Burden of mupirocin resistance among methicillin-resistant Staphylococcus aureus- Colonised patients at a tertiary care medical centre

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is an established pathogen. It can cause an array of infectious syndromes ranging from minor skin infections to severe, life-threatening conditions. This includes skin and soft tissue infections, respiratory infections, bacteremia, bone and joint infections, toxic shock syndrome and scalded skin syndrome, which are toxin-mediated. Staphylococcus aureus can be a coloniser of the skin, present specifically in the anterior nares and moist regions of the body, such as the axilla and groin. The prevalence of community-acquired MRSA has significantly increased in healthy asymptomatic individuals over the past few years. Studies have suggested that both methicillin sensitive Staphylococcus aureus (MSSA) and MRSA can be present as a coloniser of the skin.^[1-4] This serves as a reservoir of infection where these strains disseminate to the vulnerable population, particularly in a healthcare setting, and cause infection. Moreover, the colonisers are themselves at risk of a subsequent infection by these pathogens on presentation of any risk factors. A systematic review and meta-analysis on the prevalence, antibiogram, and risk factors of MRSA asymptomatic carriage in Africa^[5] identified several significant risk factors for MRSA colonisation which included a history of prior hospitalisation, prior antibiotic use, diabetes mellitus, HIV-positive status, invasive procedures and healthcare workers as the important ones. The majority of the studies have demonstrated that clinical infection with MRSA does have nasal colonisation that identifies a person as an MRSA coloniser, and this important finding initiates the clinicians to intervene with interventions such as decolonisation, continued monitoring and surveillance.

Mupirocin is an established topical antimicrobial agent which has been used for decolonisation of MRSA carriers since the 1980s. It is a relatively potent decolonising agent; immediately after completion of nasal mupirocin treatment, 81.5% to 100% of patients are successfully decolonised compared with spontaneous or vehicle-mediated decolonisation rates of 0% to 46%.^[6-8] Mupirocin acts on isoleucyl tRNA synthetase to inhibit protein synthesis. There are two phenotypes of mupirocin-resistant S. aureus, ‘low-level’ and ‘high-level’ (LL-MR and HL-MR, respectively). The working definition of LL-MR is a mupirocin minimum inhibitory concentration (MIC) of 8–256 mg/L, and the working definition of HL-MR is a mupirocin MIC of 512 mg/L. The LL-MR genotype is a mutation of the chromosomal gene ileS-2 (mupA), which encodes a resistant version of isoleucyl tRNA synthetase, while HL-MR’s genotype is a plasmid-transferrable alternative version of the same gene.^[9] People who are carriers of Staphylococcus aureus must be colonised with different strains, some MSSA, some MRSA, some LL-MR, some HL-MR, giving a perfect opportunity to perpetuate the resistant genes by horizontal gene transfer to sensitive strains and also to form new resistant strains. Interestingly, since HL-MR is plasmid-mediated, it can also be transferred to Coagulase Negative Staphylococci, which are commensal flora of human skin, and this can again act as a reservoir to perpetuate colonised MRSA strains.

As far as LL-MR is concerned, they are chromosomal in nature, which could be induced by antibiotic stress and exposure to mupirocin in vivo, such as decolonisation. This complex cycle and resilience of Staphylococcus aureus resistance mechanisms to mupirocin show that the resistance to this particular drug is unlikely to be eradicated just by restriction of its use or by decolonisation. Studies looking at the long-term efficacy of mupirocin that have focused on nasal decolonisation of S. aureus, including MRSA, have shown that initial clearance over several weeks is effective but that recolonisation occurs after 3 months.^[10] In fact, a multidisciplinary approach should be undertaken, which includes the use of standard precautions, screening programs, targeted treatment, continuous surveillance and the use of other alternative drugs. Therefore, this study was proposed to be undertaken keeping in mind the natural course of resistance mechanism to mupirocin, their prevalence in MRSA colonised patients and the future approach to tackle the same.

MATERIAL AND METHODS

This was a cross-sectional study conducted in the Department of Microbiology, Adesh Medical College & Hospital, Shahabad, Kurukshetra, Ambala. The study duration was six months, from May 2024 to October 2024. A universal sampling approach was used, and all isolates of MRSA isolated which met the eligibility criteria were included in the study (n=58). Sample size for the study was calculated by taking a study with a prevalence 6.75% and was calculated by the formula sample size (n)= Z1-α/2 PQ/d^2.[11]

Inclusion criteria included MRSA isolated from any clinical samples. Exclusion criteria included MSSA and other Staphylococcus species, other gram-positive cocci, duplicate MRSA isolation from the same patient.

Culture was performed according to the standard laboratory methods. Colonies were grown in Nutrient Agar, Blood Agar and Mac Conkey agar in a 90 mm Petri dish and incubated at 37°C for 24 hours. Staphylococcus aureus was identified using appropriate colony morphology, Gram staining, where Staphylococcus aureus will be observed as Gram-positive cocci in grape-like clusters and biochemical tests like the Catalase test and Coagulase test as per the standard operating procedures. Detection of MRSA was done by the Cefoxitin disc diffusion method. All isolates of Staphylococcus aureus were tested for MRSA by using a 30 µg cefoxitin disc by the Kirby-Bauer disc diffusion method (Clinical and Laboratory Standards Institute 2023 guidelines, M100, 33^rd Edition).

Zones of inhibition of more than or equal to 22 mm were taken as MSSA, whereas zones of inhibition of less than 21mm were labelled as MRSA. All the patients showing the growth of MRSA in any clinical specimens received for culture and susceptibility testing were screened for MRSA colonisation by taking swabs from the anterior nares, axilla and groin. All the identified MRSA isolates identified as colonisers were further duplicated for the presence of green colonies on CHROMagar MRSA after 48 hours of incubation at 37°C. The susceptibility testing for mupirocin was done by Mupirocin E-test strip disc diffusion methods as per manufacturers guidelines.

Descriptive statistics, such as percentages and proportions, will be used for statistical analysis. Microsoft Excel will be used for data entry, handling and analysis.

RESULTS

MRSA was isolated from 58 clinical samples. Among them, pus and related samples (wound swab and tissues) constituted the highest number of MRSA isolation 86.2% (50/58), followed by blood, which constituted 13.8% (8/58) of MRSA isolations. As far as the MRSA isolation rates from various departments are concerned, the highest was observed from the Surgical Departments, where in the Surgical Intensive Care Unit (ICU), the isolation rate was 19/58 (32.7%), whereas in the Orthopaedic and Obstetrics & Gynaecology departments, the isolation rate stood at 13.8% (8/58) and 12% (7/58) respectively. This was followed by the Medical ICU (MICU), where the carriage rates were observed to be 15% (9/58) in MICU and 7% (4/58) in the Critical Care Unit (CCU). Paediatrics and Neonatal ICU showed carriage rates of 10% (6/58) and 8% (5/58), respectively [Figure 1].

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Figure 1:

Methicillin-resistant Staphylococcus aureus (MRSA) ward wise distribution. ICU: Intensive care unit.

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Out of the total 58 patients in whom MRSA was isolated from various clinical samples, 18/58 (31.0%) patients showed colonisation by MRSA. Among these 18 patients, 4/58(6.9%) showed axillary colonisation, 12/58 (20.7%) showed nasal colonisation, 2/58(3.4%) showed groin colonisation, 4/58 (6.9%) showed overlapping carriage in 2 sites (nasal and axilla), and 40/58 (40%) showed no colonisation of MRSA [Figure 2]. Out of the 18 MRSA isolates that were subjected to mupirocin susceptibility, 2/18(8.6%) showed high level mupirocin resistance (MIC ≥ 512 µg/ml), 1/18(5.2 %) showed low level mupirocin resistance (MIC 8 to 256 µg/ml), and 15/18 (86.2%) were mupirocin sensitive (MIC ≤ 4 µg/ml).

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Figure 2:

Methicillin-resistant Staphylococcus aureus (MRSA) colonisation.

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DISCUSSION

Mupirocin is an important drug which has long been used for decolonisation of carriers of MRSA wherever indicated. This is particularly useful to prevent autoinfection with this potentially virulent organism when patients are admitted to any critical area, such as the ICU, or when they present with any risk factors, such as prior hospitalisation, prior antibiotic use, diabetes mellitus, HIV-positive status, or invasive surgical procedures.^[5]

In our study, we included 58 MRSA isolates, which were isolated from various clinical samples. A maximum of these MRSA isolates (86.2%) were from pus and related samples (wound swab, tissues), followed by blood (13.8%). This finding was comparable with a study done by Preeja et al. in which out of 132 MRSA isolated from various clinical samples, pus constituted the maximum number (n=111), followed by blood.^[12] Another study done by Vasumathi et al. entitled “Prevalence of MRSA and its susceptibility pattern among various clinical samples in our tertiary care hospital” also showed maximum isolation of MRSA from pus sample.^[13]. MRSA is an established pathogen commonly associated with skin and soft tissue infections. A potential reason for this association can be due to these organisms being resident flora of the skin and thus can enter into the body through minor cuts, incisions, abrasions, injuries, burns, catheters and cause various pyogenic infections when the host becomes vulnerable. Not only Hospital-acquired MRSA (HA-MRSA), but also the spread of the community acquired (CA)-MRSA, in nosocomial infections, has been well recognised now and appears to be mainly because of person-to-person contact. The nares remains the most common carrier site, followed by the skin, for HA-MRSA, while skin is the more common carrier site for CA-MRSA.^[14-18] Interestingly, the presence of the Panton-Valentine leucocidin (pvl) toxin gene makes it even more virulent, capable of tissue necrosis, a major feature seen in the soft-tissue infections.

As far as the departmental distribution of these isolates is concerned, the maximum was from the surgical department, where collectively Surgical ICU, including Neurosurgery, Orthopaedics, Obstetrics and Gynaecology, constituted 58.5% of the MRSA isolation rate [Figure 2]. As described previously, an invasive surgical procedure is one of the important risk factors for MRSA colonisation and subsequent infection. Similar trends were observed by Lohan et al.^[19]

Our study demonstrated that the carriage rate of MRSA among the patients in whom there was evidence of an active MRSA infection (n=58) to be 31.0% (n=18). This is comparable with a study done by Srivalli et al., in which out of the nasal swabs of 200 healthcare workers that were collected, 52(26%) were nasal carriers of S. aureus, and among them, 17(32.7%) were carriers of MRSA.^[20] In another study done by Mamatha et al., out of 300 inpatients sampled for nasal swabs, 148(49.3%) were culture positive for Staphylococcus aureus, and 52 (17.3%) were MRSA carriers.^[21] The majority of the studies have taken nasal swabs as the sample of choice for studying the colonisation rates. MRSA often colonises skin and soft tissue (axilla & perineum) and not just the anterior nares. Only screening the nares often underestimates the carriage rates. In a study done by Yang et al. entitled “Body site colonisation in patients with community-associated MRSA and other types of S. aureus skin infections’’ the authors found that among the 99 patients who had skin and soft tissue infections, 65 were determined to have CA-MRSA, 22 had CA-MSSA, six had HA-MRSA, and six had HA-MSSA. Among patients with CA-MRSA who had skin and soft tissue infections, 37% (24/65) were found to be MRSA colonised. Twenty-five per cent of patients (16/65) were colonised in the nares, 6% (4/65) in the axilla, 17% (11/64) in the inguinal area, and 13% (7/54) in the rectal area. Among those CA-MRSA-infected patients who were MRSA colonised, 96% (23/24) could be identified using a combination of nasal and inguinal screening alone.^[22]. More studies regarding the same were found to be scarce in the literature review. However, in our study, swabs from the nares, axilla and perineum were taken. Among 18 patients who were MRSA colonised, 4/58(6.9%) showed axillary colonisation, 12/58 (20.7%) showed nasal colonisation, 2/58(3.4%) showed groin colonisation, 4/58 (6.9%) showed overlapping carriage in 2 sites (nasal and axilla), and 40/58 (40%) showed no colonisation of MRSA [Figure 1]. This is a noticeable finding, which firstly suggests that 31.03% of these patients had a retrospective colonisation with MRSA. Interestingly, many of these patients who were found to be carriers were from Surgical Departments (Surgery=5/18, Orthopaedics=7/18, Medical ICU= 4/18, NICU&CCU=1each/18). Yang et al. hypothesised that patients who were suffering from CA-MRSA skin infections could possibly be colonised at other body sites apart from the anterior nares. And one of their important findings includes that non-nasal colonisation was much more common in patients with CA-MRSA skin and soft tissue infections compared with those with non-CA-MRSA skin and soft tissue infections (SSTI).^[22] Our study too has shown that around 10 patients (4/58(6.9%) showed axillary colonisation, 2/58(3.4%) showed groin colonisation, 4/58 (6.9%) showed overlapping carriage in 2 sites (nasal and axilla), and were colonised in the sites other than anterior nares. This finding also suggests that these patients could have acquired a CAMRSA SSTI, given the evidence that 12 of them were from Surgical Departments having SSTI. This finding compels us to adopt an aggressive approach towards including swabbing of nares, axilla, as well as perineum for screening of MRSA colonisation of Surgical patients to prevent Surgical site infections. Most of these infections are caused by the contamination of an incision with microorganisms from the patient’s own commensal flora. Nasal Mupirocin for nasal colonisers and chlorhexidine body wash for skin colonisers (axilla, groin) should be used diligently to reduce the risk of a surgical site infection.^[23,24] Asymptomatic MRSA carriers are at approximately 30-fold greater risk of SSI. However, the literature is conflicting as to whether identification of the MRSA carrier state, with targeted intervention, reduces the incidence of subsequent MRSA infection. Though universal screening of large populations is not cost-effective, targeted screening of high-risk populations, such as patients impending to undergo any invasive surgical procedure, deserves additional emphasis.^[2] In another study done by Shih et al. entitled “High prevalence nasal carriage of MRSA among long-term care facility healthcare workers in relation to patient contact”, the overall prevalence of MRSA nasal carriage rate was 6%. The prevalence of the MRSA nasal carriage was higher in healthcare workers who had worked for 5 to 10 years (12.8%) and in direct contact with patients.^[25] This throws light on the fact that healthcare workers are the major reservoir of MRSA in the form of silent asymptomatic carriers, and because of direct contact with the patients, they become the major vehicle for transmission.

Out of the 18 MRSA isolates that were subjected to mupirocin susceptibility, 2/18(8.6%) showed high level mupirocin resistance (HL-MR, MIC ≥ 512 µg/ml), 1/18(5.2 %) showed low level mupirocin resistance (LL-MR, MIC 8 to 256 µg/ml), and 15/18 (86.2%) were mupirocin sensitive (MIC ≤ 4 µg/ml). Mupirocin ointment is used either alone or with chlorhexidine as part of a comprehensive MRSA decolonisation strategy.^[23,24] Increased mupirocin use predisposes to mupirocin resistance due to antibiotic pressure, which is significantly associated with persistent MRSA colonisation. Among MRSA isolates, the presence of the qacA and/or qacB gene, encoding resistance to chlorhexidine, ranges from 65% to 91%, which, along with mupirocin resistance, is associated with failed decolonisation.^[26] The LL-MR genotype is a mutation of the chromosomal gene ileS-2 (mupA), which encodes a resistant version of isoleucyl tRNA synthetase, while HL-MR’s genotype is a plasmid-transferrable alternative version of the same gen.^[9]. Two patients who were found to be infected with an HL-MR strain were from the Cardiothoracic and Vascular Surgery and General Surgery Departments. The first one had a Surgical Site Infection post-surgery, and the other was a case of breast abscess for which incision & drainage were done. The LL-MR stain was isolated from a patient admitted to the Medical ICU, who was a known case of Type 2 Diabetes mellitus. All three of these patients had high-risk factors for MRSA colonisation. However, none of them were screened for the same, and that could have possibly resulted in infection. Two of these patients, who were infected with HL-MR strains of MRSA, have SSTI. These patients were possibly colonised with a CA-MRSA strain, which, as discussed before, has a higher propensity to cause SSTI. The LL-MR strain of MRSA was from a patient with type 2 Diabetes mellitus who was admitted to the medical ICU for two weeks and thus was possibly a HA-MRSA strain. A study done by Cadilla et al. showed a unique characteristic in the distribution of CA- and HA-MRSA genotypes that was significantly different when mupA-negative and mupA-positive MDR MRSA isolates were compared. Whereas a great majority of the mupA-negative MDR isolates had an HAMRSA genotype, the majority of mupA-positive MDR MRSA had a CA-MRSA genotype. This suggests that MDR plasmids harbouring mupA are being transferred readily into the CAMRSA genotype.^[27] The above findings potentiate that the mupirocin mupA gene has disseminated to CA-MRSA as well as HA-MRSA. However, more studies are required to duplicate the same results in the Indian setting.

CONCLUSION

Nevertheless, it is quite evident that MRSA carriage is a cause of grave concern in developing nations. Lack of proper infection control guidelines and lack of motivation to follow existing guidelines and resources available are the major hurdles to be tackled. Screening for MRSA carriage is a must in high-risk patients, along with strict decolonisation protocols. A substantial increase in mupirocin resistance, especially high- level resistance mediated by the mupA gene coupled with chlorhexidine resistance, poses a serious threat to the current MRSA decolonisation protocols. There is a need for updated surveillance strategies to include alternative agents or combinations of agents for decolonisation. Also, adoption of tailored antibiotic stewardship programs to mitigate the spread of resistant strains will go a long way.