|Year : 2018 | Volume
| Issue : 1 | Page : 124-126
Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India
Saravanan Murugesan, Stalin Mani, Indhumathy Kuppusamy, Padma Krishnan
Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Chennai, Tamil Nadu, India
|Date of Web Publication||2-May-2018|
Dr. Padma Krishnan
Department of Microbiology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu
Source of Support: None, Conflict of Interest: None
The objective of this study was to detect the association of biofilm formation with IS256 among clinical and carrier isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). A total of 71 MRSE isolates were included in this study. Phenotypic detection of biofilm formation was done by Congo red agar method. Detection of genes associated with biofilm formation (icaAD, aap and atlE) and insertion sequence IS256 was done by polymerase chain reaction. Of the 71 MRSE isolates,19/40 (47.5%) clinical isolates from hospital settings and 11/31 (35.5%) carrier isolates from community settings respectively were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes. Nearly 80% of clinical isolates were found to harbour IS256, whereas only 13% of community isolates harboured IS256.
Keywords: IS256, methicillin-resistant Staphylococcus epidermidis, multiplex polymerase chain reaction, virulence determinants
|How to cite this article:|
Murugesan S, Mani S, Kuppusamy I, Krishnan P. Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India. Indian J Med Microbiol 2018;36:124-6
|How to cite this URL:|
Murugesan S, Mani S, Kuppusamy I, Krishnan P. Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India. Indian J Med Microbiol [serial online] 2018 [cited 2018 Sep 22];36:124-6. Available from: http://www.ijmm.org/text.asp?2018/36/1/124/231665
| ~ Introduction|| |
Staphylococcus epidermidis is a common coloniser of the human skin and mucous membranes and also the most frequent cause of nosocomial infections on indwelling medical devices. Major clinical challenge in pathogenesis of methicillin-resistant S. epidermidis (MRSE) infections are that they are both multidrug resistant with an exceptional ability to form thick and multilayered biofilms.
Biofilm production is one of the main factors involved in the pathogenesis of infections, which is mediated by the production of an intercellular polysaccharide adhesin encoded by an accessory gene cluster called the intercellular adhesion (ica) operon. In addition, more genes implicated in biofilm production (i.e. bifunctional autolysin E, atlE, and accumulation-associated protein, aap) have been elucidated.,
Distinguishing invasive from commensal strains is challenging since virulence factors can occur in both; the sudden transition of the organism to a pathogenic state is the subject of intense investigations. Studies aiming at distinguishing invasive from commensal strains are needed. There are several studies including one from Chennai elucidating the pathogenicity of the S. epidermidis, which mainly focus on the detection of biofilms and ica genes. The usefulness of these virulence markers has been debated widely. Recently, it was demonstrated that the insertion sequence element IS256 was superior to ica gene detection in distinguishing clinically relevant isolates.,
Hence, the present study was designed to detect the biofilm formation and various genes involved (icaAD, aap and atlE) and the association of biofilm formation with IS256 among clinical and carrier isolates of MRSE.
| ~ Materials and Methods|| |
This study was approved by the Institutional Human Ethical Committee. A total of 281 coagulase-negative staphylococci (CoNS) isolates were collected from hospitalised patients (n = 127) and asymptomatic healthy individuals from community settings (n = 154). Initial isolation and speciation were done by standard biochemical methods.
Bacterial DNA was obtained by the boiling lysis method. Species-specific polymerase chain reaction (PCR) was done to identify S. epidermidis using recN gene.
Methicillin resistance was detected using multiplex PCR (M-PCR) (mecA and femA genes). Phenotypic production of slime by S. epidermidis was assessed by culture on Congo red agar (CRA) method. Biofilm-associated genes were amplified by M-PCR method using the published primer sequences., [Table 1]. Bacterial insertion sequence element IS256 was used as a novel molecular marker to differentiate invasive and commensal strains of S. epidermidis. PCR detection of the IS256 was performed by previously described method.S. aureus ATCC 25923, 43300 and S. epidermidis ATCC 12228, 35984 were used as the control strains in our study.
|Table 1: Primers and their sequences for various genes used in this study|
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| ~ Results|| |
Of the 127 and 154 CoNS isolates from hospitalised patients and asymptomatic healthy individuals from community settings, 40 (31.5%) and 31 (20%) isolates were found to be MRSE, respectively. All the MRSE isolates from hospitalised patients (n = 40) and community settings (n = 31) were subjected to CRA method for the detection of biofilm formation and associated genes (icaAD, aap and atlE genes) [Figure 1].
Nearly 72.5% (29/40) and 58% (18/31) of the isolates were found to be positive for biofilm formation among hospitalised patients and asymptomatic healthy individuals from community settings, respectively.
A total of 19/40 (47.5%) and 11/31 (35.5%) of the isolates from hospital and community settings were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes, respectively. IS256 was found in 80% (32/40) and 13% (4/31) of the isolates from hospital and community settings, respectively [Figure 2].
|Figure 2: Representative gel picture showing IS256 - S. epidermidis virulence marker - Gu et al., 2005|
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| ~ Discussion|| |
In this study, a total of 71 MRSE isolates were included and compared for their biofilm production and genetic traits (icaAD, aap and atlE) and the presence of IS256 element. By CRA method, it was found that biofilm production was higher among isolates in hospital settings (72.5%) than community settings (58%). Our study report was in agreement with the other previous studies., The ica operon is considered one of the main genetic determinants of biofilm formation and its detection has been suggested as a tool for discriminating invasive from contaminating strains in clinical specimens.
In the present study, the majority of the biofilm-producing isolates harboured the icaAD, aap and atlE genes. The prevalence of icaAD was found to be 77.5% and 64.5% from hospital and community settings, respectively.
The prevalence of aap was found to be 65.3% and 46.6% from hospital and community settings, respectively. The prevalence of atlE was found to be 88.8% and 28.8% from hospital and community settings, respectively.
With a few exceptions, icaAD gene was not detected. A single clinical isolate exhibited the icaAD-aap+ atlE+ genotype, which was in agreement with the previous studies. These observations seem to reflect the importance of aap + and atlE+ genes for biofilm production in S. epidermidis. These results indicate that probably some other mechanisms coexist in S. epidermidis. There was a significant association between the detection of these genes and the biofilm expression profiles, indicating a tendency of the isolates that display the strong biofilm producer phenotype to harbour the ica operon and atlE and aap concomitantly.
Previous studies suggested the ica operon as a marker to differentiate the invasive and commensal S. epidermidis isolates.,, In the present study, ica gene was present in 20/31 (64.5%) MRSE isolates from asymptomatic healthy individuals. Thus, these data indicate that despite their role of biofilm production in S. epidermidis infections, both biofilm and the genes associated with this phenotype should not be used as markers for clinical significance, as suggested previously.
Previous studies have reported IS256 as an important factor for S. epidermidis infectivity , and a marker to differentiate the invasive and commensal S. epidermidis isolates. In this study, 80% of clinical isolates were IS256 positive, whereas 13% of isolates showed the presence of IS256 in asymptomatic healthy individuals. Supporting our data, previous studies reported that IS256 is a good marker to differentiate between invasive and non-invasive isolates.,,
Hence, findings of the present study have shown that IS256 occurred more significantly in strains of clinical origin, indicating that IS256 might thus constitute a molecular marker to discriminate invasive strains from commensal strains of S. epidermidis.
| ~ Conclusion|| |
Based on this study, it is suggested that either IS256 alone or IS256 and the ica operon together can be used as molecular markers to discriminate invasive strains from commensal strains of S. epidermidis. This study demonstrated ica gene to be the predominant gene amongst the genes tested to be associated with biofilm formation among S. epidermidis isolates. The ability of S. epidermidis to form biofilms helps the bacterium to survive hostile environments within the host and may exhibit increased virulence.
The presence of ica operon and biofilm formation makes S. epidermidis among the healthy skin of asymptomatic individuals more capable of producing a chronic infection and surviving adverse conditions. Hence, measures have to be taken to reduce the risk of hospital-acquired S. epidermidis infections.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Otto M. Staphylococcus epidermidis
– The 'accidental' pathogen. Nat Rev Microbiol 2009;7:555-67.
Büttner H, Mack D, Rohde H. Structural basis of Staphylococcus epidermidis
biofilm formation: Mechanisms and molecular interactions. Front Cell Infect Microbiol 2015;5:14.
Okee MS, Joloba ML, Okello M, Najjuka FC, Katabazi FA, Bwanga F, et al.
Prevalence of virulence determinants in Staphylococcus epidermidis
from ICU patients in Kampala, Uganda. J Infect Dev Ctries 2012;6:242-50.
El Farran CA, Sekar A, Balakrishnan A, Shanmugam S, Arumugam P, Gopalswamy J, et al.
Prevalence of biofilm-producing Staphylococcus epidermidis
in the healthy skin of individuals in Tamil Nadu, India. Indian J Med Microbiol 2013;31:19-23.
de Araujo GL, Coelho LR, de Carvalho CB, Maciel RM, Coronado AZ, Rozenbaum R, et al.
Commensal isolates of methicillin-resistant Staphylococcus epidermidis
are also well equipped to produce biofilm on polystyrene surfaces. J Antimicrob Chemother 2006;57:855-64.
Gu J, Li H, Li M, Vuong C, Otto M, Wen Y, et al.
Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis
. J Hosp Infect 2005;61:342-8.
Du X, Zhu Y, Song Y, Li T, Luo T, Sun G, et al.
Molecular analysis of Staphylococcus epidermidis
strains isolated from community and hospital environments in China. PLoS One 2013;8:e62742.
Kloos WE, Bannerman TL. Update on clinical significance of coagulase-negative staphylococci. Clin Microbiol Rev 1994;7:117-40.
Iorio NL, Azevedo MB, Frazão VH, Barcellos AG, Barros EM, Pereira EM, et al.
Methicillin-resistant Staphylococcus epidermidis
carrying biofilm formation genes: Detection of clinical isolates by multiplex PCR. Int Microbiol 2011;14:13-7.
Abimanyu N, Krishnan A, Murugesan S, Subramanian G K, Gurumurthy S, Krishnan P, et al.
Use of triplex PCR for rapid detection of PVL and differentiation of MRSA from methicillin resistant coagulase negative staphylococci. J Clin Diagn Res 2013;7:215-8.
Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphylococcus epidermidis
to smooth surfaces. Infect Immun 1982;37:318-26.
Vandecasteele SJ, Peetermans WE, R Merckx R, Rijnders BJ, Van Eldere J. Reliability of the ica, aap and atlE genes in the discrimination between invasive, colonizing and contaminant Staphylococcus epidermidis
isolates in the diagnosis of catheter-related infections. Clin Microbiol Infect 2003;9:114-9.
Rohde H, Burandt EC, Siemssen N, Frommelt L, Burdelski C, Wurster S, et al.
Polysaccharide intercellular adhesin or protein factors in biofilm accumulation of Staphylococcus epidermidis
and Staphylococcus aureus
isolated from prosthetic hip and knee joint infections. Biomaterials 2007;28:1711-20.
Rohde H, Kalitzky M, Kröger N, Scherpe S, Horstkotte MA, Knobloch JK, et al.
Detection of virulence-associated genes not useful for discriminating between invasive and commensal Staphylococcus epidermidis
strains from a bone marrow transplant unit. J Clin Microbiol 2004;42:5614-9.
[Figure 1], [Figure 2]