were collected from localities in the Nile Delta region of Egypt. Isolates were cultivated, identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and antibiotic susceptibility testing was performed by the broth microdilution method. PCR amplifications were carried out, targeting resistance-associated genes. Thirty-eight Staphylococcus isolates and six Streptococcus isolates could be cultivated. Staphylococcus aureus isolates revealed a high resistance rate to penicillin, ampicillin, clindamycin, and erythromycin. The mecA gene defining methicillin-resistant Staphylococcus aureus, erm(C) and aac-aphD genes was found in 87.5% of each. Coagulase-negative staphylococci showed a high prevalence of mecA, blaZ and tetK genes. Other resistance-associated genes were found. All Streptococcus dysgalactiae isolates carried blaZ, erm(A), erm(B), erm(C) and lnuA genes, while Streptococcus suis harbored erm(C), aphA-3, tetL and tetM genes, additionally. In Streptococcus gallolyticus, most of these genes were found. The Streptococcus agalactiae isolate harbored blaZ, erm(B), erm(C), lnuA, tetK, tetL and tetM genes. Streptococcus agalactiae isolate was analyzed by DNA microarray analysis. It was determined as sequence type 14, belonging to clonal complex 19 and represented capsule type VI. Pilus and cell wall protein genes, pavA, cadD and emrB/qacA genes were identified by microarray analysis.


Introduction
Mastitis is an infection or inflammation of the mammary gland. It is the most common bacterial disease on dairy farms and leads to a reduction in milk production, with high economic losses due to high costs of treatment and the need of disposing of potentially contaminated milk. The prevention and treatment of mastitis lead to the administration of a considerable amount of antimicrobials to adult dairy cattle [1]. Microorganisms like Escherichia (E.) coli, different Staphylococcus (S.) and Streptococcus (St.) species are transmitted through colostrum to young calves and can cause gastrointestinal and pulmonary diseases. In some cases, this leads to the death of calves [2]. and streptococci in milk from cattle and buffaloes and the characterization of isolates is described concerning their phenotypic and genotypic resistance to antibiotics, because the knowledge about the situation in Egypt is limited.

Sample Collection and Cultivation
The present study was conducted in 2018 and 2019 on 50 milk samples of dairy animals from 50 different localities in Qalyubia and Monufia governorates in the Nile Delta region of Egypt. All dairy cattle and buffaloes were local Egyptian breed and kept by smallholders (1-5 animals) at different localities. They were hand-milked twice daily.
All animals were subjected to clinical examination. Animals with clinical mastitis were identified when one or more of the following signs were observed: cardinal signs of inflammation in one or more of udder's quarters, signs of systemic reaction, such as fever, depression, disturbed appetite and abnormal physical character of milk such as clot formation, discoloration, altered viscosity, aberrant smell or presence of blood. Due to the absence of observable clinical signs in animals with subclinical mastitis, the presumptive diagnosis was done based on laboratory diagnostic tests of milk samples, including the California mastitis test (CMT).
Milk samples were taken after washing and drying of the udder. Teat ends were disinfected with cotton swabs soaked in 70% ethanol. The first few streams were discarded. Approximately 10 mL of milk from each udder quarter were put into sterile tubes. Samples were transported to the laboratory on ice and stored at 4 • C for subsequent bacteriological analyses according to National Mastitis Council guidelines [17].
Isolation of bacteria from milk samples was carried out as described by the National Mastitis Council [17]. A loopful of milk sample was streaked on blood agar (Oxoid Deutschland GmbH, Wesel, Germany), supplemented with 5% sheep red blood cells and then subcultured on selective media: Mannitol Salt Agar, Edwards Medium and Brilliance ESBL Agar (Oxoid Deutschland GmbH), for the identification of expanded-spectrum beta-lactamase (ESBL)-producing microorganisms. All plates were incubated aerobically at 37 • C for 24 h. The plates were examined for colony morphology, pigmentation and hemolytic characteristics after 24-48 h.

MALDI-TOF MS
Isolates were identified using MALDI-TOF MS [18]. Briefly, bacteria from overnight cultures were suspended in 300 µL of bi-distilled water and mixed with 900 µL of ethanol (96% vol/vol; Carl Roth GmbH, Karlsruhe, Germany) for precipitation. After centrifugation for 5 min at 10,000× g, the supernatant was removed and the pellet was re-suspended in 50 µL of 70% (vol/vol) formic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Fifty microliters of acetonitrile (Carl Roth GmbH, Karlsruhe, Germany) were added, mixed and centrifuged for 5 min at 10,000× g. One and a half microliters of the supernatant were transferred onto an MTP 384 Target Plate Polished Steel TF (Bruker Daltonik GmbH, Bremen, Germany). After air-drying the material was overlaid with 2 µL of a saturated solution of α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in a mix of 50% acetonitrile and 2.5% trifluoroacetic acid (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). After air-drying spectra were acquired with an Ultraflex instrument (Bruker Daltonik GmbH, Bremen, Germany), the instrument was calibrated with the IVD Bacterial Test Standard (Bruker Daltonik GmbH, Bremen, Germany). An analysis was carried out with the Biotyper 3.1 software (Bruker Daltonik GmbH, Bremen, Germany). An interpretation of results was performed according to the manufacturer's recommendation: score of ≥ 2.3 represented reliable species-level identification; score 2.0-2.29, probable species-level identification; score 1.7-1.9, probable genus-level identification, and score ≤ 1.7 was considered an unreliable identification.

DNA Extraction
Genomic bacterial DNA was prepared from colonies with typical growth and subculture on blood agar. A loop-full of bacteria was added to 0.2 mL aliquot of lysis enhancer A2 dissolved in lysis buffer A1 (both from the StaphyType Kit, Alere Technologies GmbH, Jena, Germany), followed by incubation for 60 min at 37 • C and 550 rpm in a thermomixer.
For streptococci, 10 µL of achromopeptidase (Sigma-Aldrich Chemie GmbH; 100 units dissolved in 5 mL of phosphate-buffered saline (PBS) and stored frozen in small aliquots) and 5 µL of lysozyme (10 mg/mL bidistilled water) were used for lysis and incubated at 37 • C and 550 rpm in a thermomixer.
After lysis, the samples were processed using the High Pure PCR Template Purification Kit (Roche Diagnostics, Mannheim, Germany), according to the instructions of the manufacturer.

Microarray Analysis
For the analysis of streptococci, a microarray specifically developed and validated for St. agalactiae (Alere Technologies GmbH, Jena, Germany) was used, targeting group B streptococci virulence-associated markers and resistance-associated genes. Additionally, macrolide/lincosamide, tetracycline, and heavy metal resistance genes, genes associated with phages and gene motility were included. Protocols, data interpretation, and evaluation have been described previously [36]. Briefly, a linear and thermally synchronized primer elongation reaction was used for labeling. A mix of 1 to 2 µg of unfragmented RNA-free target DNA, 1.5 µL of a primer mixture (0.135 µmol/L each), dNTP mix, Taq DNA polymerase, and biotin-16-dUTP was amplified and labeled, using the following program: an initial denaturation at 96 • C for 5 min was followed by 55 cycles (60 s at 96 • C, 20 s at 50 • C, and 40 s at 72 • C). After washing, the hybridization of labeled DNA samples in ArrayStrips (Alere Technologies GmbH, Jena, Germany) was carried out. After washing, the microarrays were incubated with 100 µL of horseradish peroxidase-streptavidin mixture for 15 min. A repeated washing step followed. Finally, 100 µL of a precipitating substrate were added. After 5 min of incubation at room temperature (without shaking), the substrate was removed and the arrays were scanned and analyzed using the ArrayMate reader (Alere Technologies GmbH, Jena, Germany), using a specific software.

Antimicrobial Susceptibility Profiles of Staphylococci
All Staphylococcus isolates, 8 S. aureus, and 30 CoNS, were examined for their susceptibility to 22 antimicrobial agents. Table 4 shows that S. aureus isolates had high resistance rates to penicillin (87.5%), ampicillin, clindamycin, and erythromycin (75.0% each), respectively. All S. aureus isolates were fully susceptible to ceftaroline, teicoplanin, and vancomycin. Resistance rates to other antibiotics ranged between 25.0% and 62.5%.

Detection of Resistance-associated Genes in Staphylococci
All S. aureus isolates harbored the blaZ gene associated with penicillin resistance, the tetK gene associated with tetracycline resistance and valS often found in the optrA operon connected with linezolid resistance (Table 5). Other frequently detected resistance determinants were mecA associated with β-lactam resistance, erm(C) for erythromycin resistance and aac-aphD responsible for aminoglycoside resistance in 87.5% of all isolates. The erm(B) (resistance to erythromycin) and msrC (macrolide resistance) genes were also found frequently, with 75.0%. Table 5. PCR results for detection of resistance-associated genes of staphylococci. Non-Staphylococcus aureus isolates exhibited a high prevalence of resistance genes mecA, blaZ and tetK, with 96.6%, 80.0%, and 73.3%, respectively. Approximately half of the isolates harbored aac-aphD, erm(C) and erm(B) genes.

Microarray Analysis of Streptococcus Isolates
Six Streptococcus isolates of different species were tested with a microarray system. This system was developed and validated exclusively for St. agalactiae and only for this isolate (19CS0081) was a valid result obtained. The other Streptococcus isolates did not harbor more antibiotic resistance-associated genes, as detected by PCR investigation.
Isolate 19CS0081 was a St. agalactiae strain belonged to clonal complex (CC) 19. The sequence type (ST) was 14. The isolate represented capsule type VI, but it obtained a hybridization signal with one of the capsule III probes (cpsG-III), too. Belonging to the alpha antigenic cell wall protein genes, the alp-5 gene was detected. Pilus protein genes pilA1, pilB1 and pilC1 could be detected. From the group of surface proteins, the sip gene (surface immunogenic protein) was detected, as well as MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesin protein gene pavA (fibronectin-binding protein gene). Besides the antimicrobial resistance-associated genes heavy metal resistance marker cadD encoding cadmium resistance protein D and multidrug resistance transporter genes, emrB/qacA were found in the St. agalactiae isolate.

Discussion
Staphylococci are major causative agents of clinical or subclinical bovine mastitis and generate important losses in the dairy industry in Egypt [37]. They are also considered as a risk factor for food poisoning in humans [38].
In this study, 38 Staphylococcus isolates were cultivated from 50 milk samples, whereas 32 came from cattle and buffaloes with clinical signs of mastitis and 18 from subclinical mastitis cases, respectively. The prevalence of staphylococci with 75.0% and 77.7% nearly agreed with the data of Dorgham et al. [39]. The authors detected staphylococci in 68.8% and 62.5% of milk samples from Egypt. The milk came from cattle, buffaloes, and goats with clinical and subclinical mastitis. Others also reported rates of 52.0% and 67.0% Staphylococcus positive milk samples, in cases of clinical and subclinical bovine mastitis in Egypt [40].
Eight isolates (16.0%) were identified as S. aureus, which was similar to other studies on mastitis milk samples in Egypt who reported about 16.1%, 14.9% and 25.8% S. aureus positive samples, respectively [41][42][43]. Other previous studies have confirmed that S. aureus and St. agalactiae were the most prevalent causative agents of mastitis in Egypt [44][45][46].
Several Staphylococcus species, like S. warneri, S. pasteuri, S. xylosus, and others were found in the milk samples of cattle and buffaloes. A similar spectrum of species was found in milk of cattle and buffaloes with subclinical mastitis, namely S. intermedius, S. xylosus, S. epidermidis, S. hominis, S. sciuri, S. hyicus, S. lugdunensis and S. simulans [47]. These reports complete the results of investigations made in cattle herds with mastitis problems, where S. chromogenes, S. hyicus, S. simulans, S. epidermidis, S. hominis, S. haemolyticus, S. xylosus, S. warneri, S. sciuri, S. capitis, S. saprophyticus, and S. lentus were found [48][49][50]. They assured that the environment was found as a reservoir, suggesting that intra-mammary infection with such bacterial species is possibly considered as an environmental hazard.
Streptococci form a large group of organisms. Some of them are associated with bovine udder infections. The most common pathogens causing bovine mastitis are St. agalactiae, St. dysgalactiae and St. uberis [51]. In the present study, in 12.0% of milk samples, Streptococcus isolates were detected, which was similar to results from China, where 8.7% of dairy cattle found positive for Streptococcus species [52]. Atypical streptococci found and connected with mastitis were St. suis and St. gallolyticus.
More than a single pathogen was detected in some of milk samples. There were cases with two or three different Staphylococcus species, as well as mixed infections of staphylococci and streptococci. This makes it difficult to identify the true mastitis causing agent. As a result, a possible antibiotic treatment is dependent on the antibiotic resistance of the different bacteria.
Seven out of eight isolates carried the mecA gene, which is defining them as methicillin-resistant S. aureus (MRSA). This is a high rate of MRSA from milk samples and this observation might indicate an uncontrolled usage of antibiotics. The results were supported by other investigations, which described the presence of mecA gene in phenotypic β-lactam-resistant S. aureus isolates in more than half of Egyptian milk samples originating from mastitis cases [53,54]. In contrast, an investigation on milk samples from Switzerland and Germany resulted in only 2 MRSA out of 128 isolates [55]. The presence of mecA gene in S. aureus isolates from bovine milk have been reported in previous studies from India [56], China [57], Italy [58], Tunisia [59] and Brazil [60]. MRSA isolates showed not only resistance to methicillin/oxacillin and other β-lactams; they were also resistant to other antibiotics as aminoglycosides, macrolides and/or quinolones [61]. In this study, all MRSA isolates showed multidrug resistance, which is in agreement with previous reports on MRSA isolated from dairy products [37,53,62,63]. The mecB and mecC genes also responsible for methicillin resistance were not detected. They were found only sporadically in S. aureus isolates from domestic animals yet, although in a Bavarian dairy herd, multiple cases of methicillin resistant CC130 S. aureus were found harboring the mecC gene [64].
Penicillin resistance of Staphylococcus species is usually mediated by blaZ gene about enzymatic hydrolysis of β-lactam ring of antibiotics. It was detected in all S. aureus isolates and 73.3% of non-S. aureus isolates, from which a high percentage showed phenotypic resistance to penicillin. High prevalence of blaZ gene in penicillin-resistant staphylococci was reported from Egypt [53], China [57] and Brazil [65]. Similar results were obtained with Polish Streptococcus isolates [66]. Resistance to penicillin G is very important, because this antimicrobial agent is the most recommended and used antibiotic for the treatment of staphylococcal mastitis. Increasing resistance to penicillin G can be explained by the uncontrolled use of antimicrobial agents in Egypt.
Resistance to macrolides (such as erythromycin) occurred among staphylococci in this study. Resistance to this group of antibiotics is conferred via a variety of mechanisms. Three related determinants, erm (A), erm (B), and erm (C) genes, have been identified to be mainly responsible for erythromycin resistance [67]. In this study, the three erm genes were detected in 25.0% to 87.5% of the S. aureus isolates. This is comparable to the results described previously [35]. Additionally, the macrolide resistance gene msrC was prevalent and the percentage in S. aureus was much higher than in other staphylococci. The erm (B) and erm (C) genes occurred in all Streptococcus isolates, which reflects the situation reported by other authors [68][69][70]. The main cause for this result may be due to the localization of these genes on transposons and the ability of a transfer from bacteria to another, including streptococci by horizontal gene transfer [68]. Erm (TR) gene was not detected in this study, which was in agreement with the results of Dogan et al. [71]. They described the absence of the erm (TR) gene in bovine streptococci, however, it could be detected in human isolates.
Aminoglycoside resistance (including gentamicin) was frequently detected in staphylococci and is caused, to a great extent, by the presence of aac-aphD genes, as previously described for Chinese isolates [57]. Aminoglycoside resistance in streptococci is mediated by aad-6 and aphA-3 genes, resulting in the enzymatic inactivation of antibiotics. Both genes were not detected in the St. agalactiae isolate, but at least one was present in other isolates, which is equal to results from Poland [66].
Tetracycline resistance genes spread among bacteria and can be found regularly in multidrug resistant bacteria [72]. In the current study, tetK, tetL, and tetM genes were frequently detected, which corresponded with reports from China for S. aureus [57] and isolates from Egypt for other CoNS [47]. The cause is the extensive and frequent usage of tetracycline in mastitis treatment and as prophylaxis to reduce bacterial infections in general in Egypt, which leads to increased resistance to this antimicrobial [73]. Tetracycline resistance-associated genes (tetK, tetL, tetM, and tetS) in streptococci were detected in all isolates, in which St. gallolyticus harbored all four genes. All isolates carried tetL and tetM genes, which was described equally for Polish isolates [66].
Linezolid is one of the few clinically effective drugs for the control of MRSA and MRCoNS infections. Resistance to linezolid in staphylococci is still very rare, but has been increased in recent years [74]. Transferable linezolid resistance due to the presence of the cfr gene has been known since the year 2000. The cfr gene was first discovered in a bovine S. sciuri isolate and was later reported in various Staphylococcus species. In this study, the cfr gene was not detected. The result was in agreement with a study on Staphylococcus isolates from chicken meat in Egypt [75].
Linezolid and phenicol resistance can be mediated by the optrA gene, too [76]. Phenotypic resistance to linezolid was detected in 50.0% of S. aureus and 70.0% of non-S. aureus isolates and the optrA gene was frequently detected by PCR. Previously, the presence of optrA gene connected with resistance to linezolid in Staphylococcus isolates was reported [77,78]. This result is alarming because linezolid is an antibiotic with good activity against MRSA, making it desirable for the treatment of staphylococcal infections [74].
Vancomycin resistance in staphylococci is very important, because vancomycin is becoming the final choice for the treatment of MRSA infections in humans [79]. Phenotypic vancomycin resistance in S. aureus was not determined [80], however, it was detected in other staphylococci, as described before [75]. In this study, the discrepancy between phenotypic resistance and the presence of resistance genes was noticeable. More than 60% of S. aureus isolates carried the vanC1 gene, but did not show phenotypic resistance determined by the broth microdilution method. A similar situation arises with other staphylococci.
Although erm genes responsible for macrolide-lincosamide-type B streptogramin-associated resistance in Streptococcus species, lincosamide resistance was mediated by the presence of specific genes (lnu), which cause enzymatic inactivation of the drug due to nucleotide transferases. This mechanism was detected for the first time for Enterococcus faecium and thought to be exclusively present in this species, although later on, it was detected in other species like St. agalactiae and St. uberis [81,82]. In this study, the lnuA gene was detected in all streptococcal isolates, as it was identical with the results of a previous investigation [66].
Antibiotic resistance is increasing, because treatment of mastitis generally occurs by using antibiotics without testing of antibiotic susceptibilities of causative bacteria [83]. In this study, 75.0% of S. aureus isolates detected were multidrug-resistant and revealed a high phenotypic resistance to penicillin, ampicillin, and erythromycin/clindamycin, thus have been reported by [53] for S. aureus isolates and by [63], too. A similar situation was described for Egyptian CoNS isolates from cattle, buffaloes, and goats [39].
In this study, we used a microarray-based assay for the simultaneous detection of typing markers, resistance determinants and clinically relevant virulence factors in St. agalactiae. The system was developed especially for this species. For other Streptococcus species, only the probes for resistance determinants were useful, but gave no advantage in comparison to PCR assays. For St. agalactiae, it was possible to determine the ST and CC of the isolate. With DNA microarrays, it is possible to genotype isolates. The method is less cumbersome than MLST, in which an isolate is characterized after sequencing of seven house-keeping genes. Here, hybridization patterns for the St. agalactiae isolate appear to correspond to ST14 within CC19. CC19 is the most common but very diverse clonal complex. In a study about German St. agalactiae, 75% of human isolates and 32% of bovine isolates belonged to this CC [36]. The determined capsule type VI is a very rare type and was found in a German strain collection, only once in an isolate of human origin.
In Egyptian milk samples which were obtained from cattle and buffaloes of small farmers, several zoonotic bacteria like staphylococci, streptococci, E. coli, and enterococci were detected. Normally, the milk is directly consumed by farmers, sold to consumers or used for the production of soft cheese, etc., without pasteurization. The consumption of dairy products entails a health hazard, because some of the bacteria are able to infect humans or are causing agents of food poisoning. Reinforced is the risk by the increasingly occurred resistance of bacteria during the last decades.
In this study, multidrug-resistant isolates of different bacterial species were confirmed via phenotypic characterization as well as the detection of resistance determinants. One source of reinforced occurrence of resistant bacteria is to look for inexpertly and excessively use of antibiotics in veterinary medicine. Here, the consumption of antibiotics has to be reduced, veterinarians and farmers need to be trained in the use of antimicrobials, a diagnosis must be made before therapy, and last but not least, milk must be pasteurized. Additionally, governmental monitoring tools can help to reduce antibiotic usage.