Phenotypic characterization of coagulase negative staphylococci from various clinical isolates

Mamta Gour^1*, Kalpana Date², V. R. Thombare³, K. K. Gour⁴

¹Assistant Professor, ⁴Associate Professor, Department of Anatomy, Department of Microbiology, L.N.M.C. Bhopal, Madhya Pradesh, INDIA.

²Associate Professor, ³Professor and HOD, Department of Microbiology, N.K.P. S.I.M.S., Nagpur, Maharashtra, INDIA.

Email: mamtakgour@gmail.com, drkkgour@gmail.com

Abstract              Background: Despite their frequency as contaminants, coagulase-negative staphylococci (CNS) have become important nosocomial pathogens, accounting for 9% of all nosocomial infections. These infections are difficult to treat because of the risk factors and the multiple drug resistance shown by these organisms. Material and Methods: One hundred and forty CNS were isolated from various clinical samples like blood, pus, urine body fluids, urine, catheter tip, gastric lavage and wound swab. After confirming the isolates as CNS, species level identification was performed by simple, nonexpensive conventional methods and antibiotic sensitivity testing was also carried out. Result: 140 isolates could be identified to species level. Among these 140 identified CNS isolates, S. saprophyticus was the most frequently isolated 58 (41.43%) from various clinical sample, followed by S. epidermidis 45(32.14%), S. lugdunensis 23(16.43%), S. hemolyticus 11(7.86%), S. schleiferi 3(2.14%). In the present study, the susceptibility pattern of CNS species against antimicrobial agents showed that 65.71 % of the isolates were Methicillin Sensitive (MSCNS). Majority of the CNS species were sensitive to Amikacin, (94.29%), Nitrofurantoin (85.71%), Tetracycline (75.71%), and clindamycin (75%). None of the CNS species showed resistance to vancomycin and Linezolid. Conclusion: The increased pathogenic potential and multiple-drug resistance demonstrates the need to adopt simple, reliable and non-expensive methods for identifying and determining the antibiotic sensitivity of CNS.

Keywords: Antibiotic sensitivity test, CNS, S. saprophyticus, S. epidermidis, MRCNS

INTRODUCTION

Coagulase-negative Staphylococci (CNS), which are the normal skin flora, have emerged as predominant pathogens in hospital-acquired infections¹. The clinical significance of species other than S. epidermidis has been increasingly recognized in the recent years². They are rarely significant when isolated from skin, sputum and nasal swabs but may well be significant when isolated from wound swabs, pus, body fluids or blood cultures especially if foreign material is present³. Distinguishing clinically significant pathogenic strains from contaminant strains are one of the major challenges faced by clinical microbiologists³. It is important to study the clinical significance of the CNS isolate in a given clinical situation in view of deciding its pathogenic role. The present study was carried out to identify the prevalent species of CNS and their antibiogram.

MATERIALS AND METHODS

The present study was performed after obtaining the Institutional Ethics Committee clearance. A total of 140 non-repeat CNS were isolated and studied over a period of 2 year 1 month from November 2010 to December 2012 from different clinically significant specimens such as blood, pus, urine, body fluids, urine catheter tip and gastric lavage, wound swab, received from patients of all ages and any sex .The isolates were considered clinically significant when isolated in pure culture repeatedly. The isolates were identified by colony morphology, Gram stain, catalase test and coagulase test (slide and tube coagulase), susceptible to Furazolidone (100µg) and resistant to Bacitracin. Bacitracin susceptibility was performed to exclude Micrococcus and Stomatococcus species⁴. The conventional tests that are simple, easy to perform and non-expensive were chosen from the scheme of Koneman et al, 6^TH edition. These include the heat stable nucleases, ornithin decarboxylase test, PYR, acetoin, urease test, beta-galactosidase test, alkaline phosphatase test, and fermentation of glucose, sucrose, maltose, mannose, mannitol, and trehalose. Susceptibility to novobiocin and polymyxin B was performed as per the standard procedure⁵. The antibiotic sensitivity testing was performed on Muller-Hinton agar by the Kirby-Bauer disc diffusion method⁶ using a panel of antibiotics, which includes ampicillin (A), amikacin(AK), cotrimoxazole (Co), cefotaxime (Ce), cefazolin, cefoxitin, cephelexin, chloramphenicol, ciprofloxacin, clindmycin, erythromycin, linezolid, nitrofurantoin, tetracycline, gentamicin (G), and vancomycin (Va). Cefoxitin was used to detect methicillin-resistance.

OBSERVATION AND RESULTS

Table 1: Distribution of 140 isolates in different clinical sample

The simple conventional methods identified 140 CNS isolates to species level. Among these 140 identified CNS isolates, S. saprophyticus was the most frequently isolated 58 (41.43%) from various clinical samples, followed by S. epidermidis 45 (32.14%), S. lugdunensis 23 (16.43%), S. hemolyticus 11(7.86%), S. schleiferi 3(2.14%), Table 2 shows the species-wise distribution of CNS in the different clinical specimens. Urinary tract infection were most commonly due to S. saprophyticus (74.03%), followed by S. epidermidis (10.39%), S hemolyticus (10.39%) S. lugdunensis (2.60%), S. schleiferi (2.60%) Abscesses were mainly due to s.epidermidis (55.81%) followed by s. lugdunensis (39.53%), s.hemolyticus (2.33%),and s, schleiferi (2.33%), Bacteremia was caused most commonly by s. epidermidis (85.71%)followed by s. hemolyticus (14.29%). (Table 2)

DISCUSSION

Coagulase negative staphylococci (CNS) were generally regarded to be the contaminants, having little clinical significance in the past⁷. CNS are now recognized as a major cause of nosocomial infections in critically ill patients especially in intensive care units, that leads to morbidity and even mortality⁸. Because there is increasing pathogenecity of these organisms, CNS should be identified to the species level by simple, reliable and preferably inexpensive methods⁹. The overall incidence of clinically significant CNS among all the isolates from various clinical samples received in the study period was found to be 6.9 % in our study. The majority of CNS isolates were from urine (55%) and pus (30.71%).The CNS infection was more common in females (60%) than in males (40%) in the present study, which is shown in another study¹⁴ as well as our study. In present study, the commonest species isolated in clinically significant CNS was S. saprophyticus (41.43%) followed by S. epidermidis (32.14%) isolates amongst CNS. The other species isolated were S. lugdunensis (16.43%), followed by S.hemolyticus (7.86%) and S. shleiferi (2.14%). In our study, the most commonly isolated species was S. Saprophyticus (41.43%) followed by S. Epidermidis (32.14%). Similar results were seen in other studies as shown by Nord et al¹⁰ and John J F et al¹¹. As majority of samples included in our study were urine specimens and S. saprophyticus is the commonest cause of UTI. S. saprophyticus is determined to be the true urinary tract pathogen, which is found to be the second most common cause of urinary tract infection after E. coli in females¹². The reasons for the association of S. saprophyticus with urinary tract infections in young women remain unclear, but may relate to carriage of the organism in the rectum or introitus¹³. The present study revealed that, urinary tract infections by CNS were most commonly due to S. saprophyticus (74.03%) followed by S. epidermidis (10.39%), S. hemolyticus (10.39%), S. lugdunensis (2.60%) and S. schleiferi (2.60%), (Table 2). The results are similar to study of Nord et al. The incidence of methicillin resistance was 56% in this study, and many other studies have documented a still higher resistance¹⁴. None of the isolates showed resistance to vancomycin in our study. However, others have noted a reduced susceptibility to vancomycin¹⁵.

CONCLUSIONS

Recently, CNS have emerged as a potential pathogen, especially CNS are now recognized as a major cause of nosocomial infections in critically ill patients especially in intensive care units¹⁶. There is also an increased resistance among these isolates and glycopeptides have become the drug of choice in the treatment of these infections. Hence, there is a need for accurate identification of these isolates to a species level and their antibiotic sensitivity pattern to avoid decreased susceptibility to glycopeptides. In this study, the most common species identified was S. saprophyticus. Resistance to ampicillin and amoxyclav was high and none of the isolates showed resistance to vancomycin. All the methicillin resistant and methicillin sensitive strains of CNS were susceptible to Linezolid and vancomycin.

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