Hrev_master [page 26] [Healthcare in Low-resource Settings 2023; 11:11229] Prevalence of MLSB Phenotypes of Staphylococcus aureus isolates in a tertiary care hospital of Delhi Malika Grover, Nisha Goyal, Seema Gangar, Narendra Pal Singh Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, Delhi, India Abstract Against the backdrop of the ever-chang- ing Staphylococcal resistance pattern, clin- damycin remains a viable therapeutic alter- native Variation of Clindamycin drug resis- tance patterns with geographic area make inducible clindamycin resistance testing imperative for all staphylococcal isolates to avoid therapeutic failure. This was a prospective study conducted over a period of 1.5 years from January 2021 until June 2022. Prevalence of different MLSB Phenotypes of Staphylococcus aureus iso- lates was determined by standard disc diffu- sion method as per CLSI guidelines. Pyogenic samples received in the Microbiology lab that yielded Staphylococcus aureus were further tested for the presence of clindamycin resistance by disc diffusion method. Out of 6586 total pyogenic and respiratory specimens received in the lab, Staphylococcus aureus was yielded in 752 samples. On further test- ing for the MLSB phenotypes, 16.3% iso- lates were found to be iMLSB, 19.28% were cMLSB, 43.1% were of MSB type. ICR screening will reduce the unessential sub- jection of the patient to the antibiotic, and would prevent unnecessary adverse effects in the patients. Introduction Staphylococcus aureus (S. aureus) is a potential pathogen as well as a colonizer of the humans owing to the arsenal of viru- lence factors including toxins such as TSST-1 (toxic shock syndrome toxin), exfo- liative toxins (ETA and ETB), heat stable enterotoxins etc. Manifestation of Staphylococcal infections ranges from local (folliculitis, carbuncles, furuncles, impeti- go, wound infections) to systemic (endo- carditis, pneumonia, sepsis, osteomyelitis, arthritis). Localised S. aureus infections have the potential to become invasive and cause bacteremia at any stage of the infec- tion. The mainstay of treatment for these infections include cell wall inhibitors such as β-lactams, glycopeptides, DNA gyrase- inhibiting quinolones, and ribosomal inhibitors such as macrolides, lincosamides and streptogramins (MLSB). MLSB drugs are a good alternative in treating infections, especially in current times of increasing resistance. Clindamycin in particular is an important antibiotic for skin and soft tissue infections caused by S. aureus (especially MRSA i.e., Methicillin resistant Staphylococcus aureus) due to its ease of administration (available as oral/parenteral) and its property to neu- tralise toxins. It switches off production of toxins like TSST responsible for toxic shock syndrome,1 alpha toxin which is a pore forming cytotoxin leading to infections such as dermonecrosis, keratoconjuctivitis and pneumonia2 and pVL (Panton-Valentine leukocidin), which is associated with mani- festations like necrotising pneumonia, pur- pura fulminans and skin sepsis.3 The three antimicrobial classes of MLSB act by bind- ing to the 50s ribosomal subunit, thus inhibiting protein synthesis in the bacteria.4 Resistance amongst these can be conferred mainly by three mechanisms – target site modification, antimicrobial inactivation and efflux. The enzyme erythromycin ribosome methylases plays the most significant role in the resistance, by attaching the adenine residue of 23s rRNA to methyl groups, thus decreasing affinity for MLSB antibiotics. It is encoded by the erm (erythromycin ribo- some methylation) gene which is of three main types i.e., erm (A), erm (B) and erm (C); also, genes erm (F) and erm (Y) may be responsible. The other mechanisms that contribute to the cross resistance of these MLSB pheno- types include drug inactivation mediated by lun gene and active efflux mechanisms that pumps out antimicrobials from the bacteria, mediated by msr gene.5 MLSB drugs can exist as different phe- notypes – constitutive, inducible, or MSB (Figure 1): i) constitutive MLSB (cMLSB) – defined as those isolates which are clin- damycin and erythromycin resistant; ii) inducible MLSB (iMLSB) – defined as iso- lates which are clindamycin susceptible and erythromycin resistant. However, a D- shaped zone of inhibition is seen around clindamycin, with flattening towards the erythromycin disc; iii) MSB – is defined as those isolates which are clindamycin sus- ceptible and erythromycin resistant with a circular zone of inhibition around the two. Clinical and Laboratory Standards Institute (CLSI) states two methods for detecting Inducible Clindamycin Resistance (ICR), i.e., by disc diffusion and broth microdilution. Detection of inducible clin- damycin resistance in particular holds sig- nificance in clinical scenarios, wherein the S. aureus isolates exhibiting in vitro clin- damycin susceptibility will not show in vivo response on administration of the drug. This leads to unnecessary overuse of the drug in the patient, thus enhancing the risk of emer- gence of resistant strains of bacteria and putting the patient at increased risk of side effects of the drug. Improper treatment dur- ing the initial phase can also put the patient at risk for metastasis of the disease. Our current study aims at identifying the distribution of MLSB phenotypes of S. aureus isolates for better understanding of Healthcare in Low-resource Settings 2023; volume 11:11229 Correspondence:Nisha Goyal, Department of Microbiology, University College of Medical Sciences & Guru Teg Bahadur Hospital, 110095 Delhi, India. Tel.: +91.8447444427. E-mail: drnishagoyalucms@gmail.com Key words: Staphylococcus aureus, inducible clindamycin resistance, constitutive clin- damycin resistance, MSB phenotype. Conflict of interest: the authors declare no potential conflict of interest, and all authors confirm accuracy. Ethics approval and consent to participate: not applicable. This study used only the sam- ples received in the lab for routine susceptibil- ity testing and no other sample was collected for the purpose of this study. Patients were not identified or visited at any point of time. Informed consent: not applicable Patient consent for publication: not applicable Availability of data and materials: all data generated or analyzed during this study are included in this published article. Received for publication: 31 January 2023. Accepted for publication: 7 June 2023. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2023; 11:11229 doi:10.4081/hls.2023.11229 Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affili- ated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guar- anteed or endorsed by the publisher.No n- co mm er cia l u se on ly resistance patterns to crucial antibiotic of clindamycin in the management of infec- tions caused by S. aureus. Material and Methods This was a prospective study carried out over a period of one and a half year span- ning from January 2021 to June 2022 in our tertiary care hospital of Delhi. A total of 6586 samples, including pus aspirates, peri- toneal fluid, pleural fluid, synovial fluid, respiratory samples, and genital secretions were received in the microbiology lab of our hospital. The samples were cultured on Blood agar, MacConkey agar and Chocolate agar using standard laboratory protocols. Bacterial identification of the growth was done by conventional methods, using biochemical reactions (Catalase, slide and tube coagulase, Mannitol salt agar). The samples that yielded growth of S. aureus on culture were further subjected to Antimicrobial Susceptibility Testing (AST) by Kirby Bauer disk diffusion method, according to latest CLSI guidelines.6 For AST 0.5 McFarland of the strain was lawn cultured on Muller Hinton agar, followed by placement of the antimicrobial discs at a distance of 15-20 mm edge to edge from each other and incubation at 35°C±2°, ambient air. Isolates were classified as Methicillin susceptible or resistant on the basis of zone of inhibition diameters of Cefoxitin. While, presence of clindamycin resistance (consti- tutive, inducible and MSB) was determined by performing disk diffusion method, plac- ing Erythromycin (15µg) and Clindamycin (2µg) at a distance of 15-26mm from each other. Zone cut-offs for the antibiotics have been descried in the Table 1. Isolates with intermediate zone diameters were consid- ered as resistant for ICR analysis. Presence of D-zone i.e., flattening of the zone of inhi- bition adjacent to the erythromycin disc was interpreted as inducible clindamycin resis- tance, as shown in Figure 1a. Results Out of the total 6586 pyogenic and res- piratory samples received, S. aureus was isolated from 11.4% (752/6586) samples. Majority of these samples were received from the patients admitted in surgical wards. The organism was isolated more commonly from the male population (54.9%) as compared to the females (45.07%). Isolation of S. aureus was more common from adult patient population (71.8%) in comparison to the paediatric population (28.9%). Of the total S. aureus isolates 335 (44.54%) were MSSA (Methicillin sensitive Staphylococcus aureus), while 417 (55.45%) were MRSA (Methicillin resistant Staphylococcus aureus. All the strains of this gram-positive organism were tested for different MLSB phenotypes i.e., inducible, constitutive and MSB. Inducible clin- damycin resistance was found in 16.35% of the isolates; constitutive clindamycin resis- tance was observed in 19.28% of the observed isolates, while MSB phenotypes were observed in 43.08%. Percentage distri- bution of various MLSB phenotypes has been described in Table 2. Distribution of MSSA and MRSA were also observed among the MLSB phenotypes (Table 3). On application of Fischer’s exact test, no significant association was observed between methicillin susceptibility of the isolates and the constitutive and MSB phe- Article Table 1. Antimicrobial susceptibility break points (CLSI 2022). Antibiotic Susceptible Intermediate Resistant Erythromycin (15 µg) ≥23 mm 14-22 mm ≤13 mm Clindamycin (2 µg) ≥21 mm 15-20 mm ≤14 mm Cefoxitin (30 µg) ≥22 mm - ≤21 mm Table 2. Distribution of various MLSB phenotypes among Staphylococcal aureus isolates from clinical samples (n=752). Erythromycin Clindamycin D Test Phenotype No. of isolates Percentage susceptibility susceptibility Susceptible Susceptible Negative - 160 21.27 Resistant Resistant Negative cMLSB 145 19.28 Resistant Susceptible Positive iMLSB 123 16.35 Resistant Susceptible Negative MSB 324 43.08 Figure 1. Identification of various MLSB phenotypes of Staphylococcal aureus isolates from clinical samples (n=752): a) inducible MLSB (iMLSB); b) constitutive MLSB (cMLSB). [Healthcare in Low-resource Settings 2023; 11:11229] [page 27] No n- co mm er cia l u se on ly notypes, as the p value was found to be 0.0556. Association of methicillin susceptibility was established in the isolates displaying inducible clindamycin resistance. Of the total 123 isolates showing inducible clin- damycin resistance, 29.2% were Methicillin susceptible while the rest 71% were found to be Methicillin resistant (Figure 2). No significant association was observed between ICR phenotype and Methicillin susceptibility (p≥0.05). Discussion S. aureus is the most common aetiolog- ical agent of pyogenic infections. Drugs such as Trimethoprim-Sulfamethoxazole, Tetracyclines (Minocycline and Doxycycline) and Clindamycin have gained importance in present scenario of increasing drug resistance in staphylococcal isolates.7 Clindamycin, belongs to the Lincosamide group of antibiotics and pos- sesses activity against gram-positive as well as anaerobic bacteria. Its properties such as good tissue penetration, cost, spectrum and, oral bioavailability make clindamycin con- ducive to treating infections. It is thus, used for skin and soft tissue infections, with par- ticular significance in cases of CA-MRSA infections, wherein an oral treatment regi- men can suffice for the patient. This Lincosamide antibiotic is also effective in treating conditions such as pleural empye- ma, osteomyelitis and septic arthritis. Though clindamycin has several prop- erties to its advantage, there are a few chal- lenges that a clinician faces while using the drug. Pseudomembranous colitis due to Clostridioides difficile is observed in 0.1- 10% of the patients using clindamycin per- sistently1 and likelihood of failure if the strain possesses erm gene are the two main disadvantage to clindamycin use. Clindamycin resistance can either be induced or can be rendered constitutively based on the phenotype. In our study, constitutive resistance to the MLSB drugs was found to be more (19.3%) in comparison to the inducible phe- notype. ICR rates were found to be 16.35%, which were considerably higher in MRSA isolates (70.8%) than the MSSA strains. Not many studies have commented upon the reason justifying the higher prevalence of ICR in MRSA, but one possible explanation is more positivity rate for ermA in MRSA than MSSA.8 This is indicative of increased chances of treatment failure with clin- damycin in resistant infections. Table 4 compares the distribution of MLSB pheno- types in various geographical regions of our country and beyond. The presence of MSB phenotype in our study was higher in comparison to the other two variants. Similar finding was observed in the other areas of Delhi.10 Therefore, Clindamycin can be used empirically by clinicians for indicated infections with less- er chances of it turning out to be ineffective. Table 4 shows the Geographical distri- bution of MLSB phenotypes in various geo- graphical regions. In our study higher prevalence of cMLSB than that of iMLSB was observed, which was found to be in concordance with other studies conducted in the regions of Kolkata, Shimla and Nepal.4,6,7 Conversely higher prevalence of iMLSB than cMLSB was observed in other regions of Delhi and Wardha.5,8 The varying Article Table 4. Geographical distribution of MLSB phenotypes in various geographical regions. Study Year Region No. of isolates (n) iMLSB (%) cMLSB (%) MSB (%) Kumar et al.9 2010 Kolkata, India 195 16.9 23.1 16.9 Lall and Sahni et al.10 2014 Delhi, India 305 43.1 21.4 54.3 Mokta et al.11 2015 Shimla, India 350 13.71 17.14 8.28 Deotale et al.12 2017 Wardha, India 247 14.5 3.6 14.17 Adhikari et al.13 2017 Nepal 147 21 53.4 25.17 Our study 2022 East Delhi, India 752 16.35 19.28 43.08 Figure 2. Distribution of MSSA and MRSA among Staphylococcus aureus isolates exhibit- ing inducible clindamycin resistance (n=752). Table 3. MSSA & MRSA distribution amongst the constitutive and MSB phenotypes. MLSB Phenotype MSSA (%) MRSA (%) Constitutive 59 (40.7) 86 (59.4) MSB 171 (52.8) 153 (47.2) [page 28] [Healthcare in Low-resource Settings 2023; 11:11229] No n- co mm er cia l u se on ly [Healthcare in Low-resource Settings 2023; 11:11229] [page 29] geographical prevalence of different resis- tance patterns emphasizes upon the impor- tance of Clindamycin testing in all isolates. It was observed that the prevalence of clindamycin resistance (both cMLSB and iMLSB) was more in MRSA isolates in comparison to the MSSA isolates that was consistent with the findings of other studies.11,14,15 Against the backdrop of the ever-chang- ing Staphylococcal resistance pattern, clin- damycin remains a viable therapeutic alter- native. Our study may prove useful in better understanding of varying distribution of dif- ferent MLSB phenotypes of S.aureus in recent times. 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