Staphylococcal Cassette Chromosome mec Typing and Multilocus Variable Number Tandem Repeat Analysis of Methicillin Resistant Staphylococcus aureus Clinical Isolates with Vancomycin Creep Phenomenon.

Background: The association of treatment failure and mortality with vancomycin minimum inhibitory concentration creep (MIC) is a matter of serious concern in patients with severe methicillin resistant Staphylococcus aureus (MRSA) infections. The purpose of the study was to identify and characterize staphylococcal cassette chromosome mec (SCCmec) and clonal types of MRSA strains, exhibiting the vancomycin MIC creep phenomenon. Methods: A total of 3305 S. aureus strains were isolated from various clinical samples of Lahore General Hospital, Lahore, Pakistan. MRSA strains were identified by cefoxitin resistant (≤21mm) followed by mecA and mecC gene genotyping. Vancomycin MIC creep was determined by E-test. Isolates having MIC values >1.5 µg/mL were further subjected for SCCmec typing (I-V and XI) and multiple-locus variable number tandem repeat analysis (MLVA) by amplification of spa, sspA, clfA, clfB, and sdrCDE genes. A dendrogram was created based on the similarity index using bioneumerics software. Results: About 13.3% (440/3305) isolates were MRSA with 99.3% (437/440) and 0.7% (3/440) carried mecA and mecC genes, respectively. In 120 MRSA isolates, the MIC of vancomycin was >1.5µg/mL. In MRSA isolates with high vancomycin MIC (>1.5µg/mL), the most common SCCmec type was SCCmec III (38.3%), followed by SCCmec IVa (15.8%), SCCmec IIIa (13.3%,), SCCmec IVc (7.5%), SCCmec IVe (5.8%), SCCmec IVd (5.8%), SCCmec IVb (4.2%), SCCmec II (2.5%), SCCmec V (1.7%), SCCmec I (1.7%) and SCCmec XI (1.7%). MLVA revealed 60 genotypic groups of MRSA isolates having a 92% similarity index. Conclusion: SCCmec III was the most common type in genetically related MRSA isolates showing vancomycin MIC creep. The presence of SCCmec XI may further add burden to infection control measures.

Four year trend of antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus in a tertiary care hospital, Lahore.

OBJECTIVE: To determine the susceptibility pattern of methicillin-resistant staphylococcus aureus to different antibiotics. METHODS: The descriptive study was conducted at the Microbiology Department of the University of Health Sciences, Lahore, Pakistan, from August 2016 to July 2019, and comprised staphylococcus aureus samples that were processed and identified using colony morphology on blood agar, gram stain, catalase, coagulase and deoxyribonuclease testing. Screening for methicillin-resistant staphylococcus aureus was done using cefoxitin disc 30µg and oxacillin disc 1mg. Antimicrobial susceptibility was tested using the modified Kirby-Bauer disc diffusion method in line with the Clinical and Laboratory Standards Institute guidelines 2019. Data was analysed using SPSS 24. RESULTS: Of the 2704 strains processed, 402(14.86%) were found to be methicillin-resistant staphylococcus aureus. Of them, 204(50.74%) were recovered from pus, while 10(2.48%) were recovered from urine. All 402(100%) isolates were sensitive to vancomycin and linezolid, and resistant to penicillin, followed by erythromycin 306(76.11%) and sulfamethoxazole/ trimethoprim 295(73.38%). Overall, lower resistance was seen with doxycycline 145(36.06%) and clindamycin 160(39.80%). Inducible clindamycin resistance was seen in 142(35.23%) isolates. CONCLUSIONS: An efficacious susceptibility pattern of methicillin-resistant staphylococcus aureus was seen with vancomycin and linezolid, moderate susceptibility with doxycycline and clindamycin, while high resistance was observed for penicillin, erythromycin and trimethoprim/sulfamethoxazole.

Etiological Profile and Antimicrobial Patterns in Blood Culture Specimens in a Tertiary Care Setting.

Introduction Universally, blood stream infections are linked with increasing morbidity and mortality. Timely diagnosis for identification of bacterial etiology, their susceptibility pattern and choice of empiric treatment plays a vital role in management. Objective To reveal the etiological profile and antibiotic sensitivity in blood culture specimens in a tertiary care setting. Methods This descriptive study was carried out in pathology laboratory of a tertiary care hospital from August 2016 to July 2019. All the 750 blood culture bottles were processed and isolates were recognized by morphological appearance on recommended media, gram stain, and different biochemical tests using Analytic Profile Index. Antibiotic sensitivity was implemented by modified disc diffusion method as per Clinical and Laboratory Standards Institute (CLSI) principles (2019). Results Out of 750 blood samples, 212 (28.26%) were culture positive. The percentage of gram-negative bacilli (n = 105) and gram-positive cocci (n = 104) was almost same (49.52%), while candida spp. was recovered from three (1.41%) isolates. The identified gram-negative bacteria were E. coli and Acinetobacter baumannii each (19.04%), Klebsiella pneumoniae and Pseudomonas aeruginosa each (16.19%), Enterobacter cloaca (11.42%), Salmonella typhi (8.57%), Burkholderia cepacia (1.90%), and Raoultella terrigena (7.61%). Among gram-positive isolates, coagulase-negative staphylococci (79.80%), Staphylococcus aureus (6.73%), Enterococcus spp. (11.53%) and Streptococcus spp. (1.92%) were recovered. Colistin, imipenem, meropenem, and amikacin were most successful against gram-negative rods. The sensitivity to vancomycin, teicoplanin and linezolid was 100%, for gram positive organisms. Methicillin resistance was present in 84.4% Staphylococcal isolates. Conclusion Local data showing changing etiological pattern and antibiogram of isolated pathogens, along with adequate infection prevention and control measures can be useful to improve patient care, in terms of hospital stay, duration of medication and treatment cost.

Comparison of phenotypic methods with mecA gene based polymerase chain reaction for Methicillin-resistant Staphylococcus aureus detection.

OBJECTIVE: To determine methicillin resistance in staphylococcus aureus by different phenotypic methods, and to evaluate their accuracy with mecA gene polymerase chain reaction for methicillin-resistant staphylococcus aureus detection. METHODS: The descriptive cross-sectional study was conducted from January to December 2015 at the Post- Graduate Medical Institute, Lahore, Pakistan, and comprised consecutive, non-repetitive clinical isolates of methicillin-resistant staphylococcus aureus that were screened with oxacillin disk 1µg and cefoxitin disk 30µg by Kirby-Bauer method using Clinical and Laboratory Standards Institute guideline. The isolates were cultured on oxacillin screen and mannitol salt agar, and subjected to latex agglutination for penicillin-binding protein 2aand polymerase chain reaction for mecA gene. Data was analysed using SPSS 20. RESULTS: All the 105 isolates were resistant on oxacillin and cefoxitin disk diffusion test, but 95(90.47%) were positive for mecA gene by latex agglutination and polymerase chain reaction. The sensitivity of oxacillin salt agar, mannitol salt agar and latex agglutination was 94.31%, 96.73% and 98.95%, respectively. Keeping polymerase chain reaction as the gold standard, the specificity and diagnostic accuracy of latex agglutination were 77.77% and 97.14% respectively, which was the highest among all the phenotypic methods. CONCLUSIONS: Latex agglutination method can be proposed as a swiftly reliable diagnostic technique for the detection of mecA gene in methicillin-resistant staphylococcus aureus isolates in resource-constrained settings where molecular methods are limited.