This cross sectional study was conducted in a tertiary care hospital, Mangalore (South India), from January 2015 to February 2017. Ethical approval for the study was obtained from Institutional Ethical Committee (Ref: NU/CEC/Ph.D-65/2012) and informed consent was obtained from the patients. Demographic details and all other relevant clinical details were also documented.

Patients without hospital associated risk factors such as the long term admission to a medical care facility, surgery or dialysis in the previous year, previous MRSA isolation or colonisation were considered as having community associated infections and remaining cases as hospital associated infections and comparative study of CA-MSSA and HA-MSSA were statistically computed. Duplicate isolates of Staphylococcus aureus from the same patient at a different site of infection was excluded during the study period.

From the available hospital records, the prevalence of Staphylococcus aureus infections in previous years was studied. In this study, Staphylococcal strains were collected from the tertiary care hospital laboratory during the study period and the prevalence of MSSA in community and hospital associated infections were analysed. Staphylococcus aureus was isolated from various clinical samples (pus, blood, cerebrospinal fluid, body fluids, sputum, throat swab, urine). Staphylococcus aureus was isolated and identified from clinical specimens by standard laboratory procedures [21]. S. aureus was phenotypically identified by cultural characteristics on blood agar and Mac Conkey’s agar (Hi-Media) incubated at 37°C for 24-48 hours, Gram reaction, catalase test, coagulase test and mannitol fermentation [21]. Staphylococcus aureus ATCC (American Type Culture Collection) 25923 was used as the quality control strain.

Detection of methicillin resistance in S. aureus was done by disc diffusion test using cefoxitin disc (30 μg) on Muller-Hinton agar (Hi-Media, Mumbai, India) [22]. A zone of inhibition ≤21 mm was considered as methicillin resistant, and a zone of inhibition ≥22 mm was considered as methicillin sensitive, according to Clinical and Laboratory Standards Institute (CLSI) guidelines [23]. For quality control S. aureus ATCC 29213 (MSSA) and ATCC 43300 (MRSA) were used.

Antibiotic resistance profiles of S. aureus isolates against commonly prescribed antibiotics were determined by the Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Hi-Media, Mumbai, India) and the results were interpreted according to the CLSI guidelines [21]. The antibiotics used were amikacin (30 μg), ampicillin (10 μg), cephalexin (30 μg), cefoxitin (30 μg), cefotaxime (30 μg), ciprofloxacin (5 μg), clindamycin (2 μg), chloramphenicol (30 μg), co-trimoxazole (25 μg), doxycycline (30 μg), erythromycin (15 μg), gentamicin (10 μg), levofloxacin (5 μg), linezolid (30 μg), netilmycin (30 μg), oxacillin (1 μg), ofloxacin (5 μg), penicillin (10 U), rifampicin (5 μg), tetracycline (30 μg), tigecycline (15 μg), teicoplanin (30 μg) and vancomycin (30 μg).

Data analysis was done using Statistical Package for Social Sciences (SPSS) software version 20. The collected information were summarised by using frequency, percentage, mean and standard deviation. Chi-square test was used to compare the difference in antibiotic sensitivity with respect to CA-MSSA and HA-MSSA, a p-value <0.05 was considered as statistically significant.

A total of 305 S. aureus were isolated during the study period. Staphylococcus aureus were isolated from various clinical samples [Table/Fig-1]. Out of 305 Staphylococcus aureus isolated, 219 (71.8%) were CA-MSSA and 86 (28.2%) were HA-MSSA. Out of 305 MSSA cases, inpatient admissions (IP) were 64.9% and outpatient admissions (OP) were 35.1% [Table/Fig-2]. The median age of the patients was 40 years, minimum age was 1 year and the maximum age was 85 years. Frequency of isolation of Staphylococcus aureus from cases of different age groups is shown in [Table/Fig-3]. There was a significant difference between the isolation of CA-MSSA and HA-MSSA with regard to age group and IP/OP admission (p<0.05). In this study, 107 (35.1%) were females and 198 (64.9%) were males. Staphylococcus aureus were isolated mostly from Skin and Soft Tissue Infections (SSTI), (61.3%) [Table/Fig-4]. In this study, the overall mortality rate observed was 14 (4.6%). There was no significant difference between CA-MSSA and HA-MSSA with regard to gender, isolation from clinical samples and mortality rate. Patients with HA-MSSA infection had longer duration of hospital stay compared to patients with CA-MSSA infection [Table/Fig-5]. The clinical details of CA-MSSA and HA-MSSA cases are given in [Table/Fig-6].

Antibiotic resistance profiles of MSSA against a panel of 23 antibiotics were studied [Table/Fig-7]. All MSSA isolates were 100% sensitive to amikacin, oxacillin, cefoxitin, linezolid and vancomycin. MSSA showed 100% resistance to ampicillin. MSSA showed higher antibiotic resistance against ciprofloxacin (64.6%), erythromycin (43.9%) and ofloxacin (42.3%). Comparative antibiotic resistance profile of CA-MSSA and HA-MSSA is shown in [Table/Fig-8]. Both CA-MSSA and HA-MSSA showed increased susceptibility to netilmycin, tigecycline, doxycycline, tetracycline, chloramphenicol and teicoplanin. There was a significant difference between CA-MSSA and HA-MSSA resistance shown against cefotaxime and co-trimoxazole (p<0.05). Of 305 MSSA isolates, 184 (60.3%) were found to be multidrug resistant. There was a significant difference between CA-MSSA 124 (56.6%) and HA-MSSA 60 (69.8%) showing MDR (p<0.05).

This study revealed a higher prevalence of MSSA in patients attending a tertiary care hospital with community or hospital associated infections caused by Staphylococcus aureus. Several previous studies corroborate this observation [5,6,24,25]. A total of 305 MSSA were isolated during the study period. In this study, there was a significant difference between CA-MSSA and HA-MSSA with regard to age group. Higher prevalence of CA-MSSA was seen among age group 21-30 years (25.6%) and HA-MSSA from age group 41-50 years (22.1%). Authors found HA-MSSA prevalence was increasing in the older age groups with hospital associated risk factors compared to CA-MSSA found mostly in the younger and middle aged groups. Shenoy MS et al., found isolation of CA-MRSA mainly from patients of age group 21-30 years [26]. In this study, MSSA were mostly isolated from males compared to females, but no significant difference was observed between CA-MSSA and HA-MSSA with regard to gender, mortality rate and isolation from clinical specimens.

Out of 305 MSSA isolates, 71.8% were CA-MSSA which shows a higher prevalence of CA-MSSA compared to HA-MSSA 28.2%. In a study from Sikkim reported a prevalence rate of 42% in CA-MSSA and 22.7% in HA-MSSA [27]. In a study to know the epidemiology of Staphylococcus aureus in Bangalore CA-MSSA was 32% and HA-MSSA was 12% [10]. In this study, CA-MSSA were almost equally isolated from outpatients (45.7%) and inpatients (54.3%), while (91.9%) of HA-MSSA were from inpatients. The isolation of MSSA from inpatients and outpatients showed that MSSA is prevalent in the hospital and community settings. A study from Uttar Pradesh reported Staphylococcus aureus nasal carriage rate of 46.7% in children [28]. Since in this study, MSSA were isolated from cases admitted to different departments, measures have to be implemented for better patient treatment and prevent spread of MSSA in hospital and community settings. The available literature review on the prevalence of MSSA isolated from different places in India was studied and shown in [Table/Fig-9] [6,7,10,17,18,24,25,27,29].

In present study, MSSA were isolated mainly from SSTI (61.3%). A relatively lower MSSA isolation rate of 53% has been reported from Pondicherry [29], while a study from Delhi reported a higher isolation rate of 76% [24]. A study from China reported 81.2% of MSSA from SSTI [30].

Studies have reported a higher prevalence rate of MSSA compared to MRSA in India and different countries but varies worldwide [5,7]. In a study to determine the antibiotic susceptibility, virulence profile and genomic diversity among MSSA and MRSA from different parts of India from 2007 to 2015 found 70.6% MSSA and 29.4% MRSA [6]. A study from a tertiary care center in Mangalore to detect MRSA carriage in Critical Care Departments reported prevalence rate of MSSA in 15% and MRSA in 2.5% of healthcare workers [31]. A study from Mumbai reported prevalence rate of MSSA 67.3% and MRSA 29.7% [25]. A study from Northern Taiwan reported one third of Staphylococcus aureus infections in paediatric patients were due to MSSA [11].

A higher prevalence of MRSA have also been reported from several studies which has to be considered as MRSA might outnumber MSSA from the hospital and community settings [12,19]. A study from South Western India reported 52% MRSA and 48% MSSA [17,13]. Robinson DA et al., reported that the descendants of early penicillin resistant clone of Staphylococcus aureus, the phage type 80/81 have acquired methicillin resistance and re-emerged as CA-MRSA [32]. Staphylococcus aureus phage type 80/81 which caused early pandemic belonged to clonal complex 30 (CC30) [33]. A study from Singapore reported majority of CA-MRSA (SCCmec IV c) isolates belonged to sequence type (ST 30) and were highly transmissible [34]. D’Souza N et al., from Mumbai reported isolation of eleven PVL positive. MSSA isolates and all were ST 30 (CC30) and also found the presence of SCCmec IVc [4]. Mera RM et al., reported annual MRSA prevalence increased from 32.7% in 1998 to 53.8% in 2007 [35]. There could be a horizontal transfer of methicillin resistance gene from MRSA to multiple ST 30 MSSA isolates and subsequent dissemination, which can lead to outbreak of MRSA infections. Studies from South India reported increasing prevalence of MRSA compared to MSSA [17,18].

In this study, the overall antibiotic resistance profile showed MSSA were susceptible to several antibiotics. MSSA were all susceptible to amikacin, cefoxitin, linezolid, oxacillin and vancomycin. A study by INSAR group reported 100% sensitivity to vancomycin and linezolid [8]. In this study the overall resistance profile of clindamycin was found to be 20.7%. A study from Mumbai, found MSSA highly susceptible to several antibiotics compared to MRSA [25]. In this study MSSA showed 100% resistance to ampicillin and were more susceptible to chloramphenicol, doxycycline, netilmycin, tetracycline, tigecycline and teicoplanin. MDR-resistance to more than three antibiotics was found in MSSA, showing greater resistance to ciprofloxacin 64.6%, erythromycin 43.9% and ofloxacin 42.3%. Shahkarami F et al., reported resistance to tetracycline 51.5%, gentamicin 30.3% and lower resistance to ciprofloxacin 22.2% and erythromyvin 15.2% [13]. Bouchiat C et al., reported resistance to erythromycin as 54.3% and no significant difference between MSSA and MRSA [10].

Staphylococcus aureus resistance to fluoroquinolones has been found to be increasing [12]. MSSA showed a resistance of 54.5% to ciprofloxacin reported by Bouchiat C et al., from Bangalore [10]. In India, a study reported 46.6% of MSSA resistant to ciprofloxacin [8].

In this study, a significant difference was found between CA-MSSA and HA-MSSA regarding antibiotic resistance profile against cefotaxime and co-trimoxazole (p<0.05). From this study, no significant difference was found between CA-MSSA and HA-MSSA resistance against ciprofloxacin 63.5% and 67.4% and erythromycin 41.6% and 50%, respectively (p>0.05). A study from Bangalore found no significant difference between CA-MSSA and HA-MSSA regarding antibiotic resistance against erythromycin 43.8% and 41.7% and against ciprofloxacin 56.3% and 50%, respectively [10]. Kini AR et al., reported CA-MSSA with 67% resistance to erythromycin and 48% resistance to ciprofloxacin [15]. A study from Andhra Pradesh reported CA-MSSA and HA-MSSA resistance against ciprofloxacin 57.1%, and 66.7% and against erythromycin 29.3% and 17.4%, respectively and no significant difference between CA-MSSA and HA-MSSA in antibiotic resistance [12]. In this study, it was found CA-MSSA and HA-MSSA susceptible to multiple classes of antibiotics, and CA-MSSA showed increased susceptibility to gentamicin, netilmycin, rifampicin, tigecycline levofloxacin, doxycycline and chloramphenicol compared to HA-MSSA. Emerging clindamycin resistance and increasing resistance against erythromycin is of great concern as macrolides have a significant role in the treatment of Staphylococcal infections.

In this study CA-MSSA and HA-MSSA showing MDR were found. MDR shown by HA-MSSA were found to be higher compared to CA-MSSA. Both CA-MSSA and HA-MSSA showed resistance to ciprofloxacin, erythromycin, ofloxacin, ampicillin, penicillin and the emergence of clindamycin resistance. Aggarwal S et al., reported out of 109 Staphylococcus aureus isolated from a variety of infections in India, 102 (93.6%) were MDR and from the total 70.6% were MSSA, 29.4% were MRSA respectively [6] A study from Punjab reported higher prevalence of MRSA compared to MSSA and MDR [19]. Tayebi Z et al., reported 69.4% of MSSA exhibited MDR [14]. Emergence of MDR in MSSA is a matter of great concern as MDR MSSA will spread in hospitals and to the community which can create difficulty in patient treatment. Studies reporting increasing prevalence of virulent MRSA in community and hospital settings [3], there is an increased chance to transfer mobile methicillin resistant gene from MRSA to MSSA and MRSA might replace MSSA from hospital and community settings and measures to be taken to prevent MSSA becoming MDR and becoming methicillin resistant which can cause treatment failure.

In the present study, antibiotic susceptibility testing for vancomycin by Minimum Inhibitory Concenteration (MIC) method was not carried out and authors did not analyse the severity of MSSA infections caused in each clinical condition.

From this study, it was found that MSSA is increasingly prevalent in community and hospital settings with emergence of MDR. Immediate control measures have to be taken to prevent the spread of MDR MSSA in the hospital settings and to the community also to prevent the replacement of MSSA by more virulent MRSA. Strict aseptic techniques in hospitals, treatment of patients and carriers, screening of hospital staff, antibiotic stewardship and treatment plan has to be implemented immediately to prevent the spread of MSSA and emergence of MDR.