2016| Volume 2 | Issue 2 |p. 13 - 21 Ewemen Journal of

© 2016 Ewemen Resources Limited / EJMR. All rights reserved

2016| Volume 2 | Issue 2 |p. 13 - 21

Ewemen Journal of Microbial Research ISSN: 2488-9148

Available online at http://ewemen.com/category/ejmr/

Full Length Research

MOLECULAR CHARACTERIZATION OF β-LACTAMASE PRODUCING STAPHYLOCOCCUS AUREUS FROM MASTITIC COW ISOLATES

1*ISHAQ Shamsu Abdullahi and 2NAFI’U Shafiu Abdullahi

1Department of Biology, Federal Collage of Education (Technical) Bichi, Kano State, Nigeria

2Department of Science Laboratory Technology, Kano State Polytechnic, Kano State, Nigeria.

ABSTRACT

Received 21 March, 2016 Revised on the 20 April, 2016 Accepted 14 July, 2016 *Corresponding Author’s Email:

[email protected]

Beta-Lactamase producing isolates were recovered from multidrug resistance Staphylococcus aureus. The multidrug resistance Staphylococcus aureus isolates from six local governments of Kano State, North-West Nigeria were documented to be DNA encoded and resistance against β-lactam and cephalosporin. Conventional laboratory analysis showed that out of the 31 β-lactamase producers, only 13 (41.9%) were multidrug resistant to cefoxitin, ampicillin and cephalexine. The antibiotic susceptibilities of the S. aureus isolates were in the following order: ofloxacin (99.3%) > ciprofloxacin (98.6%) > gentamicin (91.5%) > cephalexine (56%) > sulphamethoxazole/trimethoprim (46.1%) > tetracycline (43.3%) > cefuroxime (33.3%) > cefoxitin (2.1%) > amoxicillin (1.4%) > ampicillin (1.4%). Resistance to three or more antimicrobials was presented in 98.6% of the S. aureus isolates. PCR assay was used to detect mecA and blaZ genes in multi-drug resistant Staphylococcus aureus isolates that were β-lactamase producers. The MDR- Staphylococcus aureus was also tested by latex agglutination for presence of PBP2a. The results revealed that none of the isolates had any gene amplification to both mecA and blaZ genes despite the various degree of resistance in antibiotic susceptibility profile test and β-lactamase production test with conventional detection analysis. It was assumed that their resistant genes were not coded for by the primers used in this study as these isolates were likely to contain other resistant genes, which were also expressed at a molecular level. This study suggests that molecular characterization might not necessary show great correlation with analytical methods. Keywords: MecA, blaZ, Multidrug resistant, Staphylococcus aureus, β-lactamase, Molecular characterization

INTRODUCTION

Mastitis is the inflammation of the parenchyma of the mammary glands of animals (Roberson et al., 1994). Bovine mastitis is a multifactorial disease that results in reduced milk production, changes in milk composition and milk discard. It imposes serious

economic losses to the farmers and the dairy industry (Ribeiro et al., 2003; Pitkala et al., 2004). Mastitis is the single most common reason for the use of antimicrobials in dairy cattle husbandry. Use of antimicrobial treatment is required for clinical mastitis,

mailto:[email protected]

Ewemen Journal of Microbial Research 2016, 2(2): 13 - 21 Ishaq and Nafi’u

www.ewemen.com Page 14

persistent infections and in heifers before calving (Taponen et al., 2006). Therefore, antimicrobial resistance of mastitis pathogens has received much interest over the past few years. Among the antimicrobial agents approved for use in bovine mastitis, β-lactams, such as penicillins and cephalosporins, play a key role. Resistance to β-lactams in Staphylococcus infections are mediated by either β-lactamases codified by blaZ gene or mecA-encoded alternative penicillin binding protein, PBP2a. This shows a reduced binding to β-lactams antibiotics currently available for mastitis therapy. Because of the importance of S. aureus as a major mastitis pathogen which is very difficult to treat, this work focused on studying Staphylococci causing clinical and subclinical mastitis in bovine and their antibiotic resistance using the methods of Esmat and Bader (1996) and El-Seedy et al. (2010). β-lactam antibiotics are frequently used for treatment of Staphylococcus aureus mastitis as well as intra-mammary infusion for preventive measures in cows. Injudicious use of antimicrobials has resulted in augmenting the bacterial resistance mechanism including the β-lactamase production. It has been reported that in divergent geographical areas a limited diversity of S. aureus strains is involved in mastitis infection (Moon et al., 2007). Staphylococcus aureus strains are capable of mutation, clonal evolution and horizontal gene transfer that increase the virulence and drug resistance (Brody et al., 2008). In view of the forgoing, this study was aimed to investigate the phenotypic and genotypic characteristics of Staphylococcus aureus involved in dairy cow mastitis in southern region of Kano State, North west Nigeria MATERIALS AND METHODS

Materials

Milk obtained from infected cows, Antibiotic susptiblity dics purchased from Oxoid LTD,England, Nutrient Agar, MacConkey Agar, Mannitol salt Agar, Muller Hinton Agar, swab sticks and sterile sample collection bottles obtained from CEMAN scientific supply, B.U.K, Road Kano. Incubator, and autoclave obtained from Department of Pharmaceutical microbiology Ahmadu Bello University, Zaria, PCR Machine, set of primers, L.B broth, and DNA extraction kits were obtained from Biotechnology Centre, Ahmadu Bello University Zaria, Nigeria. Isolation, Microscopy, Identification and Biochemical Test Thirteen (13) multidrug resistant Staph. aureus isolates

were collected from milk and wound samples from mastitic cow from six different local Government of Kano. Isolation, microscopy, identification and biochemical test of the S. aureus isolates were carried out using standard microbiological methods (Harley and Prescott, 2002; Chakraborty and Nishith, 2008). Antibiotic susceptibility pattern of the isolates were carried out using disk diffusion method (Chessbrough, 2000). Interpretation of the susceptibility results were also carried out (CLSI, 2006). The antibiotics used were: Ampicillin (AMP) (10 μg), amoxicillin (AML) (10 μg), cefalexin (CL) (30 μg), cefuroxime (CXM) (30), ciprofloxacin (CIP) (5 μg), ofloxacin (OFX) (5 μg), Gentamicin (CN) (10 μg), tetracycline (30 μg), cefoxitin (FOX) (30 μg), septrin (SXT) (25 μg). The antibiotics were the commonly prescribed antibiotic at the area where the samples were collected for the treatment of infections associated with S. aureus. Isolation and Characterization of the Isolates

One mililiter of the milk sample was aseptically transferred into a sterile 9 mL nutrient broth using automatic micropipette and the wound swab was placed in 5 mL sterile nutrient broth and vortex to dislodge the isolates into the broth. The nutrient broth mixture was capped aseptically and incubated at 37℃ of the resulting overnight broth culture was carefully streaked aseptically on dried surface of sterile mannitol salt agar (MSA), MacConkey agar and blood agar plates to give well district colonies after incubation at 37℃ for 48h. Growth from 48h incubation was observed and characteristic colour of the resulting colonies was noted. The isolates (discrete colonies with distinct colors) on MSA with characteristic deep golden yellow coloration were selected and sub-cultured into nutrient broth incubated at 37˚C for 18h. This was then inoculated onto sterile nutrient agar slants aseptically and incubated at 37℃ for 24h and then stored at refrigerating temperature of 4 ℃ for further investigations.

Preliminary identification Test for Staphylococcus species

Gram Staining

Gram’s stain technique described by Cheesbrough (2006) was used to classify the isolates into Gram positive or negative. A smear of the isolate was made on a clean glass slide and heate-fixed. The smear was stained with crystal violet for 30 seconds, fixed with lugol’s iodine for 30 seconds and decolorized with 95% ethanol for 3 seconds after which it was counterstained with dilute carbol fuchsin solution 1 minute. On



examination microscopically, the isolates that produced violet cocci (Gram positive) predominantly in clusters were selected for further identification procedures.

Biochemical Test

Catalase and Coagulase tests were carried out on all the isolates that were Gram positive cocci. Catalase differentiation screening

This was used to differentiate those bacteria that produce the enzyme catalase such as Staphylococci from non-catalase producing bacteria such as streptococci. Two milliliters (2 mL) of a 3% hydrogen peroxide solution were added on a 24hrs culture of the isolates on nutrient agar slant. Rapid effervescent of gas bubbles indicating the breaking of hydrogen peroxide into oxygen and water in the presence of enzyme catalase represents positive result (Cheesbrough 2006). Coagulase Test

This was used to differentiate Staph. aureus from Staphylococcus epidermidis and Staphylococcus saprophyticus (which do not produce coagulase) using the method described by Cheesbrough (2006).The pooled human plasma was brought out of refrigerator to attain room temperature. Two hundred microlitres (200 µL) of the plasma was added to 800 µL of 24 hr nutrient broth culture of the isolate and mixed thoroughly. The mixture was incubated at 37℃ for 3 hr. At interval of 1 hr, the test tubes were observed for clotting. Both positive control (test tube containing 24 hr of S. aureus ATCC 13709 with the plasma) and negative control (test tube containing only sterile nutrient broth) were set up alongside with the test isolates. Test isolates that were positive to the coagulase test were considered as Staph aureus and selected for further investigation. Susceptibility testing of the isolates

Preparation of Inoculum

A single isolated colony was picked using sterile wire loop and carefully streaked on the surface of sterile nutrient agar plate to give well distinct isolated colonies after incubation at 37℃ for 18 h. Standardization of the inoculums

Overnight culture (18 hr) of the isolates on nutrient agar were aseptically transferred into a sterile 5mL of sterile physiological saline and shaken and the turbidity was compared to that of 0.5 McFarland

standards corresponding to approximately 1.0 × 108 cfu / mL. This was done for each of the test bacterial isolate. The mixture was used for the susceptibility testing (Cheesbrough, 2006) Antibiotic susceptibility testing

Antibiotic susceptibility testing was carried out on each purified isolates using CLSI modified disc diffusion method as described by Cheesbrough (2006). The isolates were tested against ten (10) antibiotics that were commonly used in treating cow infection in the locality. Two milliliters (2 mL) of the standardized inoculum of each isolate were aseptically poured on a fairly dried surface of sterile Mueller Hinton agar plate to evenly cover the surface of the agar, excess was drained off and the surface of the agar was allowed to be absorbed within the agar with the Petri dish lid in place for 10 min. The antibiotic discs were aseptically distributed evenly on the inoculated plate with each disc lightly pressed down to ensure its contact with the Mueller Hinton agar. Each plate contained maximum of six different antibiotics. Within 30 min of applying the disc, the plate was inverted and incubated aerobically at 30 °C for 18 hr. After the 18 hr incubation, the diameters of the zone of inhibition for each of the isolates were measured underside of the plate to the nearest millimeter (mm) (BSOP 45, 2003). The same procedures were carried out for the other isolates. Test for β- Lactamase Production

From the antibiogram result, the isolates that showed resistance to three or more antibiotics (multidrug resistant) especially penicillins and cephalosporins were selected for β-lactamase production test using the acidimetric method (David and Derek, 2005).

Briefly, 18.6 mL of phenol-red water solution was added to the vial of 20 million units of crystalline Benzyl penicillin G using sterile syringe to which 1N NaOH was added drop wise to the acidic solution until it developed a violet colour (pH 8.5).

The reagent was then dispensed in aliquots of 0.1 mL into sterile tubes and frozen at -20˚C. The desired number of tubes were removed from the freezer and thawed at room temperature of 25˚C and with a sterile loop; colonies of the isolates were added to the test solutions to make an opaque, milky suspension. A colour change form milky to yellow indicated positive reaction while from milky to violet indicated negative reaction. A positive reaction was observed within 15 min.



Molecular Characterization of Resistant Staphylococcus aureus

Bacterial Culture Preparation

The bacteria cultures were prepared using a standard procedure (Dubey, 2009). Briefly, Luria and Bertani broth media were prepared (peptone, 10 g; NaCl, 5 g; 1N NaOH, 10 mL; yeast extract, 5 g; distill water 1 L; pH 7.0 adjusted with NaOH solution) and sterilized at 121˚C for 15 min. Single colonies were picked from isolates on Nutrient agar plate and inoculated into 5 mL Luria and Bertani (LB) broth medium and incubated overnight at 37˚C for 18 - 24 hr. Bacteria culture was harvested by centrifugation at 4˚C, 8000 rpm (6800 × g) in a microcentrifuge for 30 sec at room temperature in an Eppendorff’s tube. The supernatants were decanted and cells harvested. DNA Primers

The primers oligosequences for the selected resistance genes in the study are as shown in Table 1 below. Table 1: The DNA templates obtained were subjected to multiplex PCR using the following sets of primers.

DNA Template

Forward Primer Reverse Primer Reference

mec A 1 5' AAA ATC GAT GGT AAA GGT AAA

GGT TGG C 3'

5'AGT TCT GCA GTA CCG GAT

TTG C 3' (533bp)

Zhang et al., 2005

mec A 2 5' GTG AAG ATA TAC CAA GTG

ATT 3'

5' ATG CGC TAT AGA TTG AAA

GGA T 3‘ (143bp)

Zhang et al., 2005

mec A 3 5' GTG GAA TTG GCC AAT ACA GG

-3'

5' TGA GTT CTG CAG TAC CGG AT

3‘ (1319bp)

Zhang et al., 2005

blaZ CCT AGT AAA GCT CCG GAA 3'

CTA GTC CAT TCG GTC CA 3‘ (414bp)

Vesterholm-nielsen et al., 1999

Genomic DNA extraction

DNeasy extraction Kit (Qiagen, Germany) was used to isolate microbial genomic DNA from Staph. aureus following manufacturer’s instruction (De Neel et al., 2007). The DNA was bound to silica gel membrane by passing the lysate through a column. Contaminants were washed away with the wash solutions (AW 1 and 2) and the DNA eluted with Manufacturer’s elution buffer. Purified DNA in the flow-through eluent was stored at -20˚C. Agarose Gel Electrophoresis

Agarose gels were prepared and electrophoresis was carried out using the set of primers as described by Sambrook et al., 1989).

Amplification of Resistant Gene

Amplification of resistant gene; mecA1, mecA2, mecA3 and blaZ was carried out in a 50 μL reaction mix. The master mix was prepared in a microtube comprising of; Dream Taq™ buffer DNA polymerase (Fermenters) supplied with optimized 10× Dream Taq™ buffer, which includes 20 mM MgCl2, dNTP mix, template DNA (genomic DNA), and nuclease free water. Enough master mix was prepared for the number of reactions plus one extra reaction to compensate for pipetting errors. The mix was then aliquot into thin-walled PCR tube earlier placed on an ice and the template DNA was added. The samples were vortexed gently and spun down before been transferred to the thermocycler. PCR was performed using the thermal cycling conditions Table 2.

Table 2: PCR Cycle Conditions

PCR Cycle Step Temperature (°C)

Time No of cycles

Initial Denaturation 94-95 2 min 1 Denaturation 94-95 15-30 sec 30-40 Annealing 50-65 15-30 sec 30-40 Extension 65-72 1 min 30-40 Final Extension 72 5 min 1

RESULTS AND DISCUSSION

Susceptibility of multidrug resistance mastitis isolates of S. Aureus and antibiotic resistance pattern

Ten antimicrobial agents were used to classify the 141 S. aureus into either susceptible or resistant strain. The susceptibility results were compared with zone size interpretative chart for S. aureus using Mueller Hinton Agar (Table 3.)

The multiple drug resistance (MDR) is described as non-susceptible to at least one agent in the three or more antimicrobial categories (Magiorakos et al., 2012). It was found that 3.6% of the isolates were resistant to three (3) agents, 29.1% were resistant to 4 agents, 26.2% were resistant to 5 agents, 16.3% were resistant to 6 agents, 17.7% were resistant to 7 agents, 4.3% were resistant to 8 agents, 0.7% were resistant to 9 agents and none of the isolates were found to be resistant to all (10) agents as shown in Table 2. This agrees with the findings of Li et al (2015) predominant multidrug resistance profile from penicillin/ampicillin/ kanamycin/gentamicin/tetracycline among 46 isolates identified.



Table 3: Multiple Antibiotic Resistance of the Staph. aureus Isolates

No. of antibiotics resistant to

No. & (%)

Resistance Pattern No. of resistance associate

d with pattern

1 1 (0.7)

FOX 1

2 3 (2.1)

AMP, FOX 2

AML, FOX 1

3 5 (3.6)

AML, FOX, CXM 1

AML, SXT, FOX 1

AMP, AML, FOX 3

4

41 (29.1)

AMP, AML, FOX, CXM, 17

AMP, AML, FOX, SXT 3

AMP, AML, TET, FOX, 18

AMP, AML, CL, FOX 3

5 37 (26.2)

AMP, AML, CL, SULF, FOX, 1

AMP, AML, SXT, FOX, CXM 14

AMP, GEN, CL, TET, FOX 1

AMP, AML,FOX, CXM, TET 5

AMP, AML, TET, FOX, CXM 9

AMP, AML, TET, SXT, FOX 4

6 23 (16.3)

AMP, AML, GEN, TET, FOX, CXM

1

AMP, AML, CL, SXT, FOX, CXM 9

AMP, AML, SXT, FOX, CXM, CL 7

AMP, AML, OFL, CL, SXT, FOX 1

AMP, AML, TET, SXT, FOX, CL 4

AMP, AML,CL,TET, FOX, CXM 1

7

25 (17.7)

AMP,AML,GEN,CL,SXT, FOX, CXM

2

AMP, AML, CL, TET, SXT, FOX, CXM

10

AMP, AML, GEN, CL, SXT, FOX, CXM

8

AMP, AML, GEN, CL, TET, SXT, FOX

5

8 6 (4.3)

AMP, AML, GEN, CL, TET, SXT, FOX, CXM

6

9 1 (0.7)

AMP, AML, CIP, GEN, CL, TET, SXT, FOX, CXM

1

Key: AMP = Ampicillin, CL= Cephalexine, CIP = Ciprofloxacin, GEN = Gentamicin, OFL = Ofloxacin, TET = Tetracycline, AML = Amoxicillin, SXT = Sulphamethoxazole/trimethaprim, FOX = Cefoxitin, CXM = Cefuroxime

The susceptibility of the isolates are as follows: 98.6%, 97.9%, 91.5%, 56% and 46.1% were susceptible to ofloxacin (OFL), ciprofloxacin (CIP), gentamicin (GEN), cephalexine(CL) and sulphamethoxazole/trimethoprim (SXT), respectively. The other test antibiotics were not as effective with susceptibility of 42.6%, 33.3%, 2.1%

1.4%, and 1.4% for tetracycline (TET), cefuroxime (CXM), cefoxitin (FOX), amoxicillin (AML) and ampicillin (AMP) respectively (Table 4). Table 4: Summary of the Percentage Susceptibility of S. aureus Isolates from Cow Mastitis to Different Antibiotics.

Antibiotics Disc Strength (µg) No. (%) of isolates susceptible

AMP 10 2(1.4) CL 30 79(56) CIP 5 139(98.6) GEN 10 129(91.5) OFL 5 140(99.3) TET 30 60(42.6) AML 10 2(1.4) SXT 25 65(46.1) FOX 30 3(2.1) CXM 30 (33.3) Key: AMP = Ampicillin, CL= Cephalexine, CIP = Ciprofloxacin, GEN = Gentamicin, OFL = Ofloxacin, TET = Tetracycline, AML = Amoxicillin, SXT = Sulphamethoxazole/trimethaprim, FOX = Cefoxitin, CXM = Cefuroxime

The result indicates that 97.9% of the S. aureus isolates were multi-drug resistant which is alarming, while comparatively 52% of S. aureus isolates were reported as multi drug resistant in Ethiopia (Sori et al., 2011). This conforms with the finding of Bruno et al (2014), in his work on β-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis in Brazil. His team detected resistance to penicillin in S. aureus isolates (83/100) with Nitrocefin disks, and PCR showed 79.0%, 78.0% and 62.2% of positivity for S. aureus. Phenotypic methicillin resistance was found in high percentage among the S. aureus isolates at 97.9% which is greater than that reported by Moon et al (2007) and Kumar et al (2010) in Korea and India, respectively. Kumar et al (2011) and Kenar et al (2012) had earlier reported the emergence of drug resistance was the consequence of the improper use of antimicrobials. In this study MRSA isolates showed high resistance to ampicillin, amoxicillin and cefuroxime, which supports the findings of Weems (2001) and Gross-Schulman et al (1998), that MRSA strains are equally resistant to all β-lactam antibiotics, and show intrinsic resistance to all other penicillinase resistant penicillins, all cephalosporins such as cephalothin and cephalexin and all newer β-lactam antibiotics, such as monolactam (Shanson, 1981; Thompson et al., 1982; Hirschl et al., 1984). Determination of β –lactamase Production

One hundred and thirty-eight (138) isolates that were resistant to three or more antimicrobials were selected and tested for production of β-lactamase. Out of the



141 isolates tested, 31 (22.5%) were β-lactamase producer. Among those 31 β-lactamase producers, 13 (41.9%) were resistant to cefoxitin, ampicillin, amoxicillin and cephalexine (Table 5). This is in agreement with the work of Haran et al (2012) who reported two MRSA isolates that are resistance to β-lactams, cephalosporins, and lincosamides and were multiresistant. Table 5: Antibiotic Resistance Pattern of β-lactamase Producing Staph. aureus Isolates

Isolates Number

Β-Lactamase Test

Antibiotics Resistance Pattern

IM001 POSITIVE AMP, FOX, AML, CL IM003 POSITIVE AMP, AML, CL IM006 POSITIVE AMP, FOX, AML IM010 POSITIVE AMP, FOX, AML, CL IS013 POSITIVE AMP, FOX, AML, CL IM012 POSITIVE AMP, FOX, AML IM014 POSITIVE AMP, FOX, AML IM024 POSITIVE AMP, FOX, AML, IM026 POSITIVE AMP, FOX, AML, CL IM029 POSITIVE AMP, FOX, AML IM030 POSITIVE AMP, FOX, AML IM031 POSITIVE AMP,FOX, AML, CL IM042 POSITIVE AMP, FOX, AML, CL IM044 POSITIVE AMP, FOX, AML, CL IM047 POSITIVE AMP, FOX, AML IM054 POSITIVE AMP, FOX, AML IM076 POSITIVE AMP, FOX, AML, CL IM080 POSITIVE AMP, FOX, AML, CL IM082 POSITIVE AMP, FOX, AML, CL IM085 POSITIVE AMP, FOX, AML IM086 POSITIVE AMP, FOX, AML IM094 POSITIVE AMP, FOX, AML IM102 POSITIVE AMP, FOX, AML IM108 POSITIVE AMP, FOX, AML IM131 POSITIVE AMP, FOX, AML, CL IM135 POSITIVE AMP, FOX, AML, CL IM137 POSITIVE AMP, FOX, AML IM138 POSITIVE AMP, FOX, AML IM139 POSITIVE AMP, FOX, AML IS008 POSITIVE AMP, FOX, AML, CL Key: AMP = Ampicillin, CL= Cephalexine, CIP = Ciprofloxacin, GEN = Gentamicin, OFL = Ofloxacin, TET = Tetracycline, AML = Amoxicillin, SXT = Sulphamethoxazole/Trimethoprim, FOX = Cefoxitin, CXM = Cefuroxime

PCR analysis of the resistant S. aureus Isolates

13 (41.9%) of the 31 Beta-lactamase producing MDR isolates when subjected to DNA extraction, they were positive to β-lactamase production test and at the same time were resistant to cefoxitin, Ampicillin, amoxicillin and cephalexine (Figure 1). It could be deduced from the PCR results that, S. aureus ATCC 25922 (positive control) was Methicillin resistance (MR) carrying the mecA gene and also β-lactamase producer (blaZ gene positive PCR). Out of the 13 multidrug resistant S. aureus isolates selected for PCR, none carried the mecA gene and they did not also carry blaZ gene. This

suggests that isolates that showed no amplification for mecA and blaz genes might have other antibiotics resistance genes which are expressible using other set of primers. This agrees with the findings of Li et al (2015) who recovered One hundred and thirteen methicillin susceptible Staphylococcus aureus (MSSA), one mecA-positive and phenotype-positive MRSA, seven mecA- and mecC-negative isolates from 214 quarter milk samples on 4 dairy farms.

Figure 1: Testing for the presence of mecA and blaZ genes from Staph. aureus isolates. Lane 1: molecular size marker (1000bp DNA ladder), Lane 2: Genomic DNA from multidrug resistant isolate IM001, Lane 3: Genomic DNA from multidrug resistant isolate IM010, Lane 4: Genomic DNA from multidrug resistant isolate IS013, Lane 5: Genomic DNA from multidrug resistant isolate IM026, Lane 6: Genomic DNA from multidrug resistant isolate IM031, Lane 7: Genomic DNA from multidrug resistant isolate IM042, Lane 8: Genomic DNA from multidrug resistant isolate IM044, Lane 9: Genomic DNA from multidrug resistant isolate IM076, Lane 10: Genomic DNA from multidrug resistant isolate IM080, Lane 11: Genomic DNA from multidrug resistant isolate IM082, Lane 12: Genomic DNA from multidrug resistant isolate IS008, Lane 13: Genomic DNA from multidrug resistant isolate IM131, Lane 14: Genomic DNA from multidrug resistant isolate IM135, Lane 15: Reagent plus water as negative control, Lane 16: Genomic DNA from Staph. aureus ATCC 25922 as positive control as shown in Figure 1.

The control of the spread of S. aureus has become challenging due to the species' ability to resist to antimicrobial therapy, taking into account that it has vanquished almost every existing currently available antimicrobial agent (Hiramatsu et al., 1997). The prevalence of antibiotic resistance among strains isolated from domestic animals is increasing, raising concerns about the role of domestic animals as reservoirs of S. aureus which may become involved in human infections (Anderson et al., 2008; De Neeling et al., 2007; Khanna et al., 2008). In this study, 97.9% of the S. aureus isolates were multi-drug resistant which is alarming. This result is in accordance with the high



prevalence of β-lactamase (bla) genes recorded in community strains by Maranan et al., (1997) who reported only 5 strains (3.2%) presented resistance to oxacillin, an antibiotic used for the detection of Methicilin Resistant S. aureus (MRSA). Our findings negates the recent studies in which MRSA was recovered 39% and 10.9% of pigs and horses tested, respectively (De Neeling et al., 2007). The increasing frequency of drug resistance has been attributed to combination of microbial characteristics, selective pressure of antimicrobial use, societal and technological changes that enhance the transmission of drug resistant organisms (Orozova et al., 2008). All the isolates did not carry mecA gene while showing phenotypic resistance to cefoxitin. As resistance due to β-lactamase in Staphylococcus was reported to be mainly mediated by mecA gene which is the determinant of methicillin resistance in all Staphylococci (Kilic et al., 2006) and/or blaZ gene, the determinant of β lactamase production (Vesterholm-nelsen et al., 1999), phenotypic and genotypic methods were directed toward detection of both mechanisms. Prediction of blaZ gene presence was conducted by Ampicillin and Amoxicillin disk diffusion test (CLSI, 2006, 2008). Several advantages had been reported for genotypic methods in resistance detection compared to convectional susceptibility method, and because of the disadvantages of phenotypic tests and the advantages of genotypic methods, four pairs of primers were included in multiplex PCR (Murakami et al., 1991). The first three pairs targeted the mecA gene, the determinant of Methicillin resistance (Zhang et al., 2005) while the forth pair targeted the blaZ gene, the determinant of β-lactamase production (Vesterholm-nelsen et al., 1999; Hareri et al., 2005). Using novel multiplex PCR assay, detection of blaZ and/or mecA gene were performed through successful amplification of 414 bp and/or 533 bp, 143 bp or 1319 bp for blaz mecA 1, mecA 2, or mecA 3 genes, respectively. In the standard S. aureus ATCC 25922, both mecA and blaZ genes were detected (Figure 1). Although the isolates did not carry the mecA gene, they were phenotypically resistant to cefoxitin. Non-mecA carriage can be attributed to many reasons: The first is the production of modified intrinsic PBPs with altered affinity for methicillin (Tomasz et al., 1989), the second reason can be the inactivation of cefoxitin or methicillin by increased production of β-lactamase which can be declared by detection of blaZ gene (Swenson, 2002). Expression of mecA gene yields a penicillin binding

protein called PBP2’ with reduced affinity for β-lactam antibiotic binding. In this study, none of the β-lactamase producing isolates tested positive for mecA and blaZ genes. CONCLUSION

This study reports increasing prevalence of Multidrug resistant β-lactamase producing isolates without mecA and blaZ genes. The detected multidrug resistance, mainly to cefoxitin, amoxicillin and ampicillin demonstrated that S. aureus isolates from bovine mastitis represent a potential hazard to public health. Therefore, the results obtained were of great concern not only in regard of mastitis therapy but mainly to public health, due to the eventual occurrence of cross infections, as well as, to the possibility of transmission of resistance among the microorganisms by plasmids. The findings suggest that in Kano State, methicillin resistant S. aureus specifically multidrug resistant ones are mostly responsible for cow mastitis. Further research is therefore recommended to look into the association between the S. aureus mastitis in the cow milk and the consumers, which will help to classify the infection as zoonotic or otherwise. ACKNOWLEDGMENT

The authors are highly grateful to Dr. Sharani Habibu, Rano Zonal Veretinary Officer, Kano State and the management of Sir. Muhammad Sunusi General Hospital Kano, for their support. CONFLICT OF INTEREST

The authors declare that they have no conflict of interest. REFERENCES

1. Anderson ME, Lefebvre SL and Weese JS (2008). Evaluation of prevalence and risk factors for methicillin-resistant Staphylococcus aureus colonization in veterinary personnel attending an international equine veterinary conference. Vet. Microbiol 129: 410-417.

2. Bruno RF, Nóbrega DB, Guimarães FF, Wanderley GG and Langoni H (2014). Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis. Pesquisa Veterinária Brasileira 34(4):325-328.

3. CLSI [Clinical Laboratory Standard Institute] (2006) Performance Standards of Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animal, Approved Standard. 3rd Eds.



4. CLSI [Clinincal and Laboratory Standard Institute] (2008). Perfomance Standards for antimicrobial susceptibility testing. 18th informational supplement. CLSI document M100-S18. Wanyne, PA.

5. Chakraborty P and Nishith KP (2008) Manual of Practical Microbiology and Parasitology. New Central Book Agency Limited, West Bengal.

6. Cheesbrough M (2006) District Laboratory Practice in Tropical Countries (Part 11). Cambridge University Press, Cambridge, pp. 134-143.

7. David MZ and Daum RS (2010). Community-associated methicillin- resistant Staph. aureus: epidemiology and clinical consequences of an emerging epidemic. Clin Microbiol Rev 23: 616–687.

8. David ML and Derek FJB (2005). Detection of beta-lactamase mediated resistance. pp. 1-8.

9. De Neeling AJ, van den Broek MJ, Spalburg EC, van Santen-Verheuvel MG, Dam-Deisz WD, Boshuizen HC, van de Giessen AW, E.van D and Huijsdens XW (2007). High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet. Microbiol. 122: 366-372.

10. El-Seedy FR, El-Shabrawy M, Hakim AS, Dorgham SM, Nagwa S, Bakry MA and Osman NMN (2010). Recent Techniques used for isolation and characterization of Staph. aureus from mastitis cows. J Am Sci 6(2): 232-237.

11. Esmat M and Bader A (1996). Some studies on mastitis meteritis agalcti syndrome in cows. Vet Med J Giza 44(2): 303-309.

12. Gross-Schulman S, Dassey D and Mascola L (1998). Community –acquired Methicillin resistant Staph. aureus. J A M A 280:421-422.

13. Haran KP, Godden SM, Boxrud D, Jawahir S, Bender JB and Sreevatsan S (2012). Prevalence and Characterization of Staphylococcus aureus, Including Methicillin-Resistant Staphylococcus aureus, Isolated from Bulk Tank Milk from Minnesota Dairy Farms. J Clin Microbiol 50(3): 688–695.

14. Hareri MS, Souminen L, Rantala T, Honkanen-Buzalski and Pyorala S (2005). Comparison of phenotypic and genotypic detection of penicillin G resistance of Staph. aureus isolated from bovine intramammary infection. Vet Microbiol 106: 97-102.

15. Harley JP and Prescott LM (2002) Laboratory Exercises in Microbiology. 5th Edition, The McGraw-Hill Companies.

16. Hiramatsu K, Hanaki H, Ino T, Yabuta K, Oguri T and Tenover F (1997). Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother 40: 135-136.

17. Hirschl A., Stanek G. and Rotter M. (1984). Effectiveness of cefamandole against methicillin resistant strains of Staph. aureus in vitro and in experimental infections. J Antimicrob Chemother 13: 429-435.

18. Kilic A, Li H, Stratton CW and Tang YW (2006). Antimicrobial Susceptibility patterns and Staphylococcal cassette chromosome mecC types of, as well as panton-valentine leukocidin occurrence among, methicillin-resistant Staph. aureus isolates from children and adults in middle Tennessee. J Clin Microbiol 44: 4436-4440.

19. Kenar B, Kuyucuoğlu Y and Șeker E (2012). Antibiotic susceptibility of coagulase-negative Staphylococci isolated from bovine subclinical mastitis in Turkey. Pakistan Vet J 32: 390-393

20. Kumar R, Yadav BR and Singh RS (2011). Antibiotic resistance and pathogenicity factors in Staph. aureus isolated from mastitic Sahiwal cattle. J Biosci 36: 175-188.

21. Kumar R, Yadav BR and Singh RS (2010). Genetic determinants of antibiotic resistance in Staph. aureus isolates from milk of mastitic crossbred cattle. Curr Microbiol 60: 379–386.

22. Khanna T, Friendship R, Dewey C and Weese JS (2008). Methicillin resistant Staphylococcus aureus colonization in pigs and pig farmers. Vet Microbiol 128: 298-303.

23. Li L, Zhou L, Wang L, Xue H and Zhao X (2015). Characterization of methicillin-resistant and susceptible Staphylococcal isolates from bovine milk in Northwestern China. PLoS ONE 10(3): 1-8 e0116699. doi:10.1371/journal.pone.0116699

24. Maranan MB, Moreira B, Boyle-Vavra S and Daum RS (1997). Antimicrobial resistance in staphylococcal epidemiology, molecular mechanisms and clinical resistance. Infect Dis Clin North Am 11: 813-849.

25. Murakami K, Mincimide W, Wada K, Nakamura E, Teruoka H and Watanabe S (1991). Identification of methicillin-resistant strains of staphylococci by polymerase chain reaction. J Clin Microbiol 29: 2240-2244.

26. Moon JS, Lee AR, Kangi HM, Lee ES, Kim MN, Paik YH, Park YH, Joo YS and Koo HC (2007). Phenotypic and genetic antibiogram of methicillin-resistant Staphylococci isolated from bovine mastitis in Korea. J Dairy Sci 90(5):1176-1185.

27. Orozova P, Chikova V, Kolarova V, Nenova R, Konovska M and Najdenski H (2008). Antibiotic resistance of potentially pathogenic Aeromonas strains. Trakia J Sci 6(l1): 71-77.

28. Pitkala A, Haveri M, Pyorala S, Myllys V and Honkanen-Buzalski T (2004). Bovine mastitis in Finland 2001: Prevalence, distribution of bacteria, and antimicrobial resistance. J Dairy Sci 87: 2433-2441.

29. Ribeiro MER, Petrini LA, Aita MF, Balbinotti M, Stumpf Jr. W, Gomes JF, Schramm RC, Martins PR and Barbosa RS (2003). Relation between clinical, subclinical infectious and noninfectious mastitis in milk production units in the southern region of the Rio Grande do Sul State. Revta. Bras. Agrociência 9: 287-290.

30. Sambrook J, Fritsch EF and Maniatis T (1989). Gel Electrophoresis of DNA. In: Sambrook J, Fritsch EF and Maniatis, T (Eds.), Molecular Cloning: A Laboratory Manual, Chapter 6, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

31. Sori T, Hussien J and Bitew M (2011). Prevalence and susceptibility assay of Staphylococcus aureus isolated from bovine mastitis in dairy farms of Jimma town, south west Ethiopia. J Anim Vet Adv 10: 745-749.

32. Shanson DC (1981). Antibiotic resistant Staph. aureus. J Hosp Infect 2(11): 52-58.

33. Swenson JM (2002). New tests for the detection of oxacillin-resistance Staph. aureus. Clin Microbiol Newslett 24(21): 159-163.

34. Thompson RL, Fisher KA and Wenzel RP (1982). In-vitro activity of N-formimidoly thienamycin and other beta lactam antibiotics against methicillin resistant Staph. aureus. Antimicrob Agent Chemother 21(1): 341-349.

35. Tomasz A, Drugeon HB, de Lencastre HM, Jabes D, MacDoughl L and Bille J (1989). New mechanism for methicillin resistance in Staphylococcus aureus: Clinical isolates that lack the PBP2a gene and contain modified penicillin-binding proteins with penicillin binding capacity. Antimicrob Agents Chemother 33: 1869-1874.

36. Vesterholm-nelsen M, Larsen M and Aarestrup FM (1999). Occurance of the blaZ gene in penicillin resistant Staph. aureus isolated from bovine mastitis in Denmark. Acta Vet Scand 40: 279-286.

37. Weems JJ (2001). The many faces of Staphylococus aureus infections. Postgrad Med. 110(4):24-36.



38. Zhang K, McClure JA, Elsayed ST, Lovie T and Conly JM (2005). Novel multiplex PCR assay for characterization and concomitant subtyping of Staphylococcal cassette chromosomes mec typing I to V in methicillin-resistant Staph. aureus. J Clin Microbiol 43: 5026-5033.

Article’s Citation:

Ishaq SA and Nafi’u SA (2016). Molecular characterization of β-lactamase producing staphylococcus aureus from mastitic cow isolates. Ew J Microb Res 2(2): 13 - 21.

Documents

2016| Volume 2 | Issue 2 |p. 13 - 21 Ewemen Journal of