Sensitivity Pattern of Staphylococcus aureus Isolates from Different Sources for Methicillin, Vancomycin, β-lactamase and ESBL Production

Over the past few years, it has become hard to control the virulent strains of S. aureus because they have become resistant to various β-lactam antibiotics such as methicillin and penicillin. Such strains of S. aureus are known as methicillin-resistant S. aureus (MRSA). Methicillin-resistant S. aureus was reported in October 1960, which is now endemic in India (Ray et al., 2013). The incidence of MRSA varies from 25% in the western part of India (Patel et al., 2010) to 50 percent in South India (Gopalkrishan et al., 2010). Since then, MRSA has become endemic in hospitals and nursing homes worldwide.Beta-lactam compounds such as penicillin continue to be one of the most frequently used drugs in veterinary medicine (Pitkala et al., 2007). With the development of MRSA, vancomycin has been used as the antibiotic of choice to treat infections caused by S. aureus strains that are resistant to methicillin and oxacillin. In addition, the emergence of vancomycin-resistant S. aureus has been reported in some studies (Tenover et al., 2004; Ateba et al., 2010). The establishment of MRSA and the emergence of VRSA have great concern because these are not only resistant to methicillin but also to vancomycin, monobactams and cephalosporins through the production of ESBL (Extended-spectrum b-lactamases). Production of other Extended-spectrum b-lactamase (ESBL) enzymes leads resistance to penicillin and cephalosporins, monobactams and carbapenems antibiotics. Resistance mechanisms of staphylococci include enzymatic inactivation of the antibiotic (penicillinase and aminoglycoside-modification enzymes), alteration of the target with decreased affinity for the antibiotic (penicillin-binding protein 2a of methicillin-resistant S. aureus and D-Ala-D-Lac of peptidoglycan precursors of vancomycin-resistant strains), trapping of the antibiotic (vancomycin and daptomycin) and efflux pumps (fluoroquinolones and tetracycline) (Lowy, 2003; Pantosti et al., 2007). MRSA and VRSA strains show pathogenic and epidemiological characteristics in several ways, such as clonal evolution (Fitzgerald et al., 2001), mutation and horizontal gene transfer (Brody et al., 2008). Although there are many reasons which compromise antibiotic treatment of S. aureus infections of which resistance activity of bacteria toward antibiotics is the most important. The present study investigated the phenotypic and genotypic characteristics of all the 62 S. aureus isolates with respect to MRSA, β-lactamase, ESBL production and VRSA activities.

Sample collection
 
A total of 180 samples from various sources like human pus, animal pus, the skin of an animal, skin of human, mastitis milk, regular milk and unprocessed meat were collected. These sample sources belonged to two different localities of Bikaner (Rajasthan). The samples were collected in the morning and immediately taken to the laboratory on ice for further processing.
 
Isolation and identification of bacteria
 
The organisms were isolated and identified as described by Cowan and Steel (2003) and Quinn et al., (1994). Each sample was swabbed on nutrient agar medium and then incubated overnight at 37°C. Suspected colonies were streaked on mannitol salt agar in primary, secondary and tertiary fashion and incubated for 24 h at 37°C. Of the 180 samples, 62 isolates of S.aureus from various sources were obtained, further confirmed genotypically by 23S rRNA-based ribotyping and Proteomic based identification of S.aureus by MALDI TOF MS (VITEK MS RUO).
 
Methicillin resistance activity
 
Methicillin resistance activity among S. aureus isolates was detected by culturing them on MeReSa Agar. This method included observation of colony growth on MeReSa agar base with MeReSa selective supplement (FD229) and Cefoxitin supplement (FD259). After inoculation of testing isolate, the methicillin positive strain grew as luxuriant greenish pink color colony after incubation at 35-37°C for 18-48 hours (Alwash and Saleh, 2013).
 
Beta-lactamase activity (Acidimetric method)
 
Hydrolysis of the β-lactam ring generates a carboxyl group, acidifying un-buffered systems. The resulting acidity can be tested in tubes. The technique described by Livermore and Brown (2001) in which 2 ml of 0.5% (w/v) aqueous phenol red solution was diluted with 16.6ml distilled water and 1.2 g of benzylpenicillin was added. The pH was adjusted to 8.5 with 1M NaOH. The resulting solution, violet in color, was stored at -20°C. Before use, 100 µl of the solution was distributed into microtitre wells and inoculated with the bacterial isolates to produce dense suspensions. A yellow color within 5 min indicated β-lactamase activity.
 
Extended-spectrum β-lactamase (ESBL) activity
 
Extended-spectrum β-lactamase activity among S. aureus isolates was detected by the combined disc method described by Livermore and Brown (2001). This Method included comparing the zone given by discs containing an extended-spectrum cephalosporin with and without clavulanic acid. If an ESBL is produced, the zones are increased ≥5 mm for the discs containing clavulanic acid. This method recommends comparison of the zone given by cefotaxime 30 μg versus cefotaxime 30+Clavulanic acid 10 μg and ceftazidime 30 μg versus ceftazidime 30 +clavulanic acid 10 μg. The readymade (HiMedia) discs were used for this test as described. 

Staphylococcus aureus isolation and genotypic confirmation
 
On the basis of cultural and biochemical properties, out of 180 samples, 62 isolates, including 8 from human pus samples, 9 from animal pus, 12 from mastitis milk, 10 from normal milk, 7 from human skin, 7 from animal skin and 9  from unprocessed meat, were presumptively identified as S. aureus (Table 1). These 62 isolates were genotypically confirmed as S. aureus by 23S rRNA ribotyping using the following sequence for the Primer F-5'-ACG GAG TTA CAA AGG ACG AC-3' and R-5'-AGC TCA GCC TTA ACG AGT AC-3', producing an amplicon of 1250 bp (Fig 1) and proteomically confirmed by Maldi-tof MS. An overall recovery rate was 34.44%. These results are almost similar to those reported by Yadav et al. (2015).


 
Methicillin, β-lactamase, ESBL and vancomycin activity
 
Of the 62 isolates, phenotypically, 53(85.48%) were detected as methicillin resistant S. aureus (MRSA) on MeReSa agar base (MeReSa Selective Supplement having Methicillin 2 mg/ml+ cefoxitin 3 mg/ml in 100 ml media) (Fig 2) but none of the isolates from any source or place of sampling was detected positive by methicillin disk (5 mcg) method (Table 1). Phenotypically 100% MRSA isolates from animal pus and human skin were detected by MeReSa agar. While least number (66.66%) isolates from unprocessed meat were identified phenotypically as MRSA by MeReSa agar base method.





Beta-lactamase activity was exhibited by 44 (70.96%) isolates (Fig 3). The maximum activity was shown by 85.71% isolates from animal skin isolates, while 71.42% isolates from human skin showed least activity (Table 1). Extended-spectrum beta-lactamase (ESBL) activity was exhibited by 51 (82.25%) isolates (Fig 4) with isolates from human pus samples showed 100% activity and the least activity seen in isolates from unprocessed meat, which were 66.66% as described in Table 1.In the present study, no vancomycin-resistant S. aureus was identified by disk diffusion and E-test methods and 100% isolates from all sources were sensitive to vancomycin (Table 1). In contrast, Bhattacharyya et al., (2016) have reported seven VRSA strains from clinical and subclinical mastitis from different districts of West Bengal, India.

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Oliveira et al., (2000) reported less prevalence of β-lactamase, they studied 811 strains of S. aureus isolated from bovine mastitis in Europe and the United States. Of the strains tested, 35.6% were positive for β-lactamase on initial testing, with an additional 21.3% positive after induction of penicillin. In contrast to the present study, lower beta-lactamase production in 55.9% and 9% of the isolates from clinical mastitis was reported by Turutoglu et al., (2006) and Capurro et al., (2010), respectively. Bagcigil et al., (2012) identified 78 β-lactamase positive isolates out of 147 isolates with positive bla Zgene. Russi et al., (2008) observed similar high beta-lactamase activity in 89% of 46 penicillin-resistant strains. The present findings agree with that of Robles et al., (2014), who reported high β-lactamase activity in 100 S. aureus isolates from bovine mastitis. Marques et al., (2017) reported 100% beta lactamase producing S. aureus isolates from bovine mastitis, which is in contrast to our results. In a study on 35 S. aureus isolates from 101 milk samples obtained from clinically mastitic dairy cows in Egypt, Sayed (2014) identified 21 S. aureus strains (60%) as methicillin-resistantS. aureus (MRSA) similar to present findings. Contrary to present findings Singh et al., (2018) recovered 18 MRSA from mastitic milk and 10 MRSA from nasal swabs of dairy cattle by disk diffusion method using oxacillin.

Antibiotic resistance of S. aureus isolated from various sources. Showed high β-lactamase and Extended-spectrum beta-lactamase (ESBL) activity. Similar proportion of antibiotic resistance in S. aureus is also recorded for methicillin Increasing trend of resistance towards these antibiotics requires judicious use of antibiotics.

All authors declare that they have no conflict of interest.