Detection of Methicillin Resistance and β-lactamase Production in Staphylococcus aureus isolates: A Cross sectional study from a tertiary Care hospital

Nosocomial infections are proving to be a menace for the whole health care system, with methicillin resistant Staphylococcus aureus (MRsA) being a very notorious causative agent. Along with the role of mecA gene producing penicillin-binding protein (PBP2a), production of beta-lactamase enzyme by Staphylococcus aureus makes the organism resistant to all β-lactam agents. this study aims at phenotypic detection of methicillin resistance and β-lactamase production in all S. aureus isolates by Cefoxitin disk diffusion test and Penicillin zone-edge test, respectively. in this prospective cross-sectional study, samples were obtained from patients admitted to different departments and were processed according to the standard laboratory protocols. As per the Clsi guidelines, phenotypic detection of resistance to methicillin was done by Cefoxitin Disk Diffusion test, whereas production of beta-lactamase enzyme was phenotypically detected by penicillin zone edge test. Among 179 isolates of S. aureus , 116 (64.8%) were MRsA, whereas the remaining 63 (35.2%) isolates were methicillin-sensitive Staphylococcus aureus (MssA). Staphylococcus aureus infection among iCU and non-iCU patients were found to be 24(13%) and 155(86.6%) respectively. Among 24 iCU patients, 19 had MRsA infection, whereas among 155 non-iCU patients, 97 had MRsA infection. Out of 63 MssA isolates, only 2 (3.17%) were found to be sensitive to Penicillin by Zone-edge test, 04 isolates showed presence of β-lactamase production, while remaining 57 isolates were resistant to Penicillin. At the same time, several class of antibiotics were found to be ineffective against these MRsA isolates. Cases of methicillin-resistant and b –lactamase producing Staphylococcus aureus infections are on the rise in our hospital settings.


iNtRODUCtiON
Methicillin resistant Staphylococcus aureus infections are the major contributors among different nosocomial infection worldwide, including India. 1,2 Penicillin-resistance among Staphylococcus aureus (S. aureus) strains have been known since 1942, which further paved the way for semisynthetic penicillins, including methicillin, 3 which is used in the treatment of infections caused due to penicillin resistant S. aureus strains. With the increasing number of infections due to S. aureus, the prevalence of Methicillin resistant Staphylococcus aureus (MRSA) infections has also increased in India. 4 Since 1980, about 33% of the world's population exhibiting increased incidence of MRSA infection in hospital settings belong to the Asia pacific region. 5 It has been reported from different parts of Indian subcontinent at a rate of about 25% to 50%. 6 MRSA has been linked to the causation of several hospital acquired infections which are rapidly progressive and fatal such as pneumonia, endocarditis, necrotizing fasciitis, osteomyelitis, toxic shock syndrome and severe sepsis, whereas, various risk factors that are commonly associated with MRSA infection are prolonged hospitalization, intensive care admission, invasive procedures, hemodialysis, recent antibiotic use, HIV infection and discharge with long-term indwelling urinary catheter or long term central venous access. Earlier, MRSA was known to cause infection among hospitalized patients, commonly known as hospital-associated MRSA (HA-MRSA) infections. But recently, these infections are engulfing a large part of population which have never come in contact with the hospitals, health care workers or hospitalized patients, known as communityassociated MRSA (CA-MRSA) infections. 7 CA-MRSA is generally known for causing skin and soft tissue infections like abscesses, furunculosis and cellulitis but it also causes severe morbidities like bacteremia, septic shock, necrotizing pneumonia and osteomyelitis. 7,8 The Staphylococcal cassette chromosome mec (SCCmec) is a genomic island which when acquired, causes methicillin resistance. It expresses the mecA gene which helps in producing the penicillin-binding protein (PBP2a), making the organism resistant to all β-lactam antibiotics. 9 Recently, several other related genes like mecB, mecC, and mecD have also been discovered. 10 The mecA mediated resistance can be detected in laboratory by using Oxacillin (broth microdilution or agar dilution technique) or Cefoxitin (broth microdilution or disk diffusion technique). 11 Cefoxitin (30µg) may be used as a surrogate marker for Oxacillin in detecting MRSA, if oxacillin MIC testing is not done and can prove to be a very effective method of detection of MRSA and MSSA (Methicillin sensitive Staphylococcus aureus) isolates in resource limited settings.
There is also a second method which makes S. aureus resistant to all beta-lactams, which is by production of beta-lactamase enzyme by the organism, that hydrolytically destroy beta-lactams. 12 The gene responsible for encoding β-lactamase is blaZ, which develops such resistance. 13 Staphylococci, which are β-lactamasepositive, are resistant to penicillin, aminopenicillin, carboxpenicillin and ureidopenicillins. In the laboratory, penicillin disk diffusion zone-edge test and nitrocefin-based tests can be used for detecting β-lactamase production in S. aureus, phenotypically. However, penicillin zone-edge test has been found to be more sensitive and is recommended, if only one test is to be used for β-lactamase detection. 11 As per the Clinical and Laboratory Standards Institute (CLSI), penicillin zone edge test 14 has been recommended for screening S. aureus isolates for β-Lactamase production. 11 This study aims at phenotypic detection of Methicillin resistance and β-Lactamase production in all S. aureus isolates by Cefoxitin disk diffusion test and Penicillin zone-edge test, respectively. S. aureus isolates were obtained from various clinical samples like pus, urine, blood, conjunctival swab, ET aspirate and synovial fluid.

MAteRiAls AND MethODs
A prospective cross-sectional study of one year duration from March 2020 to February 2021, was carried out in the Bacteriology laboratory, for the detection of Methicillin Resistance and β-Lactamase production in Staphylococcus aureus. Written and informed consent was taken from patients.

inclusion and exclusion Criteria
All patients having a surgical site infection, skin ulcers or burn, skin abscess, diabetic foot ulcers and infected traumatic wound were included in the study. All patients who did not give their consent were excluded from the study.

sample Collection and Processing
Specimens were collected from various clinical departments. The specimens included in this study were swab/aspirate from soft tissue infection and abscess (pus), urine, blood, conjunctival swab, Endo-tracheal aspirate and synovial fluid. General culture media like Nutrient Agar and Blood Agar, whereas selective media like Mannitol salt agar (MSA) were used for inoculation purpose, which were subsequently incubated for 24 hours at 37°C, aerobically.
Detection of S. aureus was done following conventional methods of identification, like catalase test, tube coagulase test and mannitol salt agar. Furthermore, routine antimicrobial susceptibility test was carried out by Kirby Bauer disk diffusion method. Cefoxitin Disk Diffusion test was used for detection of methicillin resistance. Routine disk diffusion procedure was used to perform the Cefoxitin disk (30µg) test on S. aureus isolates, and isolates showing zone of inhibition of ≥ 22 mm were interpreted as MSSA or negative for mecA mediated resistance, whereas those showing zone of inhibition of ≤ 21 mm were interpreted as MRSA or positive for mecA mediated resistance. CLSI defines oxacillin resistance in S. aureus as an MIC ≥ 4µg and all strains meeting this definition are characterized as "MRSA" even though methicillin is no longer used either to test strains in the lab or as a therapeutic agent. 11

Penicillin Zone-edge test
For standard disk diffusion procedure, few bacterial colonies were used for making a normal saline suspension and the turbidity was matched up to 0.5 McFarland standard. It was further inoculated on Mueller-Hinton agar and 10-U penicillin G discs were applied, as per the CLSI guidelines. The plates were incubated at 35°C ± 2°C, in ambient air, for 16 -18 hours. Zone diameters were then measured and interpretation of susceptibility was based on CLSI (zone diameter of ≥ 29 mm) recommendations. All zone edges were assessed for sharp or fuzzy appearance. A sharp zone edge ("cliff") corresponds to the presence of b-lactamase, whereas a fuzzy zone edge ("beach") indicates the absence of β -lactamase. 11

statistical Analysis
Data were entered into MS Excel spreadsheet and SPSS version 20 (IBM Corp) was used for data analysis purpose. Descriptive statistics were presented in the form of means, standard deviation was calculated for continuous variables and frequencies for categorical variables. Data were graphically presented using columns, graphs and pie charts. A chi-square evaluation was used for group comparison for categorical data. P value < 0.05 was considered as statistically significant.    Figure 1]. The mean age of the patient was 35.08 with a standard deviation of 18.6.
Pearson Chi-square test was carried out to find any association of more incidence of MRSA infection among ICU patients. Value of Pearson's Chi-square was 2.507, degree of freedom was 1 and p value was 0.113. With a p value of p > 0.05, it is evident that there is no association between the incidence of MRSA infection among ICU patients.
As expected, all 116 MRSA isolates in our study were found to be Penicillin resistant (< 29 mm). Out of 63 MSSA isolates, only 2 (3.17%) were found to be sensitive to Penicillin by Zone-edge test having zone >29 mm with fuzzy edge at the zone indicating absence of β-lactamase production. Four of the MSSA isolates showed zone >29 mm with sharp edge at the zone indicating presence of β-lactamase production. Remaining 57 isolates had zone diameter < 29 mm [ Table 1].

DisCUssiON
In this study, analysis of 179 S. aureus isolates from different clinical samples revealed  that the overall rate of MRSA infection was as high as 64.8%, which is definitely alarming. Similar high prevalence rates have been registered by Gupta N. et al., 15 and Tiwari et al. 16 A similar study from Andhra Pradesh showed a CA-MRSA prevalence of 64.7%. 17 Other reports on CA-MRSA in India were by Shenoy et al., 18 from Mangalore, D'Souza et al. 19 24,25 The predominance in pus sample could be due to over-exposure of wound to environmental microorganisms. At the same time, presence of S. aureus as skin commensals makes the wound more prone for infection.
When gender distribution was compared, MRSA was found to be more prevalent among male patients (53.4%). Similar observations were reported by Rao et al. 26 There were no significant differences in the clinical presentations between both genders, as regards to the prevalence of MRSA.
Antimicrobial resistance pattern for other antibiotics in this study shows that MRSA strains present maximum resistance against Ciprofloxacin (71.8%), Erythromycin (70.6%), Clindamycin (59.3%), Gentamicin (36.8%) and Cotrimoxazole (34.1%). Similar findings were reported by Raj Kumar et al. 27 Almost all MRSA isolates were discovered to be sensitive to Linezolid and Vancomycin in our study, which is at par with the study done by Abbas et al. 28 In this study, no association was found between increased incidence of MRSA infections and patients admitted in ICU (p value 0.113). This suggests that MRSA infection is not confined to severely ill patients nowadays and is getting more prevalent among communities.
Among 63 isolates of MSSA, only two isolates (3.17 %) were found to be Penicillin susceptible (absence of b -lactamase) by Penicillin zone edge test. Both the isolates were from pus sample and belonged to male patient. Simultaneously, four isolates (6.34 %) were found to be b-lactamase producers. SS Richter et al 29 found around 4.46% b-lactamase producers among 448 MSSA, which is slightly lower than that observed in this study. Also, very large proportion of MSSA isolates (90.47 %) were found to be resistant to Penicillin, which is really worrisome.

CONClUsiON
High prevalence of b -lactamase production and methicillin-resistance among Staphylococcus aureus is a cause of concern. Along with the prevalence of HA-MRSA infection, there is an increase in the incidence of CA-MRSA infection as well. A patient with MRSA infection is exhibiting increased resistance to many classes of antibiotics, except Vancomycin and Linezolid, a fact which is more challenging for the health care providers and also for the clinical researchers. We recommend effective surveillance of MRSA at hospital and community level to know the exact burden and to follow all standard measures to reduce the spread of MRSA, including strict infection control and hand hygiene practices.

limitations
Major limitations of this study are small sample size, lack of molecular detection of blaZ gene and smaller study duration. Moreover, the study involves only clinical isolates from a single tertiary care center.

ACKNOwleDgMeNts
The authors would like to thank all faculty members, residents and technical staff members