Isolation, Characterisation and Antibiotic Susceptibility of Staphylococcal Isolates with Special Reference to Methicillin-Resistant Staphylococcus aureus From the Anterior Nares of Healthcare Workers in a Tertiary Healthcare Centre

This article should be cited as follows: Chaurasia M, Agrawal N, Chourasia A, Bhatnagar M, Parihar G, Rastogi V, et al. Isolation, characterisation and antibiotic susceptibility of staphylococcal isolates with special reference to methicillin-resistant Staphylococcus aureus from the anterior nares of healthcare workers in a tertiary healthcare centre. Scr Med 2021 Jun;52(2):85-95. Received: 6 March 2021 Revision received: 29 March 2021 Accepted: 29 March 2021 ARTICLE INFO (1)


Introduction
Staphylococci are ubiquitous colonisers 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, necrotising pneumonia, bacteraemia, septicaemia, acute endocarditis, myocarditis, pericarditis, osteomyelitis, encephalitis, meningitis, chorioamnionitis, mastitis, toxic-shock syndrome, scalded skin syndrome [2][3][4][5] and intravenous infections or at other sites where tubes enter the body (indwelling medical devices). 6 It is distinct from coagulase-negative staphylococci (CoNS) eg, S. epidermidis, and is more virulent despite phylogenetic similarities between them. 7,8 The key characteristics 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 those colonies that often have a golden colour when grown on solid media, while CoNS form pale, translucent, white colonies.
Staphylococcal infections occur frequently in hospitalised 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 colonised, 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 % to 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 healthcare workers who are found to be colonised 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 was to estimate the nasal carriage and antimicrobial susceptibility pattern of S. aureus and MRSA isolates among the HCWs in a tertiary healthcare centre. The prevalence of S. aureus carriers and its resistance to methicillin will help the institution develop a better MRSA infection control policy.
This descriptive study was carried out in the Department of Microbiology, Jawahar Lal Nehru (JLN) Medical College and Hospital, Ajmer, Rajasthan, India from November 2016 to December 2017. The study was approved by the Ethics Committee of JLN Medical College, Ajmer and written informed consent was obtained from all the participants.
A total of 170 HCWs aged 18 to 60 years, actively involved in healthcare provision in different departments of JLN Medical College were enrolled for the study. Each participant was interviewed using a questionnaire on general socio-demographic information, personal details and clinical symptoms. Exclusion criteria included healthcare workers not actively involved in patient care or those suffering from underlying chronic disease or respiratory tract infections, with a history of recent hospitalisation, intake of broad-spectrum antibiotics, fever or those who did not consent.

Sample collection
Nasal swabs from the anterior nares of both nostrils were collected using sterile cotton swabs with transport tubes. A swab pre-moistened with sterile saline was inserted approximately 1-2 cm Results into the anterior nares and slowly rotated against the nasal mucosa five times. 24 Both nostrils were sampled using the same swab. After collection, the swabs were re-inserted in the transport tubes, 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 (Hi-Media Laboratories Pvt Ltd Mumbai, Maharashtra, India) and incubated at 37 o C for 24 hours. After incubation, identification of genus Staphylococcus was done using standard microbiological techniques, by studying their morphology, colony characteristics and biochemical properties. Staphylococci were identified as Gram positive, catalase positive, furazolidone susceptible and bacitracin-resistant. S. aureus colonies were further identified as slide and tube coagulase positive, polymyxin B-resistant and mannitol fermenting giving yellow pigmentation on mannitol salt agar.

Detection of mupirocin-resistant Staphylococcus aureus
The MIC of mupirocin for isolation of S. aureus (Mupirocin resistance) was determined by Epsilometer test (E-test) using HiMedia, mupirocin strip (range 0.064-1024 µg/mL) and interpreted as per CLSI 2016 guidelines. 26 Isolates with mupirocin MICs ≥ 512 μg/mL were considered as high-level resistant (MuH), those with MICs 8-256 μg/mL were considered as low-level resistant (MuL), and with ≤ 4 μg/mL were considered as mupirocin sensitive.

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 find independence of attributes at 5 % level of significance (α = 0.5). The JASP 0.11.1.0 statistical package was used for statistical analysis.
In the present study, nasal swabs were randomly collected from a total of 170 HCWs from various clinical departments and screened for the study of  and 47 (43.12 %) were females. The maximum carriage rate in doctors was observed in the age group 31-40 years ie, 60 %, where 50 % were males and 10 % were females. In the 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).
In the present study Staphylococcus colonisation was detected in 159 (93.53 %) healthcare workers which comprised 34 (21.38 %) S. aureus and 125 (78.61 %) CoNS isolates. Dual colonisation with S. aureus and CoNS was observed in 10 samples. The carriage rate of S. aureus was significantly higher in nursing staff (26.60 %) as compared to doctors (10 %) (χ 2 = 5.62, p = 0.018). Professors/associate professors/assistant professors and resident doctors were found to have S. aureus nasal carriage rate 16.67 % and 7.89 %, respectively ( Figure 1). Among the antibiotics tested, all the staphylococcal isolates were susceptible only to linezolid and vancomycin (100 %). Maximum resistance was shown to penicillin G (97.49 %). Resistance to cefoxitin and oxacillin was 4.73 % and 5.92 %, respectively.
All the S. aureus isolates were found to be susceptible to linezolid and vancomycin (100 %). All S. aureus isolates showed complete resistance to penicillin G (100 %). Extremely low susceptibility was shown for erythromycin (17.71 %) and cotrimoxazole (17.65 %). Resistance to cefoxitin and oxacillin was 23.53 % (Figure 3).         57 Tiwari and Sen reported two strains of VRSA in the northern parts of India. 58 Sharma and Vishwanath studied 156 MRSA isolates which were susceptible to vancomycin by disc diffusion method but, the MIC of 18 isolates was ≥ 4 µg/mL (VISA). 59 This study showed 100 % susceptibility to linezolid and vancomycin. Vancomycin and linezolid were found to be the most sensitive drugs against S. aureus in studies by Agarwal 64 Agarwal et al 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, a few studies suggest that treatment failure may occur. This emphasizes the importance of identification of both high and low-level resistant strains. [65][66][67] Simple preventive measures like hand washing, using a sterile mask, gown and avoiding touching one's nose during work, should be reinforced in all healthcare 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 healthcare workers.

Conclusion
In the present study, very high carriage rate was detected in the anterior nares which are also the commonest site for Staphylococcus colonisation. The results obtained from the antibiogram of Staphylococci, S. aureus and MRSA isolates from colonised HCWs showed the increase in rates of resistance against various antibiotics. The present study confirms for the first 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 is scant and serves to underscore the seriousness of the problem.
A better understanding of these issues will be a key to help in 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 in the maintenance of hygiene and infection control and the effects of the use or rather, the misuse of antibiotics.

The limitations of the study
The study enrolled HCWs from a single tertiary healthcare centre, however, to generalise the results multi-centric studies are required.

Contribution of Authors
MC was 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 visualisation. All the authors collaborated and finally approved the manuscript.

Ethics 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 the Ethics Committee of the JLN Medical College, Ajmer (No 42954-85, dated 28-10-2016).

Conflict of interest
None. None.