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Year : 2006  |  Volume : 24  |  Issue : 3  |  Page : 201-204
 

Simple and economical method for speciation and resistotyping of clinically significant coagulase negative staphylococci


Department of Microbiology, University College of Medical Sciences and GTB Hospital, New Delhi - 110 095, India

Correspondence Address:
N P Singh
Department of Microbiology, University College of Medical Sciences and GTB Hospital, New Delhi - 110 095
India
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Source of Support: None, Conflict of Interest: None


PMID: 16912440

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 ~ Abstract 

An attempt was made to speciate 102 clinically significant isolates of coagulase negative staphylococci (CoNS) by a practical scheme adapted from various references. This scheme utilizes slide and tube coagulase test, urease test ornithine decarboxylase, novobiocin susceptibility and aerobic acid from mannose for assigning species group. Inclusion of one or two additional tests in a species group could identify the isolates to species level. Ninety eight (97%) isolates were conveniently identified as S. epidermidis (41%), S. saprophyticus (16.6%), S. haemolyticus (14.7%), S. hominis (14.7%), S. lugdunensis (4.9%), S. schleiferi (1.9%) and S. capitis (1.9%). Only four isolates were not identified to the species level, two of which were probably S. capitis subspecies ureolyticus / S. warneri / S. simulans . Antibiotic susceptibility testing showed maximum resistance to ampicillin (89%) followed by cefotaxime (59%) with no resistance to vancomycin. The increasing recognition of pathogenic potential of CoNS and emergence of drug resistance amongst them denotes the need to adopt simple laboratory procedures to identify and understand the diversity of staphylococci isolated from clinical material.


Keywords: Antibiotic susceptibility, clinical isolates, coagulase negative staphylococci, identification


How to cite this article:
Goyal R, Singh N P, Kumar A, Kaur I, Singh M, Sunita N, Mathur M. Simple and economical method for speciation and resistotyping of clinically significant coagulase negative staphylococci. Indian J Med Microbiol 2006;24:201-4

How to cite this URL:
Goyal R, Singh N P, Kumar A, Kaur I, Singh M, Sunita N, Mathur M. Simple and economical method for speciation and resistotyping of clinically significant coagulase negative staphylococci. Indian J Med Microbiol [serial online] 2006 [cited 2019 Jun 19];24:201-4. Available from: http://www.ijmm.org/text.asp?2006/24/3/201/26994


Coagulase negative staphylococci (CoNS) are increasingly being recognized as significant nosocomial pathogens, partly due to the growing appreciation of this group of organisms as opportunistic pathogens or due to increase in the use of transient or permanent medical devices in seriously ill and immunocompromised patients.[1] In a routine microbiology laboratory Staphylococcus is identified by a rapid screening test (coagulase test) and all non S. aureus isolates are reported as CoNS.[2],[3]

CoNS are one of the most frequent causes of nosocomial infections and are reservoirs of multiple antimicrobial resistant determinants.[4] Identification of CoNS by reference method of Kloos and Schleifer is cumbersome, time consuming and requires expensive reagents which are not routinely available in most of the clinical laboratories.[5] Several commercial kit identification systems and automated instruments are available which can identify a number of Staphylococcus species accurately but are still out of reach of most of the laboratories in developing countries. Hence, convenient, reliable and inexpensive identification methods are needed to identify most of the CoNS, commonly implicated in majority of infections, which can be utilized by most of the laboratories where automated methods are not yet available.

The present study aimed to identify the most prevalent clinical isolates of CoNS by minimum number of tests necessary and sufficient to discriminate between the species. The tests which were simple, inexpensive and easy to perform were selected from the scheme of Kloos and Shleifer to identify CoNS species group (group approach) or species.[5],[6] Further, one or two additional tests were included to complete the strain identification wherever necessary. Antimicrobial susceptibility profiles of all the isolates were done by agar disk diffusion method.[7]


 ~ Materials and Methods Top


A total of 102 consecutive non-repeat clinically significant CoNS isolates were collected from July 2004 to January 2005 in the Department of microbiology, University College of Medical Sciences and GTB Hospital, New Delhi. Strains were isolated from deep wounds, blood samples, ear swabs, CSF, ascitic fluid, synovial fluid and urine samples. The isolates were considered clinically significant when isolated in pure culture from infected site or body fluid or if the same strain was isolated twice.[1] The strains collected were initially identified by colony morphology, Gram staining, catalase, slide and tube coagulase (read after 4, 24 hours) and anaerobic acid from mannitol. Bacitracin (0.04 U) and furazolidone (100 g) susceptibilities were done to exclude Micrococcus and Stomatococcus spp.[5],[8]

Identification

All the strains which were either slide or tube coagulase negative were further identified by a scheme developed in our laboratory after reviewing a number of references.[1],[3],[6],[9],[10],[11]

The identification scheme concentrated on species groups/species commonly encountered in clinical practice: the S. epidermidis group (i.e., S. epidermidis , S. capitis subsp. ureolyticus and S. caprae ), the S. haemolyticus group ( S. haemolyticus , S. auricularis and S. caseolyticus ), the S. saprophyticus group ( S. saprophyticus subsp. saprophyticus and S. hominis subsp. novobiosepticus ), the S. warneri group ( S. warneri and S. hominis subsp. hominis ), the S. cohnii group ( S. xylosus and S. cohnii subsp. ureolyticum ), S. lugdunensis, S. shleiferi subsp . schleiferi , S. capitis subsp. capitis, S. simulans and S. cohnii subsp. cohnii .[6] This scheme involved a two step procedure [Table - 1], first step aimed to identify species group and combined slide and tube coagulase with novobiocin resistance, test for urease activity, ornithine decarboxylase and aerobic acid from mannose. If identification required additional tests, a maximum of two tests were selected from [Table - 1]: trehalose and mannitol for the S. epidermidis group, acetoin production and lactose for the S. haemolyticus group, trehalose for the S. saprophyticus group, anaerobic thioglycollate broth for the S. warneri group and xylose for the S. cohnii group.[6] All the tests were performed according to reference method.[5]

Antimicrobial susceptibility

The antibiotic susceptibility testing of all the isolates was performed on Mueller-Hinton agar by the standard disk diffusion method as per national committee of clinical laboratory standards.[7]


 ~ Results Top


Thirty-nine out of 102 strains of CoNS (38.2%) were isolated from wound infections, 29 (28.4%) from urine samples, 15 (14.7%) from blood cultures, 11 (10.7%) from catheters, 4 (3.9%) from cerebrospinal fluid and 2 (1.9%) each from synovial fluid and ascitic fluid. Ninety-five of these strains were negative for both clumping factor and tube coagulase and seven strains were slide coagulase positive and tube coagulase negative. None of the strains was slide coagulase negative and tube coagulase positive. [Table - 2] shows the CoNS species group/species by the present scheme from various specimens.

The scheme could identify 97% of CoNS isolated from clinical samples by incorporation of one or two additional tests wherever needed. S. epidermidis was the most frequent isolate and was identified if ornithine decarboxylase was positive (37 isolates) while ornithine decarboxylase negative isolates required inclusion of trehalose and mannitol for speciation. This scheme could not delineate two ornithine decarboxylase negative isolates as S. capitis subsp. ureolyticus / S. simulans / S. warneri .

S. haemolyticus , S. hominis subsp. hominis were identified with ease after the inclusion of test for acetoin production and anaerobic growth respectively. Only two isolates were unidentified because of aberrant characteristics.

The antibiotic susceptibility pattern revealed no resistance to vancomycin with 89% resistant to ampicillin followed by cefotaxime (59%), cloxacillin (25%), erythromycin (23%), ciprofloxacin (29%) and gentamicin (20%).


 ~ Discussion Top


Many laboratories do not identify clinical isolates of CoNS to the species level as they are considered normal inhabitants of skin and nares capable of causing only opportunistic infections.[1] Moreover, the conventional identification methods, though accurate, are cumbersome and employ a large battery of biochemical reactions, which often give variable results and all the tests are generally not available in most of the routine diagnostic laboratories.[3] The commercial identification methods including automated systems, are uneconomical to use for all the isolates of CoNS. As CoNS is increasingly being implicated as significant nosocomial pathogen several reviewers have emphasized the need for species identification, which is possible only by a simple, easily adaptable, inexpensive method. [1],[2],[3],[4],[5],[6] The species identification is important in monitoring the reservoir and distribution of CoNS involved in nosocomial infections and determining the etiological agent.[4],[5]

The present scheme conveniently identified the most frequently encountered clinical isolates in our hospital as S. epidermidis (41%), S. haemolyticus (14.7%), S. saprophyticus (16.6%) and S. hominis (14.7%) up to species level by incorporation of one or two additional tests. The scheme directly identified even the newly described species S. schleiferi subsp. schleiferi and S. lugdunensis using slide agglutination test for detection of clumping factor or fibrinogen affinity factor provided no slide is discarded before 10 seconds as S. lugdunensis sometimes give delayed result.[5] Consequently, the laboratories performing only slide agglutination test without tube coagulase test may misidentify S. schleiferi subsp. schleiferi and S. lugdunensis as S. aureus . This scheme could not differentiate S. capitis subsp. ureolyticus , S. warneri , S. simulans as pyroglutamyl β napthylamide (PYR) hydrolysis test, a key component for their identification, being expensive, was not included. Though PYR test has been considered crucial for the differentiation of S. warneri , S. capitis subsp. ureolyticus from S. simulans (PYR +), a few studies have reported misidentification of S. warneri as S. simulans if PYR test comes positive, thereby questioning the reliability of PYR.[6] Various workers from India have reported S. epidermidis and S. saprophyticus to be the most common isolate similar to our study.[9],[10],[12],[13] None of the reports have shown isolation of S. warneri/ S. simulans which are probably not common in India. Though a single report has mentioned isolation of S. cohnii which was not isolated in our study, however the scheme described here can identify this isolate.[9] Thus, this scheme identified 97% of CoNS upto species level.

Antimicrobial susceptibility testing showed a variable sensitivity and resistance patterns, similar to earlier reports.[2],[9],[10],[12],[13] It is therefore recommended to assess the importance of CoNS, speciate the clinically relevant CoNS to whatever level possible and perform the antibiotic susceptibility testing before any typing procedure for epidemiological studies are undertaken. This simple, inexpensive methodology will prove useful in a routine microbiology laboratory for the presumptive identification of CoNS. Further studies are needed with well characterized strains for the evaluation of the present scheme.

 
 ~ References Top

1.Kloos WE, Bannerman TL. Update on clinical significance of coagulase negative staphylococci. Clin Microbiol Rev 1994; 7 :117-40.   Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.Goel MM, Singh AV, Mathur SK, Singh M, Singhal S, Chaturvedi UC. Resistant coagulase negative Staphylococci from clinical samples. Indian J Med Res 1991; 93 :350-2.  Back to cited text no. 2  [PUBMED]  
3.Ieven M, Verhoeven J, Pattyn SR, Goossens H. Rapid and economical method for species identification of clinically significant Coagulase negative Staphylococci. J Clin Microbiol 1995; 33 :1060-3.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.Archer GL, Climo MW. Antimicrobial susceptibility of coagulase negative Staphylococci. Antimicrobial Agents Chemother 1994; 38 :2231-7.  Back to cited text no. 4  [PUBMED]  [FULLTEXT]
5.Bannerman TL. Staphylococcus, Micrococcus and other catalase positive cocci that grow aerobically, Chapter 28. In : Manual of Clinical Microbiology , 8th ed. Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH, editors. ASM Press: Washington DC; 2003. p. 384.  Back to cited text no. 5    
6.de paulis AN, Predari SC, Chazarreta CD, Santoianni JE. Five test simple scheme for the species level identification of clinically significant coagulase negative staphylococci. J Clin Microbiol 2003; 41 :1219-24.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial disk susceptibility tests:approved standards. NCCLS document. NCCLS: Wayne, Pa; 2000.  Back to cited text no. 7    
8.Baird D. Staphylococcus: Cluster forming Gram-positive cocci, chapter 11. In : Mackie and McCartney Practical Medical Microbiology , 14th ed. Collee JG, Fraser AG, Marimon BP, Simmons A, editors. Churchill Livingstone: New York; 1996. p. 245.  Back to cited text no. 8    
9.Shrikhande S, Thakkar YS, Pathak AA, Saoji AM. Species distribution of clinical isolates of Staphylococci. Indian J Med Microbiol 1996; 39 :207-10.  Back to cited text no. 9    
10.Phatak J, Udgaonkar U, Kulkarni RD, Pawar SG. Study of coagulase negative staphylococci and their incidence in human infections. Indian J Med Microbiol 1994; 12 :90-5.  Back to cited text no. 10    
11.Hebert GA Crowder CG, Hancock GA, Jarvis WR, Thornsberry C. Characteristics of Coagulase negative Staphylococci that help differentiate these species and other members of the family Micrococcaceae. Clin Microbiol 1988; 26: 1939-49.  Back to cited text no. 11    
12.Mohan V, Jindal N, Aggarwal P. Species distribution and antibiotic sensitivity pattern of coagulase negative staphylococci isolated from various clinical specimens. Indian J Med Microbiol 2002; 20 :45-6.  Back to cited text no. 12    
13.Saini S, Kaur H, Sabharwal U, Malik AK. Coagulase negative staphylococci in the urinary tract. Indian J Med Res 1983; 78 :26-8.  Back to cited text no. 13  [PUBMED]  


    Tables

[Table - 1], [Table - 2]

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