ENVIRONMENTAL AND GLOVES’ CONTAMINATION BY STA- PHYLOCOCCI IN DENTAL HEALTHCARE SETTINGS

Introduction. Each year, 37,000 people in Europe die as direct consequence of healthcare-associated infections. Staphylococcus aureus (especially methicillin-resistant -MRSA) and coagulase-negative staphylococci (CNS) are frequently isolated in these episodes. Environmental contamination by S. aureus, MRSA and CNS in dental healthcare settings is reported moderately frequently, although the associated risk for infection is


Introduction
Healthcare-associated infections (HAI) oc cur after exposure to healthcare.Each year, 4,000,000 people in Europe acquire HAIs and 37,000 of them die as the direct consequence of infection.In addition, HAIs may also inter est healthcare workers and administrative staff.The most frequent HAI types are urinary tract infections, pneumonia, surgical site infections, bloodstream infections and gastrointestinal in fections, while the most frequently isolated mi croorganisms in HAI overall are Escherichia coli, Staphylococcus aureus, Pseudomonas ae ruginosa, Enterococcus species, coagulase-neg ative staphylococci and Candida species 1 .HAIs are generally treated through antibiotic and/or surgical therapy.Therefore, HAI treatment and prevention is an important cause of emergence of highly antibiotic resistant strains, another se rious public health problem 2 .
In the specific field of dental healthcare set tings, environmental contamination is reported for methicillinresistant and methicillinsensi tive S. aureus (MRSA and MSSA), although the risk for infection associated with contami nation is not clear 35 .One problem with MRSA in dentistry is that these microorganisms may survive up to six months on clinical contact sur faces in the dental environment 6 .P. aeruginosa and Legionella pneumophila, microorganisms responsible for HAIs, also are frequently de tected in dental unit waterlines, along with oral streptococci, biological markers of bloodborne/ airborne pathogens 79 .The study of environ mental contamination and consequent infection transmission in dental healthcare settings is, therefore, essential to produce evidence-based guidelines 1012 .

Aim
The aim of the present study was to investi gate the contamination level of the environment of dental offices and of the hands of the dental staff by several types of staphylococci.

Material and Methods
One hundred thirty-six General Dental Practitioners (GDPs) working in private of fices in Rome, Italy, were considered.Details regarding their recruitment were previously described 13 ; GDPs were invited to participate before registering to Continuing Medical Edu cation courses.Participation was on a voluntary basis and there were no incentives.Data protec tion and anonymity were guaranteed.The study protocol was approved by the Review Board of the Medical and Dental Association of Rome.
Samples were collected in the midmorning/ midafternoon.GDPs must have treated at least two patients before the sampling occasion.En vironmental samples were collected from the tray in front of the patient, a clinical contact surface 10 , immediately after patient treatment and before surface cleaning, using disposable swabs previously imbibed in sterile water and passed on a 10x10 cm area of the tray.ATP bio luminescence (Lumicontrol II.PBI Internation al, Milan, Italy) was used to assess the overall contamination level, qualitatively.ATP biolu minescence is a rapid method to detect the total viable flora, with a reasonably good accuracy of 114% at levels around 100 Relative Light Units (RLUs), roughly corresponding to 2.5 colony forming units (CFU)/cm2.100 RLUs is gener ally considered the benchmark for high level of environmental cleanliness in hospitals 14,15 .
Samples which yielded RLU levels >100 were monitored using Replicate Organism De tection and Counting (Rodac) plates (Becton Dickinson Italia, Buccinasco, Italy) containing Mannitol Salt Agar (MSA -Becton Dickinson Italia), for the enumeration of staphylococci.Plates were pressed on the surface of the tray different from the area sampled with the swab for 30 s at an approximate pressure of 20-25 g/ cm 2 .Rodac plates were preferred to swab be cause they provide repeatable results and pro vide comparable levels of recovery for general bacterial contamination of a surface 16 Plates were aerobically incubated at 37°C for 48 h.Colonies grown on MSA were Gram's stained, tested for coagulase and catalase and presump tively identified using VITEK-2 "Gram-Posi tive Identification" and "Antibiotic Susceptibil ity Testing" cards (BioMérieux, Italia; Bagno a Ripoli, Italy).Further biochemical identifica tion tests were not made, therefore, microor ganisms were presumptively classified as over all staphylococci, S. aureus, coagulase negative staphylococci (CNS) and S. epidermidis, a sub group of CNS.
Following the criteria of the US Centers for Disease Control, isolated staphylococci strains were classified as methicillin-resistant on the basis of oxacillin MIC test, which implied the presence of the staphylococcal cassette chro mosome mec (SCCmec).Namely, ≥4 μg/mL for S. aureus and ≥0.5 μg/mL for CNS (guide lines available at, http://www.cdc.gov/mrsa/lab/lab-detection.html).
Right hand samples among right-hand ed GDPs (from left hand among left-handed GDPs) were collected directly from the gloved hand of GDPs soon after patient treatment and before glove removal using four Rodac plates containing the aforementioned media and fol lowing the same laboratory procedures used for the environmental samples.
Staphylococci prevalence estimates on clinical contact surfaces and on GDPs' gloved dominant hands were assessed.The association between clinical contact surface and gloved hand contamination by the various Staphylo coccus species also was assessed using the nonparametric Spearman correlation test, giving score 1 to positive samples and score 0 to nega tive samples.
GDPs were informed in the event that samples were positive for methicillin-resistant staphylococci.Environmental samples and samples from GDPs' nares were collected using sterile swabs one week after the sampling oc casion to check whether additional procedures were necessary to eradicate these microorgan isms.
A good correlation was found between positive environmental samples and positive samples from gloves for overall staphylococci, S. aureus and CNS (Table 1; p<0.0001).As for methicillin-resistant staphylococci, all the MRSA (n=2) were isolated from the environ mental sample and on GDP's glove at the same time, while one of four CNS (S. epidermidis) was isolated from both samples (Table 2).In the three sampling occasions where methicillinresistant staphylococci were isolated from the tray and from the glove of the dominant hand at the same time, the two microorganisms exhib ited the same antibiotic profile, suggesting that the strain detected on the tray was the same as the strain detected from the glove (data not in Table ).
In the six dental offices where methicillinresistant staphylococci were detected, environ mental samples, collected one week following the first sampling occasion, provided negative results regarding the occurrence of methicillin resistant staphylococci.Samples from GDPs' nares also resulted negative.[<0.0-3.5]The two methicillin-resistant S. aureus isolates and two out of 3 methicillin-resistant CNS isolates (one S. epidermi dis) were isolated on the clinical contact surface and on the GDP's gloved hand at the same sampling occasions.

Discussion
The present study is one of the papers pre sented at the workshop "Advances in Infection Epidemiology and Control in Dental Healthcare Settings", Department of Public Health and In fectious Diseases, Sapienza University, Rome, Italy on February 9th, 2013 1723 .
MRSA infection transmission is a serious public health problem and a cause for concern among dental healthcare workers and patients 24 .While MRSA infection can be transmitted to patients and to healthcare workers particularly in certain settings, such as intensive care or sur gical units 25 , there are no ascertained cases of transmission to dental healthcare workers and MRSA carriage rates among GDPs are gener ally similar to or even lower than the general population, thus suggesting that the occupa tional risk of MRSA infection is probably mini mal 3 .The question regarding the risk of acquir ing MRSA infection among dental patients is more complex.Among special patients, such as special care patients, hospitalized patients, head and neck cancer patients and oral or max illofacial surgery patients, high MRSA carriage rates are reported, as well as frequent episodes of infection and colonization, other than envi ronmental contamination of clinics and units.These elements suggest that the risk of infec tion is high among these patients.The situation among the remaining dental patients is differ ent, as there are only two reported episodes of MRSA infection, one of them due to the lack of glove use by the dentist.Carriage rate among healthy adult dental patients also is low, while there was only one case of MRSA environmen tal detection in non-special and non-surgical dental departments or offices 3,4 .These elements collectively suggest that the risk for infection is generally low.
The present study showed that MRSA spread in dental healthcare settings is possible.Indeed, MRSA were detected in two out of the 136 sampling occasions collected soon after dental therapy and in these occasions both trays and gloves from the dominant hand resulted contaminated by these microorganisms at the same time.Although no further analysis was made to ascertain whether the strains detected on the gloves and those detected on the trays were the same, it is likely that it was so, because the strain detected from the glove exhibited the same antibiotic profile as the strain detected from the tray.This assumption, along with the reported high correlation between staphylococ ci, CNS, S. aureus and S. epidermidis detected in the environment and those detected on the gloves may suggest two possible hypotheses to explain MRSA spread in the dental offices and the consequent risk for infection.First hypothe sis: GDPs were MRSA carriers and picked these microorganisms from their skin/mucosae while they were wearing gloves, spreading them in the environment.Second hypothesis: the den tal patient under treatment was MRSA carrier and was touched by GDP's gloved dominant hands, which in turn, contaminated the tray in front of the patient or, alternatively, airborne MRSA from carrier patient contaminated the tray.In favour of the second hypothesis the fact that GDPs resulted free from MRSA one week after MRSA detection in their offices, there fore, these two GDPs could only be transient carriers.Dispersion of airborne staphylococci during dental therapy also is corroborated by studies reporting CNS prevalence of 10-35% in air samples from dental clinics and offices [26][27][28] a similar detection rate as that reported in the present study (Table 1).

Conclusion
In conclusion, environmental contamina tion by methicillinresistant and methicillin sensitive staphylococci during dental therapy is possible and may pose a risk for infection, particularly among immune-depressed patients and those with open lesions in mouth.How ever, it seems likely that routine glove change between patients and cleaning/disinfection of clinical contact surfaces could be sufficient to control such a risk for infection, but to be sure that these simple methods are applied, it is nec essary that the overall level of knowledge and awareness among dental healthcare workers is improved, perhaps through specific Continuing Medical Education courses, as issues regarding infection control are generally neglected among dental healthcare workers 29 . ).
CNS: coagulase negative staphylococci (the species S. epidermidis is a member of CNS)

Table 2 .
Prevalence (95% confidence interval -95CI-between square brackets) of methicillin-resistant staphylococci isolated from the 136 clinical contact surfaces and the GDPs' gloved hands.