BACTERIOLOGICAL STUDY ON STAPHYLOCOCCAL BOVINE CLINICAL MASTITIS WITH REFERENCE TO METHICILLIN-RESISTANT STAPH. AUREUS (MRSA)

BACTERIOLOGICAL STUDY ON STAPHYLOCOCCAL BOVINE CLINICAL MASTITIS WITH REFERENCE TO METHICILLIN-RESISTANT STAPH. AUREUS (MRSA)

SAYED, S.M.

Animal Health Research Institute, Assiut Regional Laboratory

Email: smhamuda@yahoo.com

INTRODUCTION

Mastitis is one of the major challenges of the dairy industry. Staphylococcus aureus (S. aureus) is one of the most important pathogens causing mastitis in dairy cattle (Moon et al., 2007; Nam et al., 2011). Methicillin resistant Staphylococcus aureus (MRSA) has been recovered from dairy cattle in Korea (Moon et al., 2007; Nam et al., 2011 and Lim et al., 2013); Turkey (Ciftci et al., 2009); Netherland (Tavakol      et al., 2012); Iran (Ahmady and Kazemi, 2013) and Uganda (Kateete et al., 2013). Several efforts to remove this pathogen from farms are hampered by some factors, where one of these factors is antibiotic resistance. One of the major mechanisms of resistance to β-lactam antibiotics is β-lactamase producing by staphylococci. This enzyme hydrolyzes the β- lactam ring and causes inactivation of β-lactams. In the early 1950s, it has been aware of the effectiveness of penicillin in treatment of S. aureus infections because of β-lactamase producing plasmids. In 1959, methicillin, synthetic, penicillinase –resistant penicillin, was introduced and solved problems in clinical practice, for a time. However, by 1960, S. aureus strains were found to be resistant to the new semisynthetic β-lactams (methicillin, oxacillin, flucloxacillin), and became known as methicillin-resistant S. aureus (MRSA). This type of resistance was termed “intrinsic resistance” because it was not due to destruction of the antibiotic by β-lactamase (Chambers, 1997). Methicillin resistance in S. aureus is mediated by the production of an altered penicillin-binding protein (PBP2a), a transpeptidase. mecA encodes this enzyme involved in cell wall peptidoglycan synthesis. Unlike conventional PBPs of S. aureus, PBP2a does not bind to β-lactam antibiotics with high affinity (Hiramatsu et al., 2002).

It is considered that the first step in mastitis progress is adhesion of S. aureus to mammary epithelial cells and slime factor plays an important role for adhesion and colonization (Vasudevan et al., 2003). Production of slime factor also plays an important role in antibiotic resistance and it has been reported that slime producing strains are more resistant to antibiotics than non-slime producing strains (Amorena et al., 1999). Intercellular adhesion is encoded in the ica locus containing icaA, icaB, icaC, icaD genes in S. aureus strains (Yazdani et al., 2006). icaA gene encodes N-acetylglucosaminyl transferase, further, icaD plays an important role in expression of this enzyme. icaA and icaD were found to be in high prevalence among S. aureus mastitis isolates and this finding confirms that ica locus has a potential role as a virulence factor in the pathogenesis of mastitis in ruminants (Vasudevan et al., 2003).

This study was undertaken to determine the bovine mastitis Staph. Spp., their resistance to antimicrobial agents approved for its control and to determine the methicillin resistance and slime factor production of S. aureus in bovine mastitis phenotypically and genotypically for mecA, icaA and icaD genes.

MATERIALS and METHODS

Milk samples:

A total of 101 milk samples were collected from 101 cows, at various private farms in Assiut, Egypt, showing clinical signs of mastitis. All samples were taken under aseptic conditions and transferred in ice box to laboratory as soon as possible.

Isolation and identification of bacterial isolates:

Amount of 0.01 ml of each milk samples was cultured on blood agar with 5% sheep blood, Mannitol salt agar (BBL), Baird-Parker medium (Oxoid) and MacConkey agar (Biomark Lab. India) which incubated at 37°c for 48 h. The suspected colonies were identified: morphologically, by Gram's stain and biochemically confirmed by using catalase activity, coagulase test as well as Novobiocin (5 µg) and polymixin-ß sulphate (300 U) sensitivity tests, according to Quinn et al. (2004). 

Phenotypic detection of methicillin resistance:

Disc diffusion sensitivity testing was performed according to the Kirby-Bauer method, as described in the guidelines of the National Committee for Laboratory Standards (NCCLS, 2000), using discs (Bioanalyse-Turkey) containing Oxacillin (OX) 1 µg, Ampicilin (AM) 10 µg, Cefotaxime (CTX) 30 µg, Cloxacillin (CX) 1 µg, Doxycycline (DO) 30 µg, Enrofloxacin (ENR) 5 µg, Gentamicin (CN) 10 µg, Lincomycin (L) 2 µg,  Oxytetracycline (T) 30 µg, Penicillin (P) 10 µ and Trimethoprim – Sulflamethaxzole (SXT)  25 µg.  For Oxacillin susceptibility determinations, inhibition zones around the disc were measured after 24 and 48 h using the following breakpoints: susceptible (S) ≥ 18 mm; resistance (R) ≤ 17 mm (Quinn et al., 2004).

Slime production assay:

Slime production assay was performed by cultivation of ten S. aureus strains, which were methicillin resistant by phenotypic test, on Congo Red Agar (CRA) plates containing 0.8 g of Congo Red dye, 21 g Mueller-Hinton broth, 15 g granulated agar and 36 g sucrose per Liter distilled water. Strains were inoculated on CRA plates and incubated for 24-72 h at 37ºC. Slime producing strains and non-slime producing strains constitutes rough black colonies and red colonies on CRA, respectively (Yazdani       et al., 2006).

PCR for detection of mecA, icaA and icaD genes:

Detection of mecA, icaA and icaD genes was performed on those ten S. aureus isolates, which were methicillin resistant by phenotypic test, as follows:

I- DNA extraction: DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

II- Oligonucleotide Primers: Primers encoding for mecA, icaA and icaD genes were supplied from (Metabion, Germany) are listed in Table (1).

Table 1: Primers sequences, target genes, amplicon sizes.

III- PCR amplification: Primers were utilized in a 25- µl reaction containing 12.5 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 4.5 µl of water, and 6 µl of template. The reaction was performed in a Biometra thermal cycler. For mecA gene PCR, a primary denaturation step was done at 95^°C for 5 min, followed by 35 cycles of 95^°C for 45 sec., 50^°C for 45 sec. and 72^°C for 45 sec. A final extension step was done at 72^°C for 10 min, according to McClure et al. (2006). However for the icaA and icaD genes, the cycles consisted of 95ºC for 1 min, 49ºC for 1 min and 72ºC for 1 min, according to Ciftci et al. (2009).

IV- Analysis of the PCR Products: The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 15 µl of the products was loaded in each gel slot. A 100 bp DNA Ladder (Qiagen, Germany, GmbH) and 100 bp plus DNA Ladder (Fermentas, Cat.No. SM 0323) were used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.

RESULTS

Table 2: Isolated micro-organisms from mastitic milk samples.

Table 3: In vitro antimicrobial susceptibility test of isolated Staph. spp. From bovine clinical mastitis according to the agar disc diffusion method. 

Table 4: Methicillin resistant S. aureus strains tested for their phenotypic and genotypic characteristics and slime factor production.

Fig. 1: PCR results for mecA gene showing amplification of 310 bp. (L): 100 bp ladder (QIAGEN, Gmbh) (100-600 bp), 1-10 lanes for the 10 strains respectively.

Fig. 2: PCR results for icaA gene showing amplification of 1315 bp. (L): 100 bp plus ladder (Fermentas, Cat. no. SM 0323) (100-3000 bp), 1-10 lanes for the 10 strains respectively.

Fig. (3): PCR results for icaD gene showing amplification of 385 bp. (L): 100 bp plus ladder (Fermentas, Cat. no. SM 0323) (100-3000 bp), 1-10 lanes for the 10 strains respectively.

DISCUSSION

Mastitis is an important and a persistent infection producing high economies losses due to poor milk quality, reduced milk yield and increased expenditure on treatment especially staphylococcal mastitis (Abd-Elrahman, 2013) which resembled 63.37% through the current study and bacteriological examination shows that S. aureus was the main causative agent of clinical mastitis in cows (34.65%), followed by S. saprophyticus (10.89%), S. intermedius and S. epidermidis (8.91%, for each), as shown in Table (2). This result of S. aureus in close agreement with previous findings; 30, 30 and 36%, by Reddy et al. (2007); Ali et al. (2008) and Haftu et al. (2012), respectively. However, the findings (71.4%) of S. aureus by Abou-Zaid and El-Sawalhy (1999) are much higher than the present report. The lower prevalence reported by Eman et al. (2006); Moniri    et al. (2007) and Sayed & Mohamed (2008); (21.7; 21.9 and 23.6%, respectively). High prevalence of S. aureus points to poor milking hygiene as this pathogen is mainly spread during milking via milkers’ hands and towels (Bradley, 2002).

In vitro activities of Staph. spp. against 11 antimicrobial agents are summarized in Table (3). In the present work the highest rate of resistant S. aureus exhibited to Lincomycin followed by Cefotaxime, Ampicilin and Penicillin (91.43, 88.57, 68.86 and 57.14%, respectively) and MRSA resembled 60% of these isolates. The highest rate of sensitivity to Enrofloxacin and Gentamicin followed by Doxycycline (100, 100 and 91.43%, respectively), Table (3). Abd-Elrahman (2013) found that Enrofloxacin the most effective drugs against S. aureus. The highest resistance rate of S. aureus against Penicillin (47.6, 47.6, 56.5 and 66%) was reported by Calvinho et al. (2002); Gianneechini      et al. (2002); Arshad et al. (2006) and Nam et al. (2011), respectively. Vanderhaeghen et al. (2010) found an unusual high prevalence of MRSA in Belgian cases of subclinical and clinical S. aureus mastitis in cows. S. aureus can adapt rapidly to the selective pressure of antibiotics and this resulted in emergence and spread of MRSA (Deurenberg et al., 2007).

Extracellular polysaccharides, slime factor, are considered to be significant virulence factors for some staphylococci. Slime layer surrounding the S. aureus strains help in adherence and colonization of these microorganisms on the mammary gland epithelium. It is reported that slime factor production in S. aureus isolates from mastitis cause antibiotic resistance which is due to the decreased diffusion of antibiotics through the biofilm matrix and decreased metabolic activity of bacteria (Amorena et al., 1999 and Yazdani et al., 2006).

In the present study, ten MRSA strains were subjected for slime production on Congo Red Agar (CRA) plates and PCR study targeting mecA, icaA and icaD genes. Among the tested MRSA strains only five isolates (50%) were positive for mecA gene genotypically, Table (4) and Fig. (1). Moon et al. (2007) and Ciftci et al. (2009) detected that 61.9 and 30.7% of MRSA were positive for mecA gene, respectively. MRSA resistance to methicillin and other ß-lactam antibiotics is caused by the action of mecA gene (Deurenberg et al., 2007). The discrepant results between disc diffusion methods and PCR for detection of methicillin resistance may be due to another resistance mechanism such as hyperproduction of beta-lactamase, also MRSA strains show a heterogeneous character with the level of resistance varying according to the culture conditions and β-lactam antibiotic being used. Because of this heterogeneous resistance, and time consuming the detection of MRSA by phenotypic methods becomes problematic, Ciftci et al. (2009). However, PCR-based methods have shown to be a rapid and reliable approach for the identification and genotypic characterization of MRSA. Detection of mecA- based PCR methods has accepted as “gold standard” (Sancak, 2000).

The present work found that six isolates (60%) of the tested MRSA strains were slime producing positive on CRA plates in vitro, Table (4). Slime-producing S. aureus isolates from different clinical origins such as bovine mastitis (Vasudevan et al., 2003) and (Ciftci et al., 2009), wound infection (Yazdani et al., 2006) and clinical cases (Eftekhar and Dadaei, 2011) has been detected in vitro by using Congo Red Agar plates in percentages of 91.4, 37.2,  52 and 53.3%, respectively. Knobloch et al. (2002) have reported that the phenotype on CRA was found to be an unreliable indicator of slime-forming capacity among clinical isolates of S. aureus. Therefore, although CRA methods may be easier to perform than a molecular analysis of the genes implicated in biofilm production and could be performed easily in a diagnostic laboratory, it may be a poor method for determining the slime producing capacity of clinical isolates in the diagnostic laboratory (Fitzpatrick et al., 2005).

PCR methods provided a direct evidence of the genetic basis of slime production complementary to the CRA test. The ica locus consists ica A, D, B, C genes. Slime synthesis is controlled by the ica (intercellular adhesion) operon. Coexpression of icaA with icaD leads to a significant increase in activity and is related to phenotypic expression of the capsular polysaccharide (Arciola et al., 2001). In this study, slime factor production of MRSA isolates were detected by PCR targeting icaA and icaD genes and found that 5 (50%) of the tested MRSA strains were positive for both icaA and icaD genes. Six (60%) and eight (80%) isolates were positive for icaA gene and icaD gene, respectively as shown in Table (4) and Fig. (2 & 3). Ciftci et al. (2009) reported that 15 (25.42%) out of 59 S. aureus strains were positive for both icaA and icaD genes. Sixteen (27.12%) and 38 (64.41%) out of 59 strains were positive for icaA and icaD gene, respectively. Also Eftekhar and Dadaei (2011) found that 75% of MRSA carried ica operon. The icaAD gene was detected in 32% of Staphylococcal spp. (Rasha et al., 2012). While it was contrast to Arciola et al. (2001) who have reported that all strains which were positive for icaA gene were also positive for icaD gene. In addition, Vasudevan et al. (2003) and Yazdani et al. (2006) have reported that all S. aureus isolates possessed the ica locus, icaA and icaD genes. icaA and icaD genes were not be together in some isolates may due to some mutations on icaA gene, although coexpression on icaA and icaD is necessary for slime production it was considered that other genes in ica locus play role in controlling slime expression, (Ciftci et al.,2009).

In present study, among four isolates which were negative for slime production on CRA plate in vitro, one isolate was positive for both icaA and icaD genes, two isolates were positive for icaD gene and the last one was negative for both icaA and icaD genes, Table (4). Ciftci et al. (2009) found among the 37 S. aureus strains which did not produce slime factor on CRA plate in vitro, only 7 (18.9%) strains were positive for both icaA and icaD genes. Who suggests that some environmental conditions or presence of accessory genes can influence the phenotypic behavior on the Congo red agar plate, giving colonies which did not fully express the ica genes.

In this work, six isolates (60%) were positive for both methicillin resistance and slime production phenotypically and three strains (30%) were positive for all mecA, icaA and icaD genes, Table (4). Only 2 (3.39%) of 59 S. aureus strains were positive for both methicillin resistance and slime producing, phenotypically, Ciftci et al. (2009).

In conclusion, it was showed that detection of mecA gene in S. aureus isolates indicating that several cases suffering from S. aureus mastitis have an MRSA problem. Genotypic determination of mecA gene proved the most reliable method for detection of methicillin resistance. The present work paid an attention to the 3 MRSA strains (30%) were positive to all tested genes rather than slime production as the worst isolated stains all over this study (multidrug resistant, slime producing as well as carrying mecA, icaA and icaD genes). In vitro Enrofloxacin, Gentamicin and Doxycycline are the most effective drugs for Staph. spp. clinical mastitis. 

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