Bacterial insertion sequence IS256 as a potential molecular marker to discriminate invasive strains from commensal strains of Staphylococcus epidermidis

Bacterial insertion sequence IS256 as a potentialmolecular marker to discriminate invasive strainsfrom commensal strains of Staphylococcusepidermidis

J. Gua, H. Lia, M. Lib, C. Vuongc, M. Ottoc, Y. Wena, Q. Gaoa,*

aKey Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai200032, People’s Republic of ChinabCentre of Laboratory Medicine, HuaShan Hospital, Fudan University, Shanghai 200040, People’s Republicof ChinacRocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes ofHealth, Hamilton, MT 59840, USA

Received 28 June 2004; accepted 11 April 2005

01do

54

KEYWORDSStaphylococcus epi-dermidis; Molecularmarker; IS256; Poly-saccharide intercellu-lar adhesin

95-6701/$ - see front matter Q 2005i:10.1016/j.jhin.2005.04.017

* Corresponding author. Tel.: C86 2237971.E-mail address: [email protected]

Summary The skin commensal Staphylococcus epidermidis has becomeone of the most important causative agents of nosocomial infectionsassociated with medical devices. Differentiation between invasiveS. epidermidis and its commensal counterpart is crucial for clinical decisionmaking. The ica gene locus, which codes for production of polysaccharideintercellular adhesion (PIA), represents a frequently suggested molecularmarker for infectivity. Our data demonstrated that production of PIA wasnot significantly increased among clinical strains, which may explain thecontroversial results obtained previously on the correlation of ica presencewith origin from infection. Therefore, in this study, we attempted toidentify novel genes discriminating between invasive and commensal strainsbased on the comparison of genome sequences. Our results indicated thatthe bacterial insertion sequence element IS256 occurred significantly morefrequently in strains of clinical origin. Importantly, IS256 might thusconstitute a molecular marker to discriminate invasive strains fromcommensal strains of S. epidermidis.Q 2005 The Hospital Infection Society. Published by Elsevier Ltd. All rightsreserved.

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Journal of Hospital Infection (2005) 61, 342–348

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IS256 as a potential molecular marker 343

Introduction

Staphylococcus epidermidis has become one of themost important pathogens of nosocomial infectionsassociated with catheters and other indwellingmedical devices in recent years. However, sinceS. epidermidis is also a natural part of the flora onhuman skin and mucosal surfaces, it is usuallydifficult to differentiate whether an isolate rep-resents the cause of an infection or a coincidentcontamination of the sample. Rapid identificationof invasive strains that are responsible for staphy-lococcal infections is crucial and beneficial toclinical decision making.

Previous studies have established that biofilmformation is the most important pathogenic factorduring S. epidermidis infection.1–4 Therefore, thebiofilm phenotype was used as a marker todifferentiate pathogenic strains from normalinhabitants. Ziebuhr et al. found that 87% of clinicalstrains could form a biofilm on tissue culture plates,while only 11% of strains from the skin showed thisability.5 Galdbart et al. showed that biofilmformation was useful in the detection of clinicalpathogenic strains.6 However, according to manyother reports, the use of biofilm formation as amarker for pathogenicity is controversial as: (i) theassay for biofilm formation needs to be standar-dized,7 (ii) biofilm formation can be influenced byenvironmental factors,8,9 and (iii) the biofilmphenotype is subject to phase variation.5,10 Thus,biofilm formation does not appear to be an accurateand stable marker of S. epidermidis infections.

Many researchers have studied whether genesthat influence biofilm formation can be used asmolecular markers of S. epidermidis infections,such as ica, atlE, fbe, app, agr, sar and mec.6,11,12

Specifically, the ica gene locus has been investi-gated extensively. The ica operon codes for theproduction of an exopolysaccharide named poly-saccharide intercellular adhesin (PIA), which isessential for biofilm formation, immune evasionand virulence in biofilm-associated infection.5,13,14

In different studies, the ica operon was found to bepresent in 43–88% of clinical strains, whereas itoccurred in only 0–38% of strains from healthyindividuals.5,6,11,12,15,16 Therefore, ica has beensuggested as a means to discriminate invasivefrom commensal strains of S. epidermidis. How-ever, similar to biofilm formation as a phenotypicmarker, there are two problems associated with icaas a genetic marker. First, expression of ica isregulated by other genes and its function inpathogenesis is greatly influenced by environmentalfactors.17,18 Second, previous studies revealed that

the applicability of using the ica operon as a markervaried.5,6,11,12,15 As a consequence, there is aserious lack of usable markers to differentiatebetween invasive strains of S. epidermidis andtheir commensal counterparts.

The purpose of this study was to: (i) evaluate theuse of ica as a marker by ica expression studies, and(ii) seek new genetic molecular markers forinfectivity of S. epidermidis. Based on our results,we proposed the bacterial insertion sequenceelement IS256 as a novel molecular marker todifferentiate invasive and commensal strains ofS. epidermidis.

Materials and methods

Bacterial strains

In total, 117 S. epidermidis isolates were collectedfor investigation. The first group of 40 isolates wascollected from clinical samples of HuaShan Hospital(Shanghai, China): 14 recovered from the distal endsurface of the catheters of patients with inflamma-tory response focus on the area near the catheter,14 from venous blood cultures of patients with asystemic inflammatory response syndrome (bacter-aemia and sepsis), and 12 from the middle urineculture of patients with urinary tract infections.These isolates were considered to be invasivestrains. The second group contained 55 isolatesthat were isolated from the skin of students fromFudan University (Shanghai, China), who had nocontact with the hospital over a six-monthperiod. These isolates were considered to becommensal strains. The third group contained 22isolates that were isolated from the skin of themedical staff of HuaShan Hospital. These isolateswere considered to be medical staff strains. Allisolates were characterized by classic microbiolo-gical methods: Gram’s stain, catalase and coagu-lase activity on rabbit plasma (bioMerieux, Lyon,France). S. epidermidis strains were further ident-ified by biochemical characterization using theApi-Staph test (bioMerieux, Lyon, France).

Genomic DNA extraction

DNA extraction from S. epidermidis was performedas described previously with modifications.19

Briefly, overnight cultures were pelleted by cen-trifugation. The collected cells were then resus-pended in 100 mL of 0.01 M sodium phosphate buffercontaining 20% sucrose and 2.5 mg/mL lysozyme.After incubation at 37 8C for 45 min, 500 mL of lysis

J. Gu et al.344

buffer (10 mM Tris–HCl pH 8.0, 1 mM EDTA, 1% SDS)and 5 mL of 20 mg/mL proteinase K solution wereadded and incubation proceeded for a further30 min. Samples were then extracted by phenol,phenol:chloroform (1:1) and chloroform, respect-ively. The DNA was precipitated by ethanol and re-suspended in distilled water.

Polymerase chain reaction (PCR)amplification

In a previous study, several genes that have beenreported in RP62A, a biofilm-positive strain, but notfound in the genome of ATCC12228, a biofilm-negative strain, were identified.20 The respectivegene sequences were downloaded and amplificationprimers were designed for them. The 16S rRNA genewas used as an internal control. Nucleotidesequences of primers and PCR conditions arepresented in Table I. PCR reactions were performedusing PCR Mix (Tianwei Company, Beijing, China) onGeneAmp-9700 (Applied Biosystems, USA). PCRproducts were analysed by agarose gel electrophor-esis, and some were selected at random andconfirmed by sequencing (BioAsia Company, Shang-hai, China).

Repetitive element sequence-based PCR(rep-PCR)

Primer RW3A (5 0-TCGCTCAAAACAACGACACC-3 0)used for rep-PCR was designed previously by DelVecchio et al.21 Rep-PCR was performed using PCRMix (Tianwei Company, Beijing, China) and Gen-eAmp-9700 (Applied Biosystems, USA). The proto-col for PCR was as follows: preheating for 3 min at94 8C, followed by 35 cycles of 1 min at 94 8C, 1 minat 54 8C and 2 min at 72 8C, with a final extension at

Table I Primers and amplification conditions for polymera

Target genesand sequences

Primers DNA sequence (5 0–3 0)

bhp bhp-P5 TGGTATTAGGAAGCTCTCbhp-P3 ATACCAGCGTGACGCAAA

kdp kdp-P5 TGGTAGCCGATTCTAGGAkdp-P3 GGGATTAGGGCTCTATTT

IS257 IS257-P5 CTATCTAAGATATGCATTIS257-P3 TTAACTTGCTAGCATGAT

IS256 IS256-P5 AGTCCTTTTACGGTACAAIS256-P3 TGTGCGCATCAGAAATAA

icaA icaA-P5 AAGATGTTGGCTGTGATTicaA-P3 CAACAAGTTGAAGGCATA

16s rRNA 16s rRNA-P5 CGGCGTGCCTAATACATG16s rRNA-P3 CCAGAAAGTCGCCTTCGC

72 8C for 5 min. Amplified products were analysedby 1.5% agarose gel electrophoresis.

Immuno dot blot assay

To quantify PIA production, PIA was extracted byincubating cells grown for 18 h in tryptic soy brothin 0.5 M EDTA, pH 8.0 (final volume: 1:50 ofcultures) for 5 min at 100 8C. Three-microlitrealiquots of the samples were spotted on anitrocellulose membrane, air-dried and PIA wasdetected using anti-PIA antiserum as describedpreviously22 using a scanner and Total Lab Version2003 software (Nonlinear USA, Durham, NC, USA).

Results

Presence of ica but not ica expression iscorrelated with origin from infection

In this study, we first wanted to address thequestion of contradictory results in the literatureabout the applicability of ica as a genetic marker forS. epidermidis infection. To this end, a Frenchstrain collection that has previously been reportedto show a correlation between the presence of icaand infectivity6 was analysed for ica expression.Clinical strains were from the infected jointprostheses and control strains were from the skinof healthy individuals. In addition, we used our ownstrain collection to determine the distribution of icaand further putative genetic markers amongcommensal and clinical strains and among strainsisolated from hospital staff. To exclude thepossibility of contamination during strain collectionand the possibility that some strains may come fromthe outbreak of one strain, we employed an

se chain reaction (PCR) reactions

Product size(bp)

PCR conditions

AG 935 30 s 94 8C, 45 s 52 8CTC 70 s 72 8C, 25 cyclesTG 813 30 s 94 8C, 45 s 50 8CAG 60 s 72 8C, 25 cycles

GAG 576 30 s 94 8C, 30 s 51 8CGC 45 s 72 8C, 25 cyclesTG 762 30 s 94 8C, 30 s 52 8CCG 60 s 72 8C, 25 cyclesAC 832 30 s 94 8C, 30 s 49 8CTC 60 s 72 8C, 25 cyclesCA 690 30 s 94 8C, 30 s 55 8CCA 50 s 72 8C, 25 cycles

Figure 1 Representative repetitive element sequence-based polymerase chain reaction fingerprints of Staphylo-coccus epidermidis strains. Lane 1, 1 kb plus DNA ladder; Lane 2, negative control; Lanes 3–13, S. epidermidis isolates.

IS256 as a potential molecular marker 345

extensively used typing method, rep-PCR, whichdifferentiates microbes of different origins. Theresults showed that the strains in our collection haddifferent rep-PCR fingerprint patterns and thuswere essentially non-clonal (Figure 1). First, wedetermined the distribution of icaA in our samplesby analytical PCR. icaA was present in 60% (24/40)of the invasive strains, in 27% (6/22) of the strainsfrom medical staff, and in 13% (7/55) of thecommensal strains (Table II). Statistical analysisusing Chi-square test indicated that the frequencyof ica among commensal strains or strains from themedical staff group was significantly lower thanamong invasive strains, while there was no statisti-cally significant difference between the commensaland medical staff groups (Table III). In contrast,detection of ica expression by measuring PIAproduction in the French strain collection showedno significant correlation between PIA productionand origin of infection (Figure 2). Thus, our dataconfirmed a correlation between the presence ofica genes and infectivity. However, the lack ofcorrelation with PIA production might explain theproblems encountered with the use of ica as amolecular marker for infectious S. epidermidisstrains. These results prompted us to seek novelmolecular markers for infectivity of S. epidermidis.

Table II Results of IS256 and icaA gene amplificationgrouped by the origins of the isolates

Origins of strains No. ofstrains

% Positive for

IS256 icaA IS256andicaA

Invasive catheter 14 12 (86) 10 (71) 9 (64)Blood 14 13 (93) 10 (71) 10 (71)Urine 12 9 (75) 4 (33) 4 (33)Total 40 34 (85) 24 (60) 23 (58)

Medical staff skin 22 9(41) 6 (27) 5 (23)Commensal skin 55 9 (16) 7 (13) 0 (0)

IS256 represents a marker for S. epidermidisinfectivity

Based on previous work and sequence blast searchwith the TIGR (The Institute for GenomicResearch) database,20,23 two genes coding forbap-like protein (bhp) and osmolarity resistanceprotein (kdp), as well as two staphylococcalinsertion sequences (IS256 and IS257) which werepresent in the biofilm-former RP62A but absentfrom the biofilm-negative ATCC12228, were ident-ified. In order to evaluate the distribution of these

genes in clinical isolates of S. epidermidis,specific primers were designed for the fourselected genes and their distribution amongclinical isolates was determined (Table I). Seveninvasive strains and five commensal strains wereused in the preliminary screening using analyticalPCR. bhp was present in two of the seven invasivestrains and two of the five commensal strains. NoPCR products were obtained for kdp in either ofthe two groups (data not shown). All of the 12except one commensal strain were positive forIS257. IS256 was found in six of the seven invasivestrains and one of the five commensal strains.These results prompted us further to investigateIS256 as a genetic marker for virulent S.epidermidis. To this end, 117 S. epidermidisstrains, i.e. 40 invasive strains, 55 commensalstrains and 22 strains isolated from medical staff,were analysed for the presence of IS256. IS256was found in 85% (34/40) of the invasive strains,41% (9/22) of the strains from medical staff, and16% (9/55) of the commensal strains (Table II).Statistical analysis with Chi-square test showedthat the distribution of IS256 was significantlydifferent between the three groups (Table III).

Table III Comparison of the prevalence of IS256 and icaA genes in invasive, medical staff and commensal isolates

Type of isolates Genes RR 95% CI P value

Invasive vs medical staff IS256 2.08 1.24–3.49 0.001icaA 2.2 1.06–4.56 0.018

Invasive vs commensal IS256 5.19 2.82–9.57 !0.001icaA 4.71 2.26–9.85 !0.001

Medical staff vs commensal IS256 2.5 1.15–5.46 0.035icaA 2.14 0.81–5.66 0.177

RR, relative risk of finding the gene in the specified groups. The 95% CI gives the 95% confidence interval of the relative risk. The Pvalue gives the significance of the differences in prevalence of the specified gene between the studied groups.

J. Gu et al.346

To verify the applicability of IS256 as a molecularmarker for S. epidermidis infections in clinicalpractice, we compared the sensitivity and speci-ficity (sensitivity: fraction of those invasive strainsidentified as positive by the assay; specificity:fraction of those commensal strains identified asnegative by the assay) of IS256 with those of ica. Wefound that the sensitivity and specificity of IS256were 85% (34/40) and 84% (46/55), while those forica were 60% (24/40) and 87% (48/55). According toChi-square test, the difference in sensitivity ratherthan specificity between IS256 and ica was statisti-cally significant (P!0.01). The results indicatedthat the sensitivity of IS256 is better than that of icaas a molecular marker. Interestingly, when bothIS256 and ica were positive, the sensitivity ofdetecting invasive strains decreased slightly(23/40, 58%); when both were negative,

Figure 2 Polysaccharide intercellular adhesin (PIA)production in strains of clinical origin and control strains.PIA samples were isolated from the surface of cells grownto stationary growth phase by boiling with 0.5 M EDTA.PIA production was determined by immuno dot blot usinganti-PIA antisera. The results of photodigital analysis ofsamples from clinical and commensal strains are depictedas a Box and Whiskers plot. The box extends from the 25thpercentile to the 75th percentile, with a line at themedian (the 50th percentile). The difference betweenthe two groups was not statistically significant.

the specificity of detecting commensal strainsincreased dramatically (55/55, 100%).

Discussion

In recent years, S. epidermidis has become one ofthe most important pathogens involved in nosoco-mial infections. To differentiate strains that causeinfections from commensal strains has become adaily challenge for both clinical laboratories andclinicians. In previous studies, biofilm formationand several specific genes have been evaluated aspotential genetic markers for the invasiveness ofS. epidermidis. A special focus has been on the useof the ica gene locus. We demonstrated here that ina strain collection for which correlation of thepresence of ica with infectivity had been demon-strated previously, no correlation was found whenanalysing expression of the ica product PIA. Theseresults might serve as an explanation for thecontroversial results achieved with ica andprompted us to search for different markers. Weevaluated several genes that are present in theinvasive strain RP62A but not in the commensalstrain ATCC12228 as molecular markers to discrimi-nate invasive and commensal strains of S. epider-midis. We identified the bacterial insertionsequence IS256 as a potential molecular marker.The distribution of IS256 among clinical andcommensal strains was significantly different,which is in accordance with results recentlypublished by Kozitskaya et al.24 Importantly, thesensitivity and specificity of IS256 were superior tothose of the ica operon, which has been regardedpreviously as the best marker for invasiveness ofS. epidermidis.6,15 Furthermore, when combiningIS256 and ica as genetic markers, a very highdiscrimination power was achieved.

Why does IS256 represent a molecular marker todiscriminate between invasive and commensalstrains of S. epidermidis? In general, pointmutation, homologous recombination, horizontal

IS256 as a potential molecular marker 347

gene transfer and mobile DNA elements play majorroles in generating genetic and phenotypic vari-ations. IS256 is a common insertion sequence inGram-positive cocci. It was first detected as theflanking region of the aminoglycoside resistance-mediating transposon Tn4001. It was also found tobe independently present in multiple copies in thegenomes of aminoglycoside-resistant staphylococciand enterococci.25,26 Insertion and excision of IS256in the ica operon account for about 30% of phasevariation events with regard to biofilm formation,and are generally associated with more rapidphenotypic variation of S. epidermidis.10,27 In theinvasive strain S. epidermidis RP62A, there are fivecopies of IS256, two of which are parts of thecomposite transposon Tn4001, and three of whichare independent copies.

We hypothesize that the presence of multiplecopies of IS256 renders the S. epidermidis genomeflexible to adapt to varying environments. Theinsertion of IS256 may affect the expression ofcertain genes that are associated with pathogen-esis. Thus, genomic re-arrangement caused byinsertion and excision of IS256 may increase thefitness of S. epidermidis in the hospital setting.Accordingly, it was found that the frequency ofIS256 in strains from medical staff was significantlydifferent from that in commensal strains. Indeed,previous results have shown that IS256 in theinvasive strains was significantly associated withthe antibiotic resistance phenotype.24 In our study,a number of selective invasive strains also exhibitedthe multi-resistance phenotype (data not shown).All of these results suggest that invasive strains ofS. epidermidis exist specifically in the hospital andmight reflect the ability of its genome to adapt tothe environment.

In our study, the distribution of IS256 amongclinical and commensal strains was significantlydifferent, which is in accordance with resultspublished recently by Kozitskaya et al.24 However,in the data of Arciola et al.,16 the occurrence ofIS256 was only 40%, which may be due to therepresentativeness of the strains. Therefore,additional samples would be required to confirmthis result.

In conclusion, this study demonstrates thateither IS256 alone or IS256 and the ica operontogether can be used as molecular markers todiscriminate invasive strains from commensalstrains of S. epidermidis. However, little is knownabout the molecular mechanisms associated withthe preferential occurrence of IS256 in invasivestrains, which merits further investigation. Fur-thermore, since IS256 can also be found in otherspecies of coagulase-negative staphylococci,

further work is needed to determine if its associ-ation with virulence can be applied in suchorganisms.

Acknowledgements

This work was supported by the Chinese NationalNatural Science Foundation Grant (30371267), 211Project Grant-Functional Genomics of ImportantPathogenic Micro-organisms.

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