Abstract
Background:
Given the abuse of broad-spectrum agents in the treatment of clinical bovine mastitis, coagulase-negative Staphylococci (CNS) have emerged to be of clinical and epidemiological significance.
Aims:
The study aimed to identify CNS and Staphylococcus aureus in incurable clinical mastitis in 50 cattle and 90 buffaloes, determine antibiotic resistance profile, and biofilm-forming ability of CNS and S. aureus isolates.
Methods:
140 milk samples were collected from four villages in Sharkia, Egypt, for bacteriological isolation and molecular investigations.
Results:
Forty-nine Staphylococcus isolates were identified, including 11 CNS and 38 coagulase-positive S. aureus. The most recorded CNS strains were S. epidemidis (3), S. simulans (2), S. hominis (2), S. chromogen (2), S. xylosus (1), and S. warneri (1). A 63.2% of S. aureus and 27.3% of CNS isolates showed the ability to form biofilm, which was confirmed by ica PCR. S. epidemidis and S. chromogen were extensively drug-resistant, and most S. aureus isolates showed multidrug resistance (MDR). The proportion of methicillin-resistant was lower among S. aureus (84.2%), compared with CNS (90.9%).
Conclusion:
CNS present a challenge due to their uprising resistance compared with S. aureus. The appearance of CNS-MDR strains carrying ica gene leads to treatment protocol failure on bovine farms and improper control of bovine mastitis.
Key Words: Antibiotics resistance, Biofilm formation, Clinical mastitis, CNS
Introduction
Mastitis, an infectious disease affecting dairy animals, leads to significant economic losses due to reduced milk quality, treatment costs, and unresponsive cases (Wyder et al., 2011; Asli et al., 2017).
Staphylococci are the most common cause of bovine mastitis; where Staphylococcus aureus is associated with a more severe illness than coagulase-negative Staphylococci (CNS) (Bradley et al., 2007).
CNS is a global cause of bovine mastitis, with increasing frequency in dairy cattle (Klibi et al., 2018). Many CNS species, including S. xylosus, S. sciuri, S. saprophyticus, S. chromogenes, S. warneri, and S. epidermidis, have incriminated in causing mastitis (El-Ashker et al., 2020; Lee and Lee, 2022), and showed antibiotic resistance and biofilm production (Raspanti et al., 2016; Lee and Lee, 2022).
Methicillin-resistant (MR) Staphylococci are mostly caused by spread of mecA gene, which hampers treatment of MR-CNS and S. aureus infections and poses a public health hazard (Elhaig and Selim, 2015; Wang et al., 2015).
In recent years, multidrug-resistance has been observed in buffaloes and cattle infected with coagulase positive Staphylococci and CNS (Dorgham et al., 2013). Proper use of antibiotics is an essential strategy to control bovine mastitis (Klibi et al., 2018).
In Egypt, the prevalence of CNS in dairy bovine is limited, with few studies reporting CNS from milk (El-Jakee et al., 2013; El-Ashker et al., 2020; Ibrahim et al., 2022) and no reports about CNS and their biofilm-forming ability in the studied regions, so far. Therefore, the present study investigated the presence of CNS in cattle and buffalo milk with clinical mastitis in Minya al-Qamh, Sharkia, Egypt, to determine antibiotic resistance and methicillin-resistant (MR) among isolated S. aureus and CNS strains.
Materials and Methods
Ethics statement
The study protocol was approved by the Ethics Committee of Faculty of Veterinary Medicine, Suez Canal University (approval No.: 2023022).
Sampling and study area
A total of 140 milk samples were randomly taken from 50 cows and 90 buffaloes from four villages, Minya al-Qamh, Sharkia governorate (30.7°N 31.8°E), Egypt, during 2021-2022. The samples were taken from animals with clinical mastitis, showing udder redness, swelling, change in milk characteristics, and unresponsive antimicrobial treatments. Milk samples (15 ml) were collected in sterile tubes after washing the teat ends with water and rubbing with 70% ethanol and discarding the first milk streams. Then, samples were transported in an ice tank and processed in the bacteriological laboratory, Bacteriology Department, Faculty of Veterinary Medicine, Suez Canal University.
Isolation and characterization of Staphylococcus spp.
A heavy loopful of the specimen was inoculated onto nutrient agar, blood agar, Staphylococcus agar, and mannitol salt agar plates, incubated at 37°C for 24-48 h, and examined for bacterial growth and an in-vitro antimicrobial susceptibility. Isolates were identified according to previous protocols (Kalorey et al., 2007). Identified isolates were subjected to Gram staining and biochemical confirmation by the VITEK 2 system. The VITEK 2 compact instrument (bioMérieux, France) was used according to the method of Spanu et al. for accurate biochemical identification of the obtained colonies (Spanu et al., 2003; Wahdan et al., 2022).
PCR experiments
DNA was extracted using QIAamp DNA mini kits (QIAGEN, Hilden, Germany). Genus-specific PCR was used to detect Staphylococci by amplifying 570 bp of the 16S rRNA gene using 16S rRNA-F: GCA AGC GTT ATC CGG ATT T and 16S rRNA-R: CTT AAT GAT GGC AAC TAA GC (Al-Talib et al., 2009). Species-specific PCR for detection of S. aureus was performed to amplify nuc gene (270 bp) using the following primers: nuc-F: 5´-GCG ATT GAT GGT GAT ACG GTT-3´ and nuc-R: 5´-AGC CAA GCC TTG ACG AAC TAA AGC-3´ (Brakstad et al., 1992). A positive control (confirmed S. aureus from Animal Health Research Institute, Dokki, Egypt) and a negative control (sterile DNA-free water) were included. PCR amplicons were visualized on 1.5% agarose gel stained with ethidium bromide (0.5 μg/ml) and examined using gel documentation system (Biospectrum UVP, UK).
Detection of MR strains by PCR
Detection was performed by PCR targeting mecA gene (553 bp) using mecA1: 5´-AAA ATC GAT GGT AAA GGT TGG C-3´ and mecA2: 5´-AGT TCT GCA GTA CCG GAT TTG C-3´ (Murakami et al., 1991).
Detection of biofilm formation by PCR
Detection was performed by PCR targeting ica gene (1315 bp) using forward primer 5´-CCT AAC TAA CGA AAG GTA G-3´ and reverse primer 5´-AAG ATA TAG CGA TAA GTG C-3´ (Ciftci et al., 2009).
Antimicrobial susceptibility test
The antimicrobial susceptibility of recovered isolates was assessed through disc diffusion method, according to the guidelines of Clinical Laboratory Standards Institute (CLSI). Ciprofloxacin (CIP, 5 μg), imipenem (IMP, 10 μg), doxycycline (DOX, 10 μg), erythromycin (E, 15 μg), gentamicin (CN, 10 μg), ampicillin (AMP, 10 μg), tetracycline (TE, 30 μg), cefoxitin (FOX, 30 μg), vancomycin (VA, 30 μg), and clindamycin (DA, 2 μg) (Oxoid, Basingstoke, UK) were used. Results (resistant, intermediate resistance, or sensitive to antimicrobials) were given in accordance with CLSI guidelines (CLSI, 2015). Cefoxitin-resistant Staphylococci classified as MR.
Statistical analysis
The study used PAST statistical analysis 4.03 to identify genetic similarities and relatedness among confirmed isolates (analysis was based on presence of 16S rRNA, nuc, mecA, and ica genes), with Chi-square (http://vassarstats.net) calculated to evaluate significant differences between cattle and buffaloes, and MedCalc Software Ltd. used for 95% confidence intervals.
Results
Phenotypic and genotypic characterization of recovered isolates
Forty-nine (49/140, 35%) Staphylococci isolates were isolated and identified, with 38 (38/49, 77.6%) identified S. aureus, based on nuc-PCR, and 11 (11/49, 22.4%) as CNS (Table 1). Six species of CNS were classified, and their distribution is shown in Table 2, with S. epidermidis being the most prevalent. The detection rate was not-significantly higher in cattle (38%) than buffaloes (33.3%). The study found that 24 (63.2%) of S. aureus and 3 (27.3%) of CNS strains carried ica gene (Table 2). Thirty-two (84.2%) S. aureus and 10 (90.9%) CNS isolates harbored mecA gene.
Table 1.
Results of Staphylococci isolation by bacterial culture from 140 clinical mastitic milk samples
| Animals | Bacterial culture | P-value | Distribution of Staphylococci in animal samples (%) | ||
|---|---|---|---|---|---|
| Pos., n (%) | 95% CI | S. aureus | CNS | ||
| Cows (n=50) | 19 (38) | 0.3-0.6 | 0.6NS | 15 (30) | 4 (8) |
| Buffaloes (n=90) | 30 (33.3) | 0.22-0.5 | 23 (25.6) | 7 (7.8) | |
| Total (n=140) | 49 (35) | 0.3-0.5 | 38 (27.1) | 11 (7.6) | |
NS The result is non-significant at P>0.05
Table 2.
Prevalence of ica gene, and resistance profile in CNS
| CNS spp. | Number | ica gene (%) | Antibiotic resistance profile |
|---|---|---|---|
| S. epidemidis | 3 | 1 (33.3) | DA, TE, AMP, FOX, DOX, CN, VA |
| S. simulans | 2 | 1 (50) | DA, TE, AMP, CN |
| S. hominis | 2 | 0 | DA, TE, AMP, E |
| S. chromogen | 2 | 1 (50) | DA, TE, AMP, FOX, E, CN |
| S. xylosus | 1 | 0 | AMP, TE, DA |
| S. warneri | 1 | 0 | Sensitive |
| Total | 11 | 3 (27.3) |
Antimicrobial susceptibility testing and MR distribution
Table 3 shows the distribution of S. aureus and CNS isolates against ten antimicrobial agents. Over 50% of S. aureus isolates showed resistance to tetracycline, clindamycin, cefoxitin, vancomycin, and ampicillin. CNS isolates showed high resistance to ampicillin, tetracycline, clindamycin, and gentamycin. Over 50% of S. aureus strains showed multidrug resistance (MDR), with an increase in resistance among CNS strains. S. epidemidis and S. chromogen showed extensive drug-resistance (XDR). S. simulans and S. hominis showed resistance to clindamycin, tetracycline, ampicillin, gentamycin, and erythromycin. S. xylosus showed some resistance to clindamycin, tetracycline, and ampicillin. In contrast, S. warneri remains sensitive to all tested antibiotics.
Table 3.
In-vitro comparison of antimicrobial susceptibilities between coagulase-positive and coagulase-negative Staphylococcus
| Classes | Antimicrobials | S. aureus (n=38) | CNS (n=11) | ||||
|---|---|---|---|---|---|---|---|
| S (%) | I (%) | R (%) | S (%) | I (%) | R (%) | ||
| Quinolones | CIP | 29 (76.3) | 5 (13.1) | 4 (10.5) | 11 (100) | 0 (0) | 0 (0) |
| Carbapenem | IMP | 31 (81.5) | 2 (5.2) | 5 (13.1) | 9 (81.8) | 2 (18.1) | 0 (0) |
| Tetracyclines | DOX | 28 (73.6) | 8 (21) | 2 (5.2) | 7 (63.6) | 3 (27.3) | 1 (9.1) |
| TE | 5 (13.1) | 0 (0) | 33 (86.9) | 2 (18.1) | 0 (0) | 8 (72.7) | |
| Macrolides | E | 30 (78.9) | 3 (7.8) | 5 (13.1) | 7 (63.3) | 2 (18.1) | 2 (18.1) |
| Aminoglycosides | CN | 31 (81.5) | 4 (10.5) | 3 (7.8) | 5 (45.4) | 1 (9.1) | 5 (45.5) |
| Lincosamides | DA | 6 (15.7) | 2 (5.2) | 30 (78.9) | 3 (27.3) | 1 (9.1) | 7 (63.6) |
| Glycopeptides | VA | 10 (26.3) | 3 (7.8) | 25 (65.7) | 9 (81.8) | 1 (9.1) | 1 (9.1) |
| Penicillins | AMP | 9 (23.6) | 10 (26.3) | 19 (50) | 1 (9.1) | 1 (9.1) | 9 (81.8) |
| Cephalosporins | FOX | 7 (18.4) | 3 (7.8) | 28 (73.6) | 5 (45.5) | 3 (27.3) | 3 (27.3) |
S: Sensitive, I: Intermediate resistance, and R: Resistance
Distribution of 16S rRNA, nuc, mecA, and ica genes among staphylococcal isolates
The cluster (Fig. 1) showed 21 S. aureus isolates (S1, S2, S3, S6, S7, S8, S9, S11, S16, S17, S19, S21, S22, S23, S25, S26, S28, S29, S31, S33, and S35) carrying 16S rRNA, nuc, mecA and ica genes. S. aureus isolates (S4, S13, S18, S30, S37, and S38) did not have mecA gene. S. aureus isolates (S1, S2, S3, S6, S7, S8, S9, S11, S13, S16, S17, S19, S21, S22, S23, S25, S26, S28, S29, S31, S33, S35, S37, and S38) carried ica gene. Three CNS isolates (S39, S44, and S46) carried 16S rRNA, mecA, and ica genes. The tested CNS did not carry nuc gene. All CNS carried the mecA gene, but the isolate S40 did not carry mecA gene.
Fig. 1.
Classical cluster showing distribution of 16S rRNA, nuc, mecA, and ica genes among 49 Staphylococcal isolates
Discussion
Staphylococcal mastitis is a major threat among dairy animals all over the world and causes health concerns for humans (Hoque et al., 2018). In Egypt, data about CNS involved in clinical bovine mastitis is limited. The study found that 22.4% of Staphylococci isolates were CNS, with the highest resistance to ampicillin (81.8%), tetracycline (72.7%), and clindamycin (63.6%). 91% of CNS isolates were methicillin-resistant by mecA PCR.
Bacterial culture results confirmed presence of Staphylococcus sp. in 35% of milk samples from bovine clinical mastitis, indicating a considerable association between Staphylococcus infection and clinical mastitis in livestock.
Current results showed that CNS could cause bovine clinical mastitis in 7.6% of milk samples, with lower than 11.3% reported in the USA (Gillespie et al., 2009) and 50% in Finland (Pitkälä et al., 2004).
In Egypt, CNS was found in 16.6% and 59.4% of cattle and buffaloes with subclinical mastitis, while CNS was not isolated from clinical mastitis, and S. aureus was the predominant isolate in cows, buffaloes, sheep, and goats (El-Jakee et al., 2013). Other studies reported variable rates of CNS occurrence in dairy animals with clinical or subclinical mastitis, with rates of 11.76% in Damietta (Hussein et al., 2018), 12.4% in Dakahlia (El-Ashker et al., 2020), and 44.12% in Giza (Ibrahim et al., 2022).
Table 2 shows six species of CNS with varying prevalence rates. Previous reports have indicated discrepancies in CNS species patterns. In Egypt, S. sciuri being the most prevalent, followed by S. chromogenes, S. haemolyticus, S. xylosus, S. hyicus, and S. warneri in bovine mastitis (El-Ashker et al., 2020). In Argentina, S. chromogenes and S. haemolyticus were the predominant species (Raspanti et al., 2016). This may be due to the ability of some species to adapt to udder tissue or due to differences in control strategies (Raspanti et al., 2016).
In this study, 27.3% of CNS isolates showed the ability to form biofilms. Lee and Lee (2022) found 78.4% of CNS isolates from tank milk in Korea formed biofilms. While Tremblay et al. (2013) in Canada and Srednik et al. (2017) in Argentina reported >44% of CNS isolates from bovine mastitis formed a moderate to strong biofilm.
Interestingly, the capability of S. aureus to produce biofilm was higher than CNS isolates, a finding like that found in Egypt (Raheel et al., 2023).
The dominance of S. aureus among the causative agents of mastitis in cows and sheep has been reported earlier (El-Jakee et al., 2013), supporting our findings. This possibly due to its presence inside or outside the udder or a misclassification bias related to CMT (Dingwell et al., 2003). Clinical mastitis due to S. aureus has a significant local or global prevalence, with higher rates in Egypt reaching 38.3% in Ismailia (Elhaig and Selim, 2015) and 42% in Dakahliya and Damietta (Awad et al., 2017), and lower rates in Sadat, Egypt; 11.2% (Elsayed et al., 2015) and India; 50% (Nigam, 2015). These variations may be attributable to geographical and management differences, as well as the use of different methodologies (Li et al., 2017).
The infection rate of both CNS and S. aureus was higher in cattle (38%) than in buffalo (33.3%), which is similar to a previous study from Dakahlia, Egypt (El-Ashker et al., 2020). Conversely, in two studies on mastitis from Egypt, the prevalence of S. aureus was higher in buffalo (50%) than cattle (39.29%) (Elsayed et al., 2015) and CNS was lower in cattle (16.6%) than buffalo (59.4%) (El-Jakee et al., 2013). Such cattle-related findings are possibly due to the use of suboptimal hygiene practices during the milking process in the study area. The study findings present a challenge when it comes to comparing them with other studies due to variations in S. aureus’s role in mastitis, influenced by factors such as animal handling practices and hygiene levels (Jayarao et al., 2004).
Treatment and control of bovine mastitis caused by Staphylococci often fail, due to the complex nature of the organism and its increasing resistance to drugs (Wang et al., 2015). In this study, MDR S. aureus and XDR CNS were reported to have uprising resistance against tested antimicrobial groups, posing concerns in the veterinary field and highlighting potential risks.
El-Ashker et al. (2020) found that the majority of CNS showed low rates of resistance genes, while S. haemolyticus and S. warneri showed resistance to more than three antibiotics; however, in our study, S. epidermidis and S. chromogen showed resistance to more than four antibiotics, and S. warneri did not show resistance to any of the antibiotics examined. Klibi et al. (2018) found high antimicrobial resistance in CNS, primarily to beta-lactams and tetracycline in cows with mastitis, possibly due to the use of these antibiotics in Tunisia. In China and Bangladesh, S. aureus causes bovine mastitis was resistant to various antimicrobials; erythromycin, clindamycin, penicillin, trimethoprim/ sulfamethoxazole, oxytetracycline, and doxycycline (Wang et al., 2015; Li et al., 2017; Hoque et al., 2018). Moreover, several CNS strains were resistant to DA, TE, AMP, FOX, DOX, CN, and VA; possibly due to frequent and improper use of antibiotics, which may explain the failure of treatment in this study.
The mecA gene was detected in 42 isolates, confirming MR in 90.9% of CNS and 84.2% of S. aureus isolates, a higher percentage than in Korea (Lim et al., 2012) and China (Li et al., 2017). These variations may be attributable to the effects of animal populations, methodologies, and geography (Li et al., 2017).
Wide resistance to antimicrobials among S. aureus and CNS strains from clinical bovine mastitis, and the identification of most S. aureus and CNS strains as MR, poses a significant challenge to treatment (Wang et al., 2015; Shah et al., 2019). Continuous observation of antimicrobial resistance of CNS and S. aureus is required since our study reported a significant positive correlation between resistance to cefoxitin and the presence of mecA and nuc genes among recovered isolates.
The study reported a high prevalence of S. aureus and CNS in incurable mastitis cases in the Sharkia governorate, Egypt. Poor hygienic conditions lead to the detection of CNS and S. aureus, which are more resistant to several antimicrobials, indicating treatment failure. Some CNS were encoded for a specific gene responsible for adhesion intramammary. The latter finding has potential consequences regarding vets’ decisions to treat animals for mastitis, particularly if no bacterial culture analysis is performed for comparison. The restricted locality is a limitation of this study. Further studies with a larger sample size and early diagnosis of mastitis etiology could improve outcomes, treatment, and control measures.
Acknowledgements
The authors extend their appreciation to the veterinarians for collecting the samples.
Conflict of interest
The authors declare that they have no conflicts of interest.
References
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