Prevalence of virulence determinants in Staphylococcus epidermidis from ICU patients in Kampala , Uganda

Introduction: Staphylococcus epidermidis is often considered a non-pathogenic organism but it causes nosocomial infections. To distinguish invasive strains, comparative studies of patient and community isolates may offer some clues. We investigated the distribution of virulence determinants in patient isolates from Uganda. Methodology: S. epidermidis isolates were identified with the Staph API ID 32 kit. Antimicrobial susceptibility, biofilm formation and hemolysis were detected with standard procedures. Genes associated with virulence (aap, atlE, bhp, hla, hld, ica, IS256, sdrE, sea, tsst) and antimicrobial resistance (aac(6')-Ie-aph(2'')-Ia, aph(3')-IIIa, ant(4')-Ia, blaZ, mecA, vanA/vanB1) were detected by PCR. Results: S. epidermidis grew in 30 (30/50, 60%) ICU samples and 20 (20/60, 33%) community samples (one isolate per sample per patient/person). All ICU isolates (30/30, 100%) were IS256 and hld positive, 22 (22/30, 73%) were biofilm/ica positive, 21 (21/30, 70%) were hemolytic on blood agar, nine (9/30, 30%) contained atlE gene, six (6/30, 20%) hla gene, five (5/30, 17%) aap gene, and three (3/30, 10%) bhp gene. A gene encoding an aminoglycoside-modifying enzyme, aph(3')-IIIa, was highly prevalent (28/30, 93%), while blaZ (2/30, 7%), mecA (3/30, 10%), vanA (3/30, 10%) and vanB1 (3/30, 10%) were less prevalent. Of the community isolates, one (1/20, 5%) was ica positive, two (2/20, 10%) formed biofilms, and three (3/20, 15%) possessed the atlE gene. bhp, aap, IS256, hld and antimicrobial resistance genes were not detected in community isolates. Conclusions: S. epidermidis from ICU patients in Mulago Hospital is potentially virulent and could be a reservoir for antimicrobial resistant genes.


Introduction
Staphylococcus epidermidis, a common normal flora, frequently causes infections in hospitalized patients with indwelling support devices [1,2].Unlike Staphylococcus aureus, S. epidermidis lacks obvious virulence determinants and is often regarded an accidental pathogen [3].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 [1,2,4,5].Studies aiming at distinguishing invasive from commensal strains are needed [3].
While there is limited data on the molecular epidemiology of S. epidermidis infections in sub-Saharan Africa, elsewhere many investigators elucidating the pathogenicity of the organism mainly focus on detection of biofilms and intercellular adhesion (ica) genes.The usefulness of these as virulence markers has been debated widely [6,7].Recently, it was demonstrated that the insertion sequence IS256 correlates highly with aminoglycoside resistance in S. epidermidis [1,2], and its superior to ica gene detection in distinguishing clinically relevant isolates [1].Additionally, more genes implicated in biofilm production (i.e., bifunctional autolysin E, atlE; accumulation-associated protein, aap; and biofilm associated protein, bhp) have been elucidated [2,3,8].While they were demonstrated as useful in the detection of clinical strains [2], their prevalence has not been widely investigated.Since patients in intensive care units (ICU) are easily colonized/infected with nosocomial pathogens, particularly those associated with the frequent use of support devices, we aimed to determine the prevalence of a collection of virulence and antimicrobial resistance determinants in S. epidermidis from ICU patients at Mulago Hospital in Kampala, Uganda.Biofilms as well as genes encoding the staphylococcal hemolysins (hla, hld), superantigenic toxins (tsst, sea), putative adhesin serine aspartate repeat protein (sdrE), and antimicrobial resistance genes (mecA, vanA/vanB1, blaZ and the aminoglycoside modifying enzymes, aac(6')-Ie-aph(2'')-Ia, aph(3')-IIIa and ant(4')-Ia), which frequently occur in invasive isolates, were studied.

Study setting and sampling
Approval was obtained from the institution review board of Mulago Hospital.Written informed consent was obtained from the participants.This cross-sectional study was conducted from December 2007 to June 2008, on 50 patients in the Mulago Hospital ICU and 60 healthy participants from Makerere University.Duplicate samples from ICU patients included catheter tips (24/50, 48%), blood (16/50, 32%), swabs (7/50, 14%) and aspirates (3/50, 6%, two pleural and one bronchial).Catheter tips were aseptically excised and transported to the laboratory in culture bottles with 10 ml tryptic soy broth.After incubating overnight at 37°C, samples were vortexed and aliquots streaked on blood agar, and incubated overnight at 37°C.For blood and bronchial samples, 2 ml each was injected into blood culture bottles and incubated at 37°C in an automated blood culture system (BACTEC 9120).Wounds (skin, ears and eyes) were sampled by aspirating pus with a sterile syringe or using cotton swabs with Amies transport medium and incubated at 37°C overnight on blood agar.Nasal swabs from randomly selected healthy subjects were similarly processed.After standard microbiological procedures, S. epidermidis was identified with the Staph API ID 32 system (Biomerieux, Lyon, France), and confirmed by PCR [9].Antimicrobial susceptibility testing (penicillin 10U, oxacillin 5µg, clindamycin 2µg, erythromycin 15µg, tetracycline 30µg, ciprofloxacin 5µg, trimethoprim/sulfamethoxazole 1.25/23.75µg,chloramphenicol 30µg, rifampicin 5µg, gentamicin 10µg and vancomycin 30µg) was performed with the disc diffusion method following standard guidelines [10].

Detection of virulence determinants
Biofilms and hemolysis: Biofilms were detected with the microtiter plate method [11] and the biofilm unit calculated according to Amaral et al. [12].Briefly, assays were performed in triplicate in TSB/1% glucose in 96-well polystyrene flat-bottom tissue culture plates.Isolates were incubated at 37ºC overnight with gentle shaking and standardized to OD 600 = 0.005 with normal saline.Then 50 µl of standardized cells mixed with 150 µl TSB/1% glucose were incubated at 37ºC for 17 hours.After washing three times with sterile water and staining with crystal violet for 15 minutes, cells were washed again with sterile water and incubated at room temperature for one hour in 95% ethanol, and the biofilms were measured with a spectrophotometer at OD 570 .The biofilm forming S. epidermidis RP62A and its non-biofilm forming variant (ATCC 12228) were used as controls.Hemolysis was determined on blood agar plates supplemented with 5% sheep blood.
Virulence and antimicrobial resistance genes: Since virulence and antimicrobial resistance tend to co-exist in invasive strains [1], molecular assays to detect genes encoding/associated with these phenotypes were performed.DNA was extracted with the MasterPure purification kit (Epicenter, Madison, USA).ica, IS256, hla, hld, tsst and sea genes, which frequently occur in invasive strains, as well as bhp, aap and atlE, were detected by PCR.Each PCR sample contained 20 pmoles each of forward (fwd) and reverse (rev) primers, 1.5U Taq polymerase (Thermo Scientific, Surry, UK), Custom PCR-Master Mix (Thermo Scientific, Surry, UK), template DNA and nuclease-free water, in 10µl reaction volume.Details of primer sequences are shown in Table 1.Positive (S. epidermidis DNA template) and Negative controls (water, none-S.epidermidis DNA template) were always included in the reactions depending on the amplification target.icaSe1 and icaSe2 primers amplified a 639bp product containing icaA, icaD and icaB genes when amplified under the following conditions: 94°C, 5 minutes; (94 o C, 1 minute, 60 o C, 1 minute and 72°C, 1 minute) x 30 cycles; 72°C, 10 minutes.To detect genes encoding aminoglycosidemodifying enzymes (AME) [3[3], PCR of aac( 6

Discussion
In this study, we report a high prevalence of virulence/antimicrobial resistance determinants in S. epidermidis from the Mulago Hospital ICU.Since catheter-related staphylococcal infections are common in this setting [21], ICU patients could be at risk of infection with intractable pathogens.
Genes involved in biofilm production (a major virulence determinant relevant for colonization of surfaces/biomaterials) have been suggested as potential markers for clinically relevant strains [11].Consequently, the majority of the ICU isolates were biofilm/ica positive, in agreement with previous reports [1,2]; however, the usefulness of ica and biofilms is curtailed by their concomitant presence in commensal isolates [7].Furthermore, all ICU isolates were IS256 positive while 73% were biofilm/ica/IS256 positive, supporting the recent association of these factors with clinically relevant strains [1].Nine ICU isolates were biofilm/atlE/ica positive and five were biofilm/aap/atlE/ica positive.The biofilm unit (BU) was calculated using negative control values with the formula A1/A2, where A1 is the test value while A2 is the negative control value.Isolates with BU > 2x the negative control value were considered biofilm producers and were classified as follows: weak, 0.182 < BU < 0.364; moderate, 0.364 < BU < 0.728; strong, BU > 0.728 [12].Isolates M1 to M30 were from the ICU, while M31 to M50 were from the community.de Araujo et al. reported a concomitant presence of ica, atlE and aap genes as being strongly associated with biofilms [7].Two of the three bhp positive ICU isolates were concomitantly positive for ica, aap and atlE genes, while one was aap/atlE negative but biofilm/ica positive.Three community isolates were atlE positive, of which one was biofilm/atlE/ica positive and another biofilm/atlE positive.Although previously detected at high prevalence in commensal strains [7], atlE and aap genes were less common in community isolates.Biofilms are formed in two steps: an initial adherence of bacteria to inert surfaces (involving the AtlE protein [7]) and biofilm accumulation.In the second phase, bacteria connected to the polymer surface produce and accumulate the biofilm, which is thought to be the main mechanism for bacterial adherence to plastic surfaces and of auto-aggregation.In S. epidermidis, the ica operon encodes enzymes for the biosynthesis of polysaccharide intercellular adhesin (PIA), which, together with an additional protein, AAP, appear necessary for biofilm accumulation [7].Furthermore, an alternative pathway involving BAP protein is responsible for biofilm production in ica-negative isolates [8].Although BAP occurs in animal S. aureus, a BAP homolog, BHP, exists in human S. epidermidis and can induce biofilms in absence of PIA [8].
gene.Six isolates exhibiting near complete hemolysis on blood agar concomitantly contained hld and hla, while one contained the staphylococcal enterotoxin a (sea), hla and hld genes.The S. aureus hld is similar to that of S. epidermidis; it is thermostable, damages membranes of mammalian cells, and possibly causes severe enteritis [22].The hla gene encodes a dermanecrotic, neurotoxic toxin that is also responsible for abscess formation.Although prevalent in ICU isolates, the enterotoxigenicity of coagulase negative staphylococci is still debatable.Nevertheless, expression of toxin genes was demonstrated in S. epidermidis [22].While S. epidermidis is considered a reservoir of antimicrobial resistance genes for S. aureus, the presence of homologues of S. aureus toxin genes in S. epidermidis may contribute to a repertoire of virulence determinants yet to be elucidated [22].
All the three AME encoding genes [20] were detected in ICU isolates with the most prevalent being aph(3')-IIIa (only two isolates tested negative), while aac(6')-Ie-aph(2'')-Ia and ant(4')-Ia were less prevalent.AME are highly associated with the IS256 element, a component of Tn4001 that mediates gentamicin resistance by the product of the aac(6')-Ie-aph(2'')-Ia gene [1].Arciola et al. reported full association between the presence of IS256 and resistance to gentamicin [1].While aac(6')-Ieaph(2'')-Ia was detected in all IS256-positive isolates in a previous study [2], it was detected in only 27% of the ICU isolates.In staphylococci, aminogylcoside resistance highly correlates with methicillin resistance, due to genetic linkage between resistance determinants [20].However, in this study, mecA was detected in only three ICU isolates, contrasting with the high prevalence of AME.Probably we did not succeed in detecting mecA in the majority of the isolates.
In conclusion, S. epidermidis from the Mulago Hospital ICU is potentially virulent and could be a reservoir of antimicrobial resistance genes.This sub-Saharan African study supports recent reports from industrialized settings that virulence/antimicrobial resistant determinants are co-present in clinical S. epidermidis, and confer selective advantage for colonization/survival in hospital settings [1].Conclusive comparison requires similar sample types but this was not possible due to difficulty in obtaining consent; furthermore, such a comparison assumes that control subjects are healthy.Due to financial constraints, robust tools such as pulse field gel electrophoresis or multilocus sequence typing, which determine isolate relatedness, were not utilized.We hope future studies will take these omissions into consideration.

Figure 1 .
Figure 1.Prevalence of virulence/antimicrobial resistance determinants in S. epidermidis