Prevalence of and risk factors associated with the presence of Staphylococcus aureus in the chronic wounds of patients treated in primary health care settings in Brazil

Introduction: Wounds can be colonized by methicillin-resistant Staphylococcus aureus (MRSA). Methods: We evaluated the prevalence of S. aureus and MRSA in the wounds of patients treated at Basic Health Units in Brazil and identifi ed risk factors associated with their presence. Results: The prevalence rates of S. aureus and MRSA were 51.5% and 8.7%, respectively. There was a correlation between the presence of S. aureus in wounds and nostrils (p<0.01). A positive association was detected between S. aureus infection and previous benzylpenicillin use (p=0.02). No associations were observed for MRSA. Conclusions: Multidrug-resistant pathogens are present in primary healthcare settings in Brazil.

Wounds are a risk factor for colonization by pathogenic bacteria, including multidrug-resistant microorganisms and methicillin-resistant Staphylococcus aureus (MRSA) 1 .MRSA interferes with wound healing, increases the severity of the lesions, and increases the risk of other types of infection such as pneumonia or bacteremia.Moreover, infected lesions act as reservoirs and sources of infection of other individuals.
Most published studies of MRSA have been performed in the hospital setting; few have investigated MRSA in primary care facilities.The present study was conducted in the basic health units (BHU) of primary care facilities, where almost 80% of medical consultations take place.These BHU, coupled with secondary and tertiary care facilities, form the Brazilian healthcare system.Thus, it is crucial to investigate the profi le of pathogens such as S. aureus that infect the wounds of patients managed in this setting.Given the large number of individuals with infected wounds and the increasing dissemination of MRSA in hospitals and communities, this study aimed to establish the prevalence of and to identify related risk factors for methicillinsensitive Staphylococcus aureus (MSSA) and MRSA strains in the wounds of patients attended to at the BHU.This cross-sectional study included patients attending any of the 17 BHU in the City of Botucatu, São Paulo, Brazil, for wound care during the period 2010-2013.We included all patients with pre-existing and new wounds (colonized or infected) of any etiology, regardless of patient age.Patients who reported having been hospitalized or having undergone surgery or invasive procedures up to one year prior to sample collection, were excluded.
Deoxyribonucleic acid (DNA) was extracted using the Illustra Kit (GE Healthcare, USA) according the manufacturer's instructions.S. aureus isolates were classifi ed as MRSA based on the results of real-time polymerase chain reaction (PCR) assays for mecA and pvl in a 20-µL volume containing 10µL of Fast SYBR®Green Master Mix (Applied Biosystems, Belgium), 0.6µL of each primer (250nM), 4µL of template, and 4.8µL of MilliQ water.The following thermocycling reaction was performed in a StepOnePlus TM system (Applied Biosystems): 30-s pre-heating at 60°C, followed by 20 s at 95°C, 40 cycles of denaturation at 95°C for 1 s, and annealing at 60°C for 20 s.International reference strains, such as ATCC (American Type Culture Colletion) 33591 (mecA positive), ATCC 25923 (mecA negative), and ATCC 49775 (pvl positive), were included as controls.The primers have been described elsewhere 7 .A previously described multiplex PCR protocol 8 was used to characterize mec.The following controls were used for staphylococcal cassete chromosome mec (SCCmec) typing, the COL strain for type I, N315 for type IA, PER34 for type II, AN546 for type III, HU25 for type IIIA, MW2 for type IV, and JCSC for type V.
A structured questionnaire was used to obtain socioeconomic and demographic data.All information was obtained from patient records or from the patients themselves.Colonized and infected wounds were distinguished based on clinical signs and symptoms of infection.Wounds were classifi ed as infected if at least two of the following signs were observed: Fever; local erythema, edema, pain, or heat; and increased amount of purulent secretions 9 .
For the univariate analysis, we used EPI-INFO for Windows, v.7 (Centers for Disease Control and Prevention, USA).Nonparametric tests for proportions (χ 2 or Fisher's exact test, as appropriate) were performed for comparisons of categorical variables.Continuous variables were compared using Student's t-test or the Mann-Whitney U test.Multivariate analysis was performed using IBM SPSS Statistics, Version 20.0 (Armonk, NY: IBM Corp).The outcomes of interest were the presence of S. aureus or MRSA, independent of the sample collection site.Logistic regression models were built via backward stepwise variable selection.The criterion for including and retaining variables in the model was a p-value <0.05.The results are expressed as odds ratios (OR) with 95% confi dence intervals (CI).
In the multivariate analysis, we observed negative associations between the presence of S. aureus and age (OR, 0.94; 95% CI, 0.90-0.98;p <0.01), amoxicillin use (OR, 0.16; 95% CI, 0.04-0.60;p <0.01), and ciprofl oxacin use (OR, 0.28; 95% CI, 0.08-0.98;p = 0.04); a positive association was observed with benzylpenicillin use (OR, 3.81; 95% CI, 1.23-11.82;p = 0.02).No risk factors for MRSA were identifi ed in the multivariate analysis.Nasal swabs were collected from only 74 (43.3%) patients; S. aureus and MRSA were detected in 28.4% and 5.4% of these samples, respectively.S. aureus was isolated from the wounds and nasal cavities of 41 (55.4%) and 21 (28.3%) of these patients, respectively.Associations were found between nasal carriage of S. aureus and its presence in the wound (p <0.01) and between nasal carriage of MRSA and its presence in the wound (p <0.01).Associations between antimicrobial drug resistance and the presence of the pvl gene in MSSA and MRSA isolates from wounds, were investigated.Univariate analysis demonstrated positive associations between resistance to levofl oxacin (OR, 68; 95% CI, The results in this study highlight a poorly addressed issue: The presence of multidrug-resistant pathogens in primary care facilities.We observed prevalence rates of 51.5% for S. aureus and 8.7% for MRSA; resistance to macrolides, lincosamides, aminoglycosides, and quinolones in sensitive and resistant isolates; and a higher frequency of SCCmec type IV MRSA, followed by type II.Studies addressing the prevalence of MRSA in primary care units are sparse 10 .In Brazil, only two such studies have been conducted; both were performed by the same research group in the City of Goiãnia, Goiás.The fi rst was a microbiological and antimicrobial assessment of the microorganisms present in chronic leg ulcers; S. aureus was the major species identifi ed (65%), and 20.5% of the isolates were resistant to oxacillin and cefoxitin 11 .The second study demonstrated the presence of antimicrobial-resistant S. aureus and coagulase-negative staphylococci isolates from venous ulcers of 68 patients during the period 2009-2010 12 .In both studies, prevalence was calculated using the number of samples collected, which equaled the number of ulcers, rather than the study population, making it impossible for the reader to calculate the real prevalence of S. aureus and MRSA.Moreover, no risk factor analysis was performed.
The prevalence observed in our study was lower than that observed in hospital-based studies, such as the study by Forcade et al. 10 -performed in 10 primary care clinics in Texas, USA between 2001 and 2010 -in which MRSA was isolated from 73 (63%) of 119 patients with skin and soft tissue infections.Almeida et al. 1 investigated the prevalence of S. aureus and MRSA in patients in a small hospital in Northeastern Brazil; they reported that 20% and 6.4% of wounds were colonized by S. aureus and MRSA, respectively.The same study also described associations between wound colonization and nasal carriage, recent antibiotic use, and ward admission.Those data agree with our results, which revealed a positive association between nasal carriage of both S. aureus and MRSA and their respective presence in the wound.We also observed that in 31%  of patients, S. aureus isolates from the nasal and wound swabs had the same resistance profi le.
The MRSA isolates in this study carried type II [n = 6 (33%)] and type IV [n = 12 (67%)] chromosomal cassettes and showed high levels of resistance to the quinolones, macrolides, and lincosamides.However, in the present study, pvl was not detected in methicillin-resistant strains, only in methicillinsensitive strains, a fi nding consistent with the low prevalence of pvl in MRSA isolates in Brazil, which are mainly type IV.The fi ndings also demonstrate that sulfamethoxazole-trimethoprim remains a good treatment choice, as the MRSA isolates showed 100% susceptibility.Other community-based studies of patients in the same region have described the same characteristics in circulating clones 13 .
Age was independently associated with a reduced risk of S. aureus presence, with a 6% decrease in the risk of carrying S. aureus for every additional year of age.One possible explanation is that as age increases, patients are exposed to factors (not assessed in this study) that prevent S. aureus colonization; conversely, it may be attributed to increased ecological competition with other microorganisms.The use of amoxicillin (usually with concomitant use of clavulanate) and ciprofl oxacin is also protective against acquisition of S. aureus; fl uoroquinolone exposure is related to an increased risk of methicillin-resistant, not methicillinsensitive, isolates 14 .Prior benzylpenicillin use was a risk factor for S. aureus carriage; benzylpenicillin is one of the most commonly used drugs in primary care, especially for the treatment of chronic wound infection.Consistent with our results, Staphylococcus spp.worldwide currently show high resistance (≥80%) to penicillin G.After analyzing nearly 29,000 nasal swabs from healthy individuals from eight European countries, van Bijnen et al. 15 described an association between frequent penicillin prescription and high odds of nasal carriage of resistant S. aureus.We also observed a high frequency of penicillin use during our analysis of patient records.Penicillin results in Streptococcus death and S. aureus permanence.Some of the limitations of this study include the lack of identification of other putative pathogens; difficulty in differentiating infection from colonization in chronic wounds, an issue that remains controversial; failure to include certain characteristics, such as lesion diameter, in the questionnaire; and not collecting nasal samples from all study participants.However, ours is one of the fi rst studies on the prevalence of S. aureus and MRSA in patients with chronic wounds managed at various BHU in Brazil for a period of almost three years.Our results point to a high prevalence of S. aureus and the presence of MRSA isolates that are resistant to several classes of antimicrobials, suggesting the possible dissemination of these pathogens within the primary care services in the city studied.These fi ndings call attention to the circulation and potential reservoir of resistant strains in patients without the usual risk factors or exposures particular to the hospital setting.They also demonstrate the need for more research in to be conducted in these types of healthcare facilities to understand the prevalence and circulation dynamics of multidrug-resistant microorganisms, and the possible existence of healthcare-related infections.

TABLE 1 :
Univariate and multivariate analysis of risk factors for carriage of Staphylococcus aureus and MRSA in patients attending basic health units in Botucatu, Sao Paulo State, Brazil.
MRSA: methicillin-resistant Staphylococcus aureus; S.: Staphylococcus OR: odds ratio; CI: confi dence interval.*Data refer to monthly family income in minimum wages.**Regular consumption of alcoholic beverages, more than twice a week.*** Unit of measure: months.All data are expressed as n (%), except where specifi ed.Signifi cant results are highlighted in bold.

TABLE 2 :
Characterizati on of MRSA isolates from pati ents att ended to at the BHUs of the City of Botucatu, Sao Paulo State, Brazil.