Study on Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococci Co-colonization in Patients of Intensive Care Units of Tertiary Health Care Facilities (Hospitals).

Abstract

Aim of this study was to isolate, identify and characterize methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) from ICUs of three Allied Hospitals of Rawalpindi Medical College, Rawalpindi, Pakistan. The study involved the investigation of co-colonization/ co-existence, antibiotic resistance and molecular identification. Samples from 216 cases were taken from Paeds intensive care units (PICUs) and Medical intensive care units (MCUs) of three hospitals over the period of 16 months. A total of 432 samples (2 sample/patients) were taken from nasal cavity and peri-rectum area of each patient. Each sample was processed for isolation of staphylococci and enterococci. The bacterial isolates were selectively enumerated on Mannitol salt agar (MSA) for S. aureus and Bile aesculin agar (BAA) for enterococci revealed 82 as Staphylococcus aureus and 159 as enterococci. Nasal cavity was the best site for the isolation of staphylococci and peri-rectum was the best area for the isolation of enterococci. Standard morphological, phenotypic and biochemical tests were done for identification of S. aureus and enterococci. Furthermore API Staph (bioMerieux, Germany) was used to confirm identification of 82 isolates of S. aureus. Molecular identification was done by amplification of 16S rRNA, staph 16S, nuc gene, coagulase gene, mecA gene for S. aureus to discriminate it as MRSA and Methicillin-sensitive S. aureus (MSSA) whereas ddl E. fa e calis, ddl E. faecium, vanA and vanB were used to identify enterococci as Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and as vancomycin-resistant E. faecalis and vancomycin-resistant E. faecium. Both the simple and multiplex PCR techniques were used for the identification of these two types of isolates. Antibiotic susceptibility tests revealed that all of the MSSA and MRSA isolates were sensitive to vancomycin, teicoplanin, quinupristin/dalfopristin and linezolid but MRSA were found to be resistant to, amoxicillin/clavulanic acid, cephradine, ciprofloxacin, levofloxacin, gentamicin, erythromycin, imipenem and tetracycline. For enterococci! VRE were resistant to all tested antibiotics except linezolid, quinupristin/dalfopristin and teicoplanin. Whereas vancomycin-susceptible Xl enterococci (VSE) were resistant to cephalexin, cefoxitin, cephalothin, cephradine, ciprofloxacin, erythromycin, gentamicin, levofloxacin and tetracycline. MIC was performed to determine the sensitivity and resistance of all isolates using a selection of commonly used antibiotics such as ciprofloxacin, oxacillin, tetracycline and vancomycin. Vancomycin was in the range of 1-4 mg/L for all isolates whereas for VRE it ranged from 64-512 mg/L. Absence of known virulence determinants along with antibiotics resistance was confirmed by amplification through PCR for critical virulence genes mainly, methicillin-resistant gene, mecA and vancomycinresistance types-A (vanA) and type-B (vanB). The isolated MRSA, MSSA, VSE and VRE are promising candidate for further investigation of their resistance characterization, clonality and molecular typing. In this study patient stayed an average 6.72 (SO ± 9.11) days in ICUs department. S. aureus was isolated more frequently from nasal samples and majority of the patients were from two age groups <1 year and >35 years in which S. aureus were isolated more from age group >35 years. Patients of MICU were more colonized with S. aureus and dominant category of colonization was male patients. Patients from rural areas were colonized more with nasal MRSA while MSSA were more f om urban patie ts. Majority patients were from lower social class with equal isolation of nasal MRSA from this and middles class patients. There were no specific association found among clinical outcomes and isolation of S. aureus. Enterococci were more frequent from peri-rectal samples than from nasal and there were only few cases where both nasal and peri-rectal samples of the same patient had VRE isolates. Nasal VRE isolation was random from all age groups while majority of nasal VSE isolates were from age group >35 years and age group <1 year. Among gender enterococci isolation was random and from both ICU (MICU and PICU) it was almost equal. Urban patients had the major portion of nasal and peri-rectal VRE and VSE than rural patients. Lower and middle class patients were more colonized with enterococci in both the sites. In disease categories, patients with pneumonia and miscellaneous disease group were more colonized with enterococci than other categories. xu Combination therapy of antibiotics was more frequently used for treatment of admitted patients. No particular association was seen with the treatment of antibiotics and isolation of MRSA and VRE. There was some relation seen in case of vancomycin treatment and VRE isolation which need further experiments for confirmation.