Multidrug drug resistance in Staphylococcus aureus isolates from Clinical Specimens in Northern India

Staphylococcus aureus is a well-known human pathogen that is primarily associated with nosocomial infections causing variety of diseases with increasing resistance to antibiotics. The goal of this study was to determine the prevalence and multi drug resistance patterns of both methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus (MSSA) in the studied population. Out of 60 S. aureus isolated, 27 (45.00%) were found to be methicillin resistant, while 33 (57.14%) were methicillin susceptible. The incidence of MRSA in different samples was recorded with maximum in pus (55.55%), followed by wound swabs (50.00%), urine (45.95%) ear swab and blood samples (25.00%) each. MRSA isolates showed marked antibiotic resistance (over 70.00%) against oxacillin, penicillin, nalidixic acid, ampicillin, amoxicillin, and cefpodoxime. All MRSA isolates were foundto bemultidrug resistant with 14.81% isolates showing resistant to 14 drugs. The MSSA isolates tested were also highly resistant to penicillin (81.00%), ampicillin (90.00%), and amoxicillin (88.00%) with 6.06% isolates showing multi drug resistance to maximum of 11 antibiotics tested.


INTRODUCTION
Multi drug resistance is very common in Staphylococcus aureus clinical isolates worldwide, particularly in developing countries.S. aureus is a causative agent of wide variety of diseases.It is also involved in causing urinary tract infections (UTI) (Akerele and Ahonkhai, 2000).Methicillin-resistant S. aureus (MRSA) has established itself to be one of the more often wide spread and stable nosocomial pathogens of the late 20th century (Nimmo et al., 2000).MRSA is relatively ubiquitous and is the cause of many community, endemic and epidemic nosocomial colonization and infections.They cause surgical site infections, bacteraemia, lower respiratory tract infections, urinary tract infections, etc. (Duguid et al., 1978).MRSA is of concern not only, because of its resistance to methicillin, but it is also resistant to many other antibiotics (Vidhani et al., 2001).It is a very common pathogen in hospital admitted patients globally and now become a challenge for clinicians to curing infections caused by S. aureus; as common public infections are increasing in various regions and countries (Layton et al., 1995).
In Africa, US, Southern European countries, Asia and South America, MRSA prevalence has been recorded in a varied trend as high in some countries and low in others (Bell et al., 2002).National Nosocomial Infection Surveillance System (NNIS) reported that 50.00% of hospital acquired infections in ICUs in the USA are due to MRSA (Salgado et al., 2003).MRSA is now endemic in India.The incidence of MRSA varies according to the region, lower in west (Patel et al., 2010) and higher in South India (Gopalakrishnan and Sureshkumar, 2010).MRSA prevalence is rapidly increasing with the time in India.In a study conducted in Indore, the MRSA has been increased significantly from 1992 to 1999 (Verma et al., 2000).Similar findings were also observed in Uttar Pradesh where the incidence of MRSA was significantly increased from 1988 to 2008 (Mathur et. al., 1994;Tiwari et al., 2008).
Infection with MRSA strains, which are resistant to wide range of antibiotics, is directly involve with producing diseases and death (Capitano et al., 2003).Most of the MRSA strains are multi-drug resistant; however, many of the β-lactams are ineffective against a significant proportion of S. aureus isolates (Guignard et al., 2005;Llarrull et al., 2009).Resistance in MRSA is related to a chromosomal mecA gene that is responsible for the production of an abnormal penicillin binding protein called PBP2a or PBP21.Penicillin-binding proteins are membrane-bound enzymes, which targets for all β-lactam antibiotics (Weems, 2001).The mecA gene complex also has insertion sites for transposons and plasmids that facilitate acquisition of resistance to other antibiotics.Thus, S. aureus is commonly resistant to non β-lactam antibiotics, such as erythromycin, clindamycin, gentamicin, co-trimoxazole and ciprofloxacin (Chambers, 1979;Chambers, 2001).The emergence of multi drug resistant MRSA is a serious and ongoing concern.The ultimate goal of our research work is to determine the prevalence of methicillin-resistant S. aureus from clinical specimens and to suggest an appropriate and potent drug for treatment to the patients suffering from MRSA in Northern India.

Samples collection
A total of 330 clinical specimens consisting of pus swabs, wound swabs, urine, ear swabs and blood samples were collected between January 2013 and December 2014 from the diagnostic centers of Lucknow in Era's Lucknow Medical College and Hospital, Lucknow, and Integral Institute of Medical Sciences and Research, Lucknow in Northern India.
Sterile cotton wool swabs moistened with Stuart's Transport Medium were used to collect the specimens and inoculated into freshly prepared slant of nutrient agar and mannitol salt agar.The clean-catch midstream urine samples were collected directly into sterile disposable universal bottles.The cultures were incubated at 37±1°C for 24 h (Kolawole and Shittu, 1997).

Isolation and identification of the isolates
The specimens collected were directly streak onto mannitol salt agar (Hi-Media).The plates were incubated at 37°C for 24 h.Plates were observed for smaller colonies that were yellow due to mannitol fermentation on mannitol salt agar medium.S. aureus colony was confirmed by Gram staining, catalase test, coagulase test, mannitol fermentation, gelatin liquefaction, and β-hemolysis on blood agar (Washington et al., 2006).

Determination of antimicrobial resistance
Pure isolates of identified S. aureus were subjected to antimicrobial susceptibility testing using the disc diffusion method as recommended by Kirby Bauer method according to the recommendations of Clinical Laboratory Standard Institute (CLSI, 2010), formerly National Committee for Clinical Laboratory Standards (NCCLS) (2002), using the following antibiotics discs obtained from Hi-Media Laboratories Pvt.Ltd, Mumbai: methicillin (MET) 5 μg, oxacillin (ox)1 μg, penicillin G (PEN) 10 IU, erythromycin (ERYTHRO) 5 μg, nalidixic acid (NA) 30 μg, kanamycin (KAN) 30 μg, nitrofurazone (NR) 100 μg, tetracycline (TET) 10 μg, polymyxin B (PB) 300 μg, ciprofloxacin (CIP) 5 μg, ampicillin (AMP) 10 μg, ofloxacin (OF) 5 μg, sulphadiazine (SZ) 300 μg, amoxicillin (AMX) 10 μg, and cefpodoxime (CPD) 30 μg.All isolates were grown in Brain Hearth Infusion broth (Biotech Laboratories, United Kingdom) and incubated at 37°C for 6 h until the turbidity of 0.5 McFarland standards was achieved.The isolates were then swabbed onto Muller Hinton Agar (Amershan, England) and allowed to dry for 15 min.The antibiotics discs were placed on the centre of the agar plates with the aid of sterile pointed tip forceps and incubated at 37°C for 24 h.The presence of a clear zone around the antibiotic disc is measured with meter rule.S. aureus strains were tested for methicillin resistance using the disc diffusion method (Bauer et al., 1966).S. aureus isolates are considered to be resistant to methicillin if the inhibition zones are <10 mm while susceptible if the zones of inhibitions were ≥ 10 mm.

Multiple antibiotic resistances (MAR) indexing
The MAR index profile was performed on both MRSA and MSSA isolates.The MAR index was calculated according to the formula: No. of antibiotics to which all isolates were resistant/No. of antibiotics tested × No. of isolates as recommended by Downing et al. (2011).Sampling site based MAR index was calculated by the same formula modified by the total number of isolates from a sampling site as described (Riaz et al., 2011).

Statistical analysis
Statistical analysis in the present study was done by using Microsoft Office Excel 2007, SPSS version 12 (Statistical Package for Social Sciences).t-test was performed to check the significance of the data by considering (p value=0.05).

RESULTS
Out of 330 samples studied, S. aureus was isolated from 60 samples (18.18%).The results showed that the highest prevalence of S. aureus was found in wound sample (25%) as compared to others.Others are ear swab and blood (20.00% each), pus swab (18.00%) and  urine (17.1%).Out of the total 60 strain of S. aureus isolated from different samples, 27 (45.00%)were found to be resistant to methicillin, while ear swab and blood samples had the least distribution for MRSA (Table 1).
The multidrug resistance data for MSSA isolates was also observed.Maximum number of isolates (90.9%) showed resistance against ampicillin followed by 87.88, 81.88, 51.51, 45.50, 30.30, 27.27, 21.20 and 12.12% against amoxicillin, penicillin G, cefpodoxime, sulphadiazine, erythromycin, nalidixic acid, ofloxacin, kanamycin, tetracycline, polymyxin B, ciprofloxacin, nitrofurazone and oxacillin, respectively.A high occurrence rate of MSSA was recorded as compared to MRSA from clinical specimens but all the isolates of the MRSA showed a significantly higher multidrug resistance.
Multidrug resistance patterns in 27 MRSA and 33 MSSA isolates were also recorded.Both isolates of MRSA and MSSA showed a variety of resistance patterns.Besides four isolates of MRSA, each of the isolates demonstrated a single resistance pattern against different number of antibiotics.However, each of the MSSA isolates was observed with a unique and single multidrug resistance pattern.Out of the 27 MRSA isolates, 3.7% showed resistance to 7, 8, 9, 10, 11, 13, and 14 antibiotics at a time in different combinations, respectively, while 7.4% exhibited resistance to 8 and 14 antibiotics at a time in two different combinations, respectively.Of the 33 MSSA isolates, 3.03% showed resistance to 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 antibiotics at  A varied trend of MAR index was also observed among the MRSA isolates with 3.7% of the isolates showing a MAR of 0.46 to 0.93 range against different number of antibiotics.Also MAR of 1.06 and 1.86 were recorded by 7.4% isolates.In the case of MSSA isolates, 3.03% isolates demonstrated 0.13 to 0.73 MAR range (Tables 3  and 4).

DISCUSSION
S. aureus is a well-known human pathogen that is primarily associated with nosocomial infections in human causing variety of diseases with increasing resistance to β lactam antibiotics.MRSA strains have become a global cause of concern, because of their continuous increasing resistance nature against anti-staphylococcal drugs (Mulla et al., 2007).In India, the methicillin resistant S. aureus strain's importance as a problem has been recognized relatively late (Rajaduraipandi et al., 2006).In India, the MRSA prevalence is not uniform and it varies in different parts of the country.A Delhi hospital report of 2001 showed that the prevalence rate of MRSA was 51.6%, whereas in the same hospital, a study was conducted in 2008 where a prevalence rate of 38.44% reported (Tiwari et al., 2008).Dar et al. (2006) in their study at Aligarh, India reported 35.1% of S. aureus isolates resistant to methicillin.Rajaduraipandi et al. (2006) in Tamil Nadu reported that 31.1% of S. aureus isolates from clinical samples were methicillin resistant.Sangeeta et al. (2013) in their study reported that the prevalence rate of MRSA strains was 42% in 2008 and 40% in 2009.27 MRSA and 33 MSSA strains were  (Anupurba et al., 2003, Tiwari andSen, 2006;Muralidharan, 2009;Arora et al., 2010).A study was conducted to find the prevalence rate of MRSA in Eastern Uttar Pradesh and AIIMS in New Delhi and the prevalence rate of MRSA was 54.85 and 44%, respectively (Anupurba et al., 2003;Arti et al., 2008).Maximum frequency of the MRSA isolated were from the pus samples, that is, 5 out of (55.55%) followed by wound swab (50%), urine (45.95%), ear swab and blood samples (25%) as shown in Table 1.This is similar with studies by Anupurba et al. (2003) and Vidya et al. (2010).Maximum incidence of MRSA was recorded from pus, reported by Tiwari et al. (2009) and also by Anupurba et al. (2003).Our result differs with other studies where wound swabs were the main source (Rajaduraipandi et al., 2006).In this study, resistance nature of MRSA to different antibiotics was more than MSSA strains.The present study shows high resistance to ampicillin, amoxicillin, oxacillin, and penicillin.This result is similar with other studies (Tiwari et al., 2009;Vidya et al., 2010) Kumari et al. (2008) who reported that 32.6% of the S. aureus strains were resistant to ciprofloxacin.There is a difference between antibiogram of MRSA and MSSA isolates.Maximum resistance was seen with ampicillin and amoxicillin (Figure 1).This result is similar with that of Shamsadh et al. (2011).The highest level of resistance of S. aureus strain has been observed with ampicillin (100%), amoxicillin (100%) and both penicillin and oxacillin (92.59%), which is in accordance with the reports of Tiwari et al. (2009).More than 70% resistance was also seen with nalidixic acid and cefpodoxime (70.37%).The high frequency of resistance observed with kanamycin (67.7%), nalidixic acid and cefpodoxime (70.37%), penicillin (92.59%), ampicillin and amoxicillin (100%), could be attributed to their use in treatment of diseases in animals and humans.Resistant bacteria may transfer resistance genes to other bacteria and become important in the spread of antibiotic resistance.Indiscriminate use of antimicrobial agents and antibiotic sale behavior (for example, sale of antibiotics without prescription, sale of under dose and substituting brands) enhances the development of drug resistance (Indalo et al., 1997).In our findings, all the MRSA isolates showed high percentage of multidrug resistance against 7 to 14 antibiotics in 25 different patterns of combination (Tables 2 and 3).Similar trend of resistance was observed in MSSA isolates (Tables 2 and 4).A study was conducted by Majumder et al. (2001) from Assam which reported 23.2% of the MRSA isolated from clinical samples were found to be multidrug resistant.Higher percentage of multidrug resistant MRSA was also reported by Anupurba (Assadullah et al., 2003).Vidhani et al. (2001) in their study also reported a higher percentage of MRSA isolated from high risk patients admitted in burns and orthopedic units.So, the awareness should be created about the route of MRSA transmission in the hospital environment and the risk of its infections in the community.

Table 1 .
Occurrence rate of S. aureus (MRSA and MSSA) isolates from various clinical specimens.
P. value = Indicates statistically significance difference between methicillin resistance and sensitive strains.NS: Not significant, S: Significant.