Isolation, characterization and antibiotic susceptibility of staphylococcal isolates, with special reference to methicillin resistant Staphylococcus aureus, from anterior nares of health care workers in a tertiary health care centre.

Background: Staphylococcus aureus (S.aureus) and its resistant form Methicillin resistant S. aureus (MRSA) is one of the most common nosocomial pathogen, causing a wide range of infections in humans. The anterior nares are the main ecological niche for S.aureus. Nasal carriage of S. aureus acts as an important reservoir of infection among the colonized health care workers and they transmit the infection to the community. The aim of the present study is to estimate nasal colonization of S.aureus (with special reference to MRSA) in health care workers (doctors and nursing staff) and its antibiotic susceptibility pattern. Methods: A descriptive study was planned in the Department of Microbiology, JLN Medical College, Ajmer (Rajasthan, India) after due approval from institutional ethics committee. A total of 170 health care workers of either sex and ages between 18 to 60 years were screened for S.aureus. Identication was done using standard microbiological techniques, by studying their morphology, colony and biochemical characteristics. MRSA was detected by cefoxitin disc diffusion test, oxacillin disc diffusion test, minimum inhibitory concentration (MIC) of oxacillin by E-test and oxacillin screen agar test. The observations were described in proportions and chi-squared test was used to nd independence. Statistical signicance was considered at 5%. Results: Among 170 samples, 159 (93.53%) samples (50 doctors and 109 nursing staff) had staphylococci colonization. Out of 159 isolates, 34(21.38%) isolates were S.aureus. Further, 8(5.03%) S. aureus isolates were resistant to both cefoxitin and oxacillin and had oxacillin MIC values ≥ 4 µg/ml and were considered MRSA. All the MRSA belonged to nursing staff [Males: 5.50% , Females :1.83%]. All S. aureus and MRSA isolates were found sensitive to linezolid. In addition, 0.63% MRSA was found vancomycin intermediate S. aureus (VISA) and all MRSA isolates were sensitive to mupirocin (minimum inhibitory concentration ≤ 4 µg/ml). Conclusion: Screening and treatment of health care workers colonized with MRSA should be an important component of hospital infection control policy. These measures will prevent spread of infection to patients and community and thereby reduce the morbidity, mortality and health care costs associated with nosocomial infections.


Introduction
Staphylococci are ubiquitous colonizers of skin and mucosa and highly successful opportunistic pathogens. S. aureus is one of the most harmful species of staphylococci encountered [1] . It is one of the most pathogenic bacterial species in humans causing a wide variety of infections ranging from mild skin and soft tissue infections (furuncles, carbuncles etc) to severe life-threatening infections like chronic bone infections, necrotizing pneumonia, bacteremia, septicemia, acute endocarditis, myocarditis, pericarditis, osteomyelitis, encephalitis, meningitis, chorioamnionitis, mastitis, toxic-shock syndrome, scalded skin syndrome [2][3][4][5] and intravenous or other sites where tubes enter the body (indwelling medical devices) [6] . It is distinct from coagulase-negative staphylococci (CoNS) e.g. S. epidermidis, and is more virulent despite phylogenic similarities between them [7,8] .
The key characters of S. aureus are colony pigmentation, production of free coagulase, clumping factor, protein-A, heat-stable nuclease, lipase and acid production from mannitol [3] . The species aureus, refers to the fact that colonies often have a golden color when grown on solid media, while CoNS form pale, translucent, white colonies.
Staphylococcal infections occur frequently in hospitalized patients and have severe consequences, despite antibiotic therapy [9] . S. aureus are generally susceptible to β-lactam antibiotics, but extensive use of this class of drugs has led to increasing emergence of resistant strains [10] . The most notable example is the emergence of Methicillin-resistant Staphylococcus aureus (MRSA), which was reported just one year after the introduction of methicillin [11] . Also known as "a superbug" MRSA has become a major problem in most medical institutions because it is creating life-threatening situations [11] . MRSA is a major healthcare-associated (HA-MRSA) as well as a community-associated (CA-MRSA) infection [6] .
Healthcare Workers (HCWs) constitute an important reservoir of S. aureus. Nasal carriage of S. aureus acts as an important reservoir of infection among those colonized, who may then transmit the infection to co-workers and others in the community [12] . Approximately 20% of individuals are persistent carriers, about 60% are intermittent carriers and 20% almost never carry S. aureus [13] . Several studies have reported that the rate of the nasal carriage of S. aureus among the HCWs ranges from 16.8%-56.1% [14][15][16][17] . Studies conducted in different hospital settings worldwide including India, have reported the prevalence of MRSA in HCWs in the range of 5.8% to 17.8% [18-,22,9,12] . The growing problem in India is that MRSA prevalence has increased from 12% to 80.83% [23] . The health care workers who are found to be colonized with S.aureus are advised to apply mupirocin ointment in their anterior nares and they should be retested for the nasal carriage of S.aureus after 3 months of treatment [9] .
The aim of the present study is to estimate the nasal carriage and antimicrobial susceptibility pattern of Staphylococcus aureus and MRSA isolates among the HCWs in a tertiary health care centre. The prevalence of S.aureus carriers and its resistance to methicillin will help the institution develop a better MRSA infection control policy.

Materials And Methods
This descriptive study was carried out in the Department of Microbiology, Jawahar Lal Nehru Medical College and Hospital, Ajmer from November 2016 to December 2017. The study was approved from the Ethics Committee of J.L.N. Medical College, Ajmer and written informed consent was obtained from all the participants.
A total of 170 health care workers (HCWs) aged 18 to 60 years, actively involved in health care provision in different departments of J.L.N. Medical College were enrolled for the study. Each participant was interviewed using a questionnaire concerning general socio-demographic information, personal details and clinical symptoms. Exclusion criteria included health care workers not actively involved in patient care or suffering from underlying chronic disease or respiratory tract infections, history of recent hospitalization, intake of broad spectrum antibiotics, fever or those who refused consent.

Sample collection
Nasal swabs from anterior nares of both nostrils were collected using sterile cotton swabs with transport tube. A swab pre-moistened with sterile saline was inserted approximately 1-2 cm into the anterior nares and slowly rotated against the nasal mucosa ve times [24] . Both nostrils were sampled using the same swab. After collection swabs were re-inserted in the transport tube, labeled properly and transported to the laboratory within 30 minutes of collection for further processing.

Sample processing
All the specimens were inoculated on 5% sheep blood agar, nutrient agar and MacConkey agar (HiMedia Laboratories Pvt. Ltd. Mumbai, Maharashtra, India) and incubated at 37 o C for 24 hours. After incubation, identi cation of genus Staphylococcus was done using standard microbiological techniques, by studying their morphology, colony characteristics and biochemical properties. Staphylococci were identi ed as Gram positive, catalase positive, furazolidone susceptible and bacitracin resistant. S. aureus colonies were further identi ed as slide and tube coagulase positive, polymyxin B resistant and mannitol fermenting giving yellow pigmentation on mannitol salt agar.
Antimicrobial susceptibility testing Antibiotic susceptibility was studied by modi ed Kirby-Bauer disc diffusion method [25] on Mueller Hinton Agar plates (120 mm diameter) using commercially available antibiotic discs (HiMedia  [26] . Detection of methicillin-resistant Staphylococcus aureus (MRSA) All con rmed S. aureus isolates were tested for detection of methicillin resistance by four different methods. Kirby-Bauer disc diffusion method using oxacillin 1µg and cefoxitin 30 µg discs (HiMedia Laboratories, Mumbai, Maharashtra, India) [27] , minimum inhibitory concentration (MIC) testing of oxacillin by E-test and growth on Oxacillin Resistance Screening Agar (ORSA) plates [28] as per CLSI 2016 guidelines [26] . Zone of inhibition of size of ≤10 mm was taken as resistant, 11-12 as intermediate and ≥13 mm as sensitive for oxacillin. Zone of inhibition of size of ≤21 mm was taken as resistant, and ≥ 22 mm as sensitive for cefoxitin. On oxacillin E-test, an MIC of ≤2 µg/ml was considered susceptible and ≥ 4 µg/ml as resistant. Any growth on oxacillin screen agar was considered as methicillin (oxacillin) resistant.
Statistical analysis: The descriptive statistics for quantitative data was expressed as mean and standard deviation and qualitative data was expressed as proportions. Chi-squared test was used to nd independence of attributes at 5% level of signi cance (. The JASP 0.11.1.0 statistical package was used for statistical analysis.

Results
In the present study, nasal swabs were randomly collected from a total of 170 health care workers (HCWs) from various clinical depart ments and screened for the study of Staphylococcus colonization. Out of 159 subjects, 50 were doctors and 109 were nursing staff. Out of 50 doctors, 37 (74%) were males and 13 (26%) were females. Among 109 staff, 62 (56.88%) were males and 47 (43.12%) were females. In doctors, the maximum carriage rate was observed in the age group 31-40 years i.e. 60% , where 50% were males and 10% were females. In nursing staff group, maximum carriage was seen in 18-30 years age group where 20.18 % were males and 18.35 % were females accounting for a total of 38.53 % carriage rate in their group ( Figure 1).  (Figure 1).
Antimicrobial sensitivity pattern of Staphylococcus isolates by disc diffusion method is shown in Figure  2. Ten subjects had concomitant colonization of S. aureus and CoNS. Therefore antimicrobial susceptibility testing was done for 169 Staphylococcus isolates.
Among the antibiotics tested, all the staphylococcal isolates were susceptible only to linezolid (100%). Maximum resistance was shown to penicillin G (97.49 %). Resistance for cefoxitin and oxacillin was 4.73 % and 5.92 % respectively.

Discussion
Staphylococcus aureus is a common component of the skin ora, and 30% to 50% of healthy adults are colonized with it at any given time. The primary site of colonization of S. aureus in humans are the anterior nares [2,29]. Hospital workers have higher rates of MRSA nasal colonization than the general population [30]. In the present study 21.38% subjects had S. aureus colonization. Among HCWs around the globe, the nasal carriage rates of S.aureus have been reported as 14% in Nigeria, 27.5% in Turkey, 31.1% in Iran, 33.4% in France and 39.3% in Spain [31]. The growing problem in India is that MRSA prevalence has increased from 12% to 80.83% [23].
In a total of 159 subjects (50 doctors and 109 nursing staff) S. aureus and CoNS appeared in 34 and 125 samples respectively. Dual colonization of S. aureus and CoNS was observed in 10 samples. However there was no dual isolation from any of the swab in a study conducted by Vinodhkumaradithyaa, Uma et al. 2009 [22] . The prevalence of the S. aureus nasal carriage was higher among the male HCWs (13.21%) than the females (8.18 %). Similar observation was reported by Rongpharpi, Hazarika et al. (2013) [9] . The carriage rate of S. aureus was signi cantly more in nursing staff 26 [32] . Shobha, Rao et al. (2005) found none of the health care workers colonized with S. aureus [33] while a study from south India showed 9.3% S.aureus colonization [34] .
On MRSA detection using oxacillin disc diffusion, cefoxitin disc diffusion and MIC of oxacillin by E-test, 8 (23.53%) isolates were found to be MRSA. In our study, 8 S. aureus isolates that were resistant to both cefoxitin and oxacillin had oxacillin MIC value ≥ 4 µg/ml and 26 isolates that were sensitive to both oxacillin and cefoxitin had MIC values ≤ 2 µg /ml. Oxacillin screen agar could detect only 6 (17.65 %) isolates instead of 8 detected by other three methods. Hence it is recommended that all four methods be used for detection of oxacillin resistance. Pramodhini, Thenmozhivalli et al. (2011) found oxacillin disc diffusion method to be less sensitive for the detection of MRSA [35] . Mohanasoundaram and Lalitha (2008) obtained 100 % concordance in disc diffusion method and oxacillin MIC using agar dilution methods [36] .
In the present study, 97.04% staphylococcal isolates and 100% S. aureus and MRSA were found to be resistant to penicillin G.  2017) where penicillin was found to be 100% resistant to all strains of S. aureus [37,32] [39] . All the MRSA isolates were resistant to penicillin as reported by Agarwal, Singh et al. (2015) [12] . Ampicillin and amoxyclav showed a resistance of 31.95% and 37.28 % for staphylococci and 55.88% and 67.65% for S. aureus. Out of 8 MRSA isolates, 6 (75%) and 8 (100%) isolates were found to be resistant to ampicillin and amoxyclav respectively. Bhatiani, Yadav et al. 2017 has reported a 100% resistance to ampicillin [32] while 88.57% and 82.0% resistance to ampicillin by S. aureus isolates was observed by Rongpharpi, Hazarika et al. (2013) and Jindal, Malhotra et al. (2016) in studies conducted among HCWs respectively [9,40]. Study conducted at a tertiary care hospital in Iran reported 89.4% resistance among MRSA [41] . 34.91% Staphylococcus isolates, 64.71% S. aureus isolates and all MRSA isolates were resistant to cephalexin while in another study 73.7% MRSA isolates were found to be resistant to cephalexin in a similar study [42] . 24.85% and 15.38% staphylococcal, 50% and 38.24% S.aureus, 87.50% and 75.00%. MRSA isolates were resistant to gentamicin and netilmicin respectively. In studies by Hauschild, Sacha et al. (2008) and Schmitz, Fluit et al. (1999) 24.4% and 23% resistance was shown in S. aureus isolates to the above aminiglycosides [43,44] . In our study of the 34 S. aureus isolates, 38.24% were resistant to at least one of the two aminoglycosides tested. Hauschild, Sacha et al. (2008) reported that 38.1% S.
aureus were resistant to one of the aminoglycosides tested [43] . 42.60% Staphylococcus isolates and 58.82 % S. aureus isolates were resistant to cipro oxacin. Lower incidence of resistance(10.4%) was reported by Tahnkiwale, Roy et al. (2002) [45] , 41% by Duran, Ozer et al. (2012) [38] and 90% by a Mexican study on 211 isolates [46] . In Europe resistance by region showed a 5.6% resistance in northern, 6.2% in central and 23.6% in southern region [47] . Resistance to o oxacin was shown by 18.34% Staphylococcus isolates and 32.35 % S. aureus isolates. Levo oxacin resistance stood at 9.47% and 11.76% for Staphylococcus and S. aureus isolates respectively. However, 87.50 % , 75.00 % and 12.50 % MRSA isolates showed resistance to cipro oxacin, o oxacin and levo oxacin respectively. In contrast, Agarwal et al. reported 50% MRSA isolates resistant to cipro oxacin and 21.4% for levo oxacin [12] Erythromycin resistant Staphylococcus often has cross resistance to other macrolides, lincosamide and streptogramin type B (MLS B ) [48] . In the present study erythromycin resistance was seen in 85.80 % and 85.29 % Staphylococcus and S. aureus respectively. However a lower resistance to erythromycin ranging between 66.66%-67.9 % has been observed by Bhatiani [32,37,49] . Clindamycin resistance was shown in 36.69% and 50% Staphylococcus and S. aureus respectively while in a study by Verma, Joshi et al. (2000) [23] , erythromycin and clindamycin resistance was found to be 52.8% and 48.28% respectively in S. aureus isolates. 25.44 % Staphylococcus and 38.24 % S. aureus isolates respectively were tetracycline resistant. A higher resistance was reported by Shittu and Lin (2006) and Duran, Ozer et al. (2012) who reported 55.9% and 35.6% resistance for S. aureus isolates respectively [50,38]. In our study, 75.00% (6/8) MRSA isolates were found to be resistant to tetracycline which is much higher as reported by Agarwal, Singh et al.
In the present study, a total of 55.62% (94/169) Staphylococcus isolates and 58.82% S. aureus isolates showed resistance to quinupristin dalfopristin. All the MRSA isolates (8/8, 100%) were found to be resistant to quinupristin dalfopristin, while in a study only 5.56 % MRSA isolates were reported as resistant to quinupristin dalfopristin [53] .
In this study, 99.41% Staphylococcus isolates were found sensitive and only one (0.59 %) reported resistant (intermediate resistant) to vancomycin. S. aureus isolates showed 97.06 % sensitivity to vancomycin and only one (2.94%) reported resistant (VISA). However, no vancomycin resistance was observed in MRSA isolates in a study conducted at Kasturba Medical College, Hospital, Mangalore [54] . Complete sensitivity to vancomycin of S. aureus isolates was reported by Anupurba, Sen et al. (2003) and Datta, Gulati et al. (2011) [55,56] .
Our study showed 100 % susceptibility to linezolid. Vancomycin and linezolid were found to be the most sensitive drugs against S. aureus in studies by Agarwal, Singh et al. (2015) and Bhatiani, Yadav et al. (2017) [12,32] . Golan, Baez-giangreco et al. (2006) reported a signi cant trend in increased MRSA linezolid resistance from 2002 onwards [60] . Linezolid, a member of the new oxazolidone class of antibiotics is highly active in vitro against MRSA and has excellent oral bioavailability and constitutes the drug of choice against MRSA infection besides vancomycin. Our study supported this.
Resistance to mupirocin is being reported from across the globe with the prevalence of 0.5% in Nigeria to 14.6% in India [50 ,61] . Rapid resistance to mupirocin has been reported among some strains of S. aureus isolated from various hospitals. In the present study of 34 S. aureus isolates, sensitivity to mupirocin was 88 % with isolates having MIC < 0.5 µg/ml. Mohajeri, Gholamine et al. (2012) reported 100% sensitivity to mupirocin in the nasal carriage isolates of the patients [62] . Though mupirocin resistance was not seen in the S. aureus isolates in the study by Mohajeri, Gholamine et al. 2012), the MIC of 9.2 % of the isolates was as high as 4 µg/ml which was very close to a low level resistance (8 µg/ml) [62] . In the study by Saderi, Emadi et al. (2011), 6 strains had MIC > 4 µg/ml [63] . Nagarajan et al., observed all MRSA showed high level mupirocin resistance and inducible clindamycin resistance [64] . Agarwal, Singh et al (2015) reported that 4 (2%) isolates were found to be mupirocin resistant of which three isolates were high levels resistant [12] . In the presence of mupirocin resistant strains, treatment with mupirocin may be ineffective, especially with high-level resistance strains. Although low-level mupirocin resistant strains can be controlled by normal dosage schedule of mupirocin but few studies suggest that treatment failure may occur. This emphasizes the importance of identi cation of both high and low-level resistant strains [65, 66 ,67] . Simple preventive measures like hand washing, using sterile mask, gown and avoiding touching one's nose during work, should be reinforced in all health care settings. This study reiterates the need for periodic surveillance, early and accurate detection and treatment of MRSA carriers. This should be accompanied with appropriate hospital infection control measures, to prevent the nasal carriage of MRSA in hospital health care workers.

Conclusion
In the present study very high carriage rate was detected in the anterior nares which is also the commonest site for Staphylococcus colonization . The results obtained from the antibiogram of Staphylococci, S. aureus and MRSA isolates from colonized HCWs showed the increase in rates of resistance against various antibiotics. The present study demonstrates for the rst time the presence of MRSA in HCWs working in this hospital and demonstrates the prevalence of the antibiotic resistance amongst them. Vancomycin resistance in Staphylococcus species is beginning to emerge as a clinical threat, yet the attention it has received serves to underscore the seriousness of the problem.
A better understanding of these issues will be a key to helping the prevention and treatment of these infections in the future and in containing the spread of these from HCWs to patients and vice versa. All the HCWs should be periodically educated and trained about the maintenance of hygiene and infection control and the effects of the use or rather, the misuse of antibiotics.

Declarations
Limitations of the study -The study enrolled health care workers from a single tertiary health care center, however, to generalize the results multi-centric studies are required.
Con icts of interest-There are no con icts of interest for the authors.
Funding: Not applicable Authors' contribution: MC involved in planning, concept design and hypothesis generation, NA did data collection, AC did data assembly, literature review and manuscript writing, MB helped in statistical analysis, GP and VR helped in data interpretation and literature review, AT helped in manuscript writing and data visualization. All authors commented and nally approved the manuscript.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by Ethical Committee of the JLN Medical College, Ajmer (No.42954-85 dated 28 -10 20-16).

S.aureus
Tests used for detection of MRSA