Effectiveness of Epidermal Growth Factor Loaded Carboxymethylcellulose (EGF-CMC) Hydrogel in Biofilm Formation in Wounds of Diabetic Patients: A Randomized Clinical Trial.

Diabetic patients frequently develop wounds, which can be colonized by bacteria, mainly Staphylococcus aureus and Pseudomonas aeruginosa, with the ability to form biofilms. This study aimed to evaluate the colonization and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa in chronic wounds of diabetic patients treated with a bioactive dressing (EGF-CMC), which consisted of a 2% carboxymethylcellulose (CMC) hydrogel loaded with epidermal growth factor (EGF). This randomized clinical trial was conducted with 25 participants: 14 treated with EGF-CMC hydrogel and 11 treated with CMC hydrogel for 12 weeks. Participants with type 2 diabetes mellitus were selected. All had diabetic foot ulcers or chronic venous ulcers. Swab collections were performed on weeks 1, 6, and 12. The laboratory analyses included the identification of strains, microbial quantification, virulence gene investigation, and the evaluation of biofilm formation. In total, 13 S. aureus strains and 15 P. aeruginosa strains were isolated. There were no statistically significant differences regarding bacterial loads and virulence genes. However, EGF-CMC-hydrogel-treated wounds were colonized by strains with lower biofilm formation abilities. The probability of isolating biofilm-producing strains from CMC-hydrogel-treated wounds was 83% greater than the probability of isolating biofilm-producing strains from EGF-CMC-treated wounds.

Blood Cell Count and the Presence or Absence of Infection in Venous Ulcers Treated With Platelet-Rich Plasma.

INTRODUCTION: In general, chronic wounds are colonized by bacteria; however, when microorganisms start to multiply at higher levels, wounds can become infected, causing prolongation of the inflammatory phase and retardation of collagen synthesis and epithelialization. OBJECTIVE: The objective of this study was to evaluate the presence of infection in venous ulcers after 12 weeks of treatment with autologous platelet-rich plasma (PRP) and determine global white blood cell counts. MATERIALS AND METHODS: This case series study involved a sequential sample of 17 patients with venous ulcers treated with PRP for 12 weeks. Descriptive and inferential statistical analysis was performed using the McNemar test and χ² test. RESULTS: At baseline, 10 patients (58.8%) had wound infection. During the sixth week of treatment with PRP, only 3 patients (17.6%) continued to exhibit wound infection. After 12 weeks of PRP treatment, only 1 patient (5.9%) continued to exhibit wound infection. McNemar and χ² tests used to assess the presence of infection in the intervention group produced a P value of .0039 for a comparison of baseline and week 6 and a P value of .0078 for a comparison of baseline and week 12. These results demonstrated significant differences from baseline at both 6 weeks and 12 weeks of treatment, with greater significance at 12 weeks. There was no relationship between global white blood cell count and the presence of infection. CONCLUSION: After intervention with PRP, 94% of patients experienced improvement concerning the infection of ulcers.

Clinical and Microbiological Outcomes Associated With Use of Platelet-Rich Plasma in Chronic Venous Leg Uclers: A Randomized Controlled Trial.

PURPOSE: To evaluate the susceptibility profiles of Staphylococcus aureus and Pseudomonas aeruginosa strains identified in chronic venous ulcers treated with platelet-rich plasma (PRP) and petrolatum gauze or petrolatum gauze alone and to quantitatively evaluate the bacterial load and biofilm-forming capacities of the detected S. aureus and P. aeruginosa strains. DESIGN: Randomized controlled trial. SUBJECTS AND SETTING: The convenience sample included 36 participants; 18 were allocated to the PRP combined with the petrolatum gauze group, and 18 were allocated to the control group, which was treated with petrolatum gauze alone. METHODS: Thirty-six patients presenting with chronic venous ulcers were consecutively randomized to the PRP group (n = 18) or the petrolatum gauze control group (n = 18). We followed participants for 3 months during treatment and collected swab cultures from their wounds during weeks 1, 6, and 12 or until the wounds healed. The samples were analyzed using mass spectrometry. Antimicrobial susceptibility tests were performed using disk diffusion. RESULTS: P. aeruginosa was identified in 39 (39%) of 100 samples, and S. aureus was detected in only 10 (10%) samples collected over the study period. At the end of the 12-week treatment period, the wound infections reduced in both the PRP (P = .0078) and control groups (P = .01). The microorganisms were susceptible to most of the tested antimicrobials. The PRP did not increase the bacterial load in the wounds. All S. aureus strains identified showed biofilm-forming capacities and were classified as weak biofilm producers. All P. aeruginosa strains produced biofilm, with 17 strains being classified as weak, 14 as moderate, and 8 as strong biofilm producers. CONCLUSIONS: The PRP plus petrolatum gauze did not increase bacteriological growth or the microbial load in chronic venous ulcers compared with petrolatum gauze alone and could be a considered as an advanced treatment option for these types of chronic wounds.

Ciprofloxacin-Resistant Kerstersia gyiorum Isolated From a Chronic Wound in Brazil: A Case Report.

The presence of Kerstersia gyiorum in lower leg wounds has been reported in case studies from several countries. OBJECTIVE: This study evaluated the antimicrobial susceptibility profile of K gyiorum isolated from a chronic wound. METHODS: An 85-year-old woman with chronic venous insufficiency presented to an intermediate care unit in Niteroi City, Rio de Janeiro, Brazil, with an instep chronic wound of 14 cm² with wound duration of 6 months. K gyiorum was identified by matrix-assisted laser desorption ionization-time of flight, confirmed by 16S rRNA partial sequence analysis, and classified as resistant for ciprofloxacin by reagent strips(minimum inhibitory concentration [MIC] = 32 µg/mL) and the broth macrodilution method (MIC = 8 µg/mL). Intermediate resistance for ciprofloxacin was verified by microscan (MIC = 2 µg/mL). CONCLUSION: The authors identified the first, to their knowledge, lower leg wound with K gyiorum in Brazil and verified that it was ciprofloxacin resistant.

Monitoring and Molecular Characterization of Staphylococcus aureus Isolated from Chronic Wounds.

OBJECTIVE: Researchers analyzed chronic wounds treated with 2% hydrogel to determine whether the presence of methicillin-resistant Staphylococcus aureus (MRSA) is related to the presence of clinical signs of infection. METHODS: Thirty-five patients were recruited for this descriptive study using a quantitative approach. Staphylococcus aureus was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Antibiotic susceptibility was determined using a disk diffusion test according to Clinical and Laboratory Standards Institute standards. Polymerase chain reaction, pulsed-field gel electrophoresis, and multilocus sequence typing were performed. Statistical analyses were performed using Spearman correlation coefficients for the variables MRSA and clinical signs of infection. MAIN OUTCOME MEASURES: The identification of MRSA or methicillin-sensitive S aureus (MSSA), presence or absence of an infection in the wound, and molecular characterization of bacteria were measured. MAIN RESULTS: Of the 35 patients analyzed, 8 (22.9%) were classified as having an infection in their wounds. Spearman ρ indicated a strong positive correlation between the increase in the number of clinical signs of infection and MSSA (P =.84), but only a moderate positive correlation with MRSA (P =.60). The S aureus clonal pattern was unique for each of the major bacteria isolated. Global MRSA sequence-type clones (ST-1 and ST-72) were detected in 2 patients. CONCLUSIONS: Compared with those colonized by MSSA, chronic wounds colonized by MRSA did not display a strong correlation with the presence of a greater number of clinical signs of infection.

Methicillin-resistant Staphylococcus aureus in minas frescal cheese: evaluation of classic enterotoxin genes, antimicrobial resistance and clonal diversity.

This study aimed to investigate classical enterotoxin (sea to see) and mecA genes, by polymerase chain reaction and anitimicrobial susceptibility, by disk diffusion test of Staphylococcus aureus isolated from minas frescal cheese (MFC). All methicillin-resistant S. aureus (MRSA) isolates were investigated for the presence of Panton-Valentine leukocidin (PVL) genes and clonal diversity. Thirty-one S. aureus were isolated from four MFC samples. Seven (22.6%) S. aureus carried mecA gene and two of them carried enterotoxin genes seb/sec and sea/seb. Five (16.1%) S. aureus isolates showed induced resistance to clindamycin and nine (29%) were resistant to multiple -antibiotics (MDR), among these, six were MRSA. No MRSA isolates presented the PVL genes. Four MRSA were grouped into three clones and three isolates were not typable by pulsed field gel electrophoresis. MRSA isolates showed, by multilocus sequence typing, sequence types ST1, ST5, ST72 and ST4304 (new ST) and S. aureus protein A (spa type) t127, t568 and t2703. These data suggest that MFC may constitute a risk to the consumer because of its potential for staphylococcal food poisoning; however it might, also, become one of MRSA and MDR strains disseminator, including clones usually found in the hospital environment.

Prevalence, Antimicrobial Susceptibility, and Clonal Diversity of Pseudomonas aeruginosa in Chronic Wounds.

PURPOSE: Our purposes in this study were to (1) identify Pseudomonas aeruginosa strains collected from swabs of chronic wounds, (2) evaluate the susceptibility of P. aeruginosa strains to various antimicrobials, (3) detect the presence of virulence factors exoenzyme S (exoS) and exoenzyme U (exoU) in P. aeruginosa strains, and (4) evaluate wound colonization by P. aeruginosa via pulsed-field gel electrophoresis (PFGE). DESIGN: Descriptive research using a quantitative approach. SAMPLE AND SETTING: Swabs from 43 adults with chronic wounds treated in an outpatient setting in Niterói City, Brazil, were included using convenience sampling. METHODS: Swabs were collected at 2 points during treatment, 30 to 45 days apart. P. aeruginosa isolates were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Antimicrobial susceptibility testing was performed using the disk diffusion method. The presence of exoS and exoU genes was evaluated using polymerase chain reaction. Genotyping diversity was determined through PFGE. RESULTS: Forty-eight P. aeruginosa isolates were detected in chronic wounds, and 3 were multidrug resistant (6%). Resistance to aztreonam and ciprofloxacin was observed in 48% and 27% of isolates, respectively. The presence of the exoS gene was verified in 54% of isolates, and 27% were positive for the exoU gene. In most wounds, P. aeruginosa strains had the same genetic characteristics at the 2 time points analyzed, indicating that the wound beds remained colonized. CONCLUSIONS: P. aeruginosa was present in 75% of tested chronic wound samples, and the same clones persisted for more than 1 month. In addition, most bacteria contained virulence genes that were associated with high potential to establish infection. The use of silver in chronic wounds may be associated with multidrug resistance in P. aeruginosa; therefore, it is important to avoid colonization by these bacteria.

Molecular characterization of multidrug-resistant (MDR) Pseudomonas aeruginosa isolated in a burn center.

OBJECTIVE: The aim of this study was to characterize molecularly multidrug-resistant (MDR) Pseudomonas aeruginosa isolates collected from burn center (BC) patients and environment in a hospital localized in Rio de Janeiro city, RJ, Brazil. METHODS: Thirty-five P. aeruginosa isolates were studied. The antimicrobial resistance was tested by disk diffusion method as recommended by CLSI. The assessment of virulence (exoS and exoU) and resistance (blaPER-1, blaCTX-M, blaOXA-10, blaGES-1, blaVIM, blaIMP, blaSPM-1, blaKPC, blaNDM and blaSIM) genes were achieved through PCR and biofilm forming capacity was determined using a microtiter plates based-assay, as described previously. Genotyping was performed using Multilocus sequence typing (MLST). RESULTS: High rate of P. aeruginosa (71.4%; 25/35) were classified as MDR, of them 64% (16/25) were related to clone A, the most prevalent clone found in the BC studied. A total of eight carbapenems resistant isolates were detected; three belonging to clone A and five carrying the exoU virulence gene. In addition, clone A isolates were also biofilm producers. Two new sequence types (ST) were detected in this study: ST2236, grouped in to clone A; and ST2237, classified in the different clones, displaying carbapenem resistance and exoU virulence gene. CONCLUSION: The high prevalence of biofilm producers and multiresistant P. aeruginosa isolates in BC indicates that prevention programs need to be implemented to avoid infection in highly susceptible patients.

Magistral drugs in hospitalized newborns and children / Medicamentos magistrais em recém-nascidos e crianças hospitalizados

Abstract Objective: Study the use of magistral oral solutions and suspensions in infants and children at a university hospital. Methods: This is a descriptive study based on the analysis of the assessed hospital's magistral drug request forms regarding the patients in the neonatal ICU, Obstetrics, Pediatrics and Pediatric Emergency from January 2012 to December 2013. The frequency of drug requests and dispensation was evaluated and the consumption of each active ingredient of the preparations was expressed as number of “infant defined daily dose” (iDDD) and of iDDD/100 bed-days. Results: A total of 657 forms were analyzed - a monthly average of 27 pediatric preparations. The neonatal ICU accounted for 69.6% of these requests. Twenty-one drug items were used, of which the most common were folinic acid (88 requests), sulfadiazine (85) and captopril (73). The consumption of the active principle in these preparations varied in number of iDDD, from 7.5 (hydralazine) to 16,520.0 (folic acid), and in number of iDDD/100 bed-days in the neonatal ICU, from 0.1 (zinc sulfate) to 146.1 (folic acid). Conclusions: The constant consumption of magistral oral solutions and suspensions by newborns and children of the assessed hospital indicates the need for such preparations as a pediatric therapeutic alternative in this hospital.

Resumo Objetivo: Estudar o uso de soluções e suspensões orais magistrais em recém-nascidos e crianças de um hospital universitário. Métodos: Foi feito um estudo descritivo a partir da análise dos formulários de solicitação de manipulação do hospital estudado referentes aos pacientes da UTI-neonatal, obstetrícia, pediatria e emergência pediátrica de janeiro de 2012 a dezembro de 2013. As frequências das solicitações e dispensações desses medicamentos foram avaliadas e o consumo de cada princípio ativo das preparações foram expressos sob a forma de número de infant defined daily dose (iDDD) e de iDDD/100 leitos-dia. Resultados: Foram analisados 657 formulários - média mensal de 27 preparações pediátricas. A UTI-neonatal foi responsável por 69,6% dessas solicitações. Foram usados 21 itens de medicamentos, destacou-se o uso de ácido folínico (88 solicitações), sulfadiazina (85) e captopril (73). O consumo de princípio-ativo nessas preparações variou, em número de iDDD, de 7,5 (hidralazina) a 16.520 (ácido fólico) e em número de iDDD/100 leitos-dia da UTI-neonatal, de 0,1 (sulfato de zinco) a 146,1 (ácido fólico). Conclusões: O consumo constante das soluções e suspensões orais magistrais pelos recém-nascidos e crianças do hospital estudado indica a necessidade dessas preparações como opção terapêutica pediátrica nesse hospital.

Magistral drugs in hospitalized newborns and children. / Medicamentos magistrais em recém­nascidos e crianças hospitalizados.

OBJECTIVE: Study the use of magistral oral solutions and suspensions in infants and children at a university hospital. METHODS: This is a descriptive study based on the analysis of the assessed hospital's magistral drug request forms regarding the patients in the neonatal ICU, Obstetrics, Pediatrics and Pediatric Emergency from January 2012 to December 2013. The frequency of drug requests and dispensation was evaluated and the consumption of each active ingredient of the preparations was expressed as number of "infant defined daily dose" (iDDD) and of iDDD/100 bed-days. RESULTS: A total of 657 forms were analyzed - a monthly average of 27 pediatric preparations. The neonatal ICU accounted for 69.6% of these requests. Twenty-one drug items were used, of which the most common were folinic acid (88 requests), sulfadiazine (85) and captopril (73). The consumption of the active principle in these preparations varied in number of iDDD, from 7.5 (hydralazine) to 16,520.0 (folic acid), and in number of iDDD/100 bed-days in the neonatal ICU, from 0.1 (zinc sulfate) to 146.1 (folic acid). CONCLUSIONS: The constant consumption of magistral oral solutions and suspensions by newborns and children of the assessed hospital indicates the need for such preparations as a pediatric therapeutic alternative in this hospital.

Balneotherapy is a potential risk factor for Pseudomonas aeruginosa colonization

ABSTRACT The practice of immersion in burn patient has been abandoned in many parts of the world but in Brazil it is still common. The aim of this study was to ascertain if balneotherapy is a risk factor for Pseudomonas aeruginosa colonization in thermally injured patients. Eighteen patients from a Burn Center were studied for 14 weeks for Pseudomonas aeruginosa. Samples were collected by swabbing the exudate of wounds, before and after giving bath to the patients and from balneotherapy table. Pulsed-field gel electrophoresis was used to determine bacterial genetic relatedness. Thirty-seven P. aeruginosa isolates were detected from 292 swabs collected from patients' burn surface area and from the balneotherapy table. Profile analysis of P. aeruginosa DNA fragmentation showed 10 clones among the 37 strains analyzed. Type A is the most prevalent clone, with 23 strains distributed into eight subtypes. These were present in the swabs collected, before and after the patients' bath, from the surface of the bath table, suggesting that there was cross-contamination between the patients in different ways. This work demonstrates that balneotherapy is a risk factor in the Burn Center studied, because the same clone was found among P. aeruginosa isolates collected at various points and times.

RESUMO A prática de balneotarapia em paciente queimado foi abandonada em muitas partes do mundo, mas no Brasil ainda é comum. O objetivo deste estudo foi verificar se a balneoterapia é um fator de risco para a colonização por Pseudomonas aeruginosa em pacientes queimados. Dezoito pacientes internados em um Centro de Queimadura (CQ) foram acompanhados por 14 semanas. Amostras foram coletadas do exsudato de feridas, antes e depois do banho dos pacientes e também da mesa onde a balneoterapia foi realizada. A relação genética entre as cepas de P. aeruginosa foi determinada pela electroforese em gel de campo pulsado. Trinta e sete cepas foram detectadas a partir de 292 swabs coletados de área de superfície das feridas dos pacientes e da mesa de balneoterapia. Análise de fragmentação do DNA das 37 P. aeruginosa mostrou a existência de 10 clones. O tipo A foi o clone mais prevalente, com 23 cepas distribuídas em oito subtipos. Estas estavam presentes nas lesões dos pacientes antes e após o banho e na mesa onde o banho foi realizado, sugerindo contaminação cruzada inter e intra-pacientes e pacientes e mesa de banho. Este trabalho mostra que a balneoterapia é um fator de risco para colonização por P. aeruginosa, no CQ estudado, pois um mesmo clone da bactéria foi encontrado nos isolados coletados em vários pontos e épocas diferentes.

Production of metallo-ß-lactamase among Pseudomonas aeruginosa strains isolated in the State of Sergipe, Brazil.

INTRODUCTION: Acquired production of metallo-ß-lactamases is an important mechanism of resistance in Pseudomonas aeruginosa. The objective of this study was to investigate the production of metallo-ß-lactamase and the genetic diversity among ceftazidime-resistant P. aeruginosa isolates from State of Sergipe, Brazil. METHODS: Metallo-ß-lactamase was investigated using the disk approximation test and polymerase chain reaction (PCR). Genetic diversity was evaluated by pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 48 (51.6%) isolates were resistant to ceftazidime. Six (12.2%) of these were positive for metallo-ß-lactamase production. Only two (4.1%) of the ceftazidime-resistant isolates carried the bla SPM-1 gene. CONCLUSIONS: Production of metallo-ß-lactamases was not the main mechanism of resistance to ceftazidime and carbapenems among P. aeruginosa strains in Sergipe, Brazil.

Influence of papain in biofilm formed by methicillin-resistant Staphylococcus epidermidis and methicillin-resistant Staphylococcus haemolyticus isolates

Methicillin-resistant Staphylococcus epidermidis (MRSE) and methicillin-resistant Staphylococcus haemolyticus (MRSHa) are important coagulase-negative staphylococci. They are often isolated from bacteremia in humans mainly due to their ability to form biofilm on the surfaces of medical devices. Papain is a complex mixture of proteolytic enzymes and peroxidases extracted from the latex of Carica papaya and it is recognized by accelerating the healing process of wounds. This study aimed to evaluate the ability of the MRSE and MRSHa isolates to produce biofilms. Besides this, the ability of papain to inhibit the formation of biofilms or to disrupt the ones already formed by those bacteria was analyzed. Thirty MRSHa and 30 MRSE were isolated from bacteremia and used in this study. It was observed that papain has ability to reduce biofilms formed by MRSE (p < 0.06) and by MRSHa (p = 0.0005). In addition, papain was able to disrupt mature biofilms made by MRSE (p = 0.014). No antibacterial activity of papain was observed for any isolates of MRSE and MRSHa tested. Papain has been demonstrated as a potential product for reducing biofilm.

Staphylococcus epidermidis resistente à meticilina (MRSE) e Staphylococcus haemolyticus resistente à meticilina (MRSHa) são importantes estafilococos coagulase negativa. São frequentemente isolados em bacteremia humana, principalmente devido à capacidade de formar biofilmes nas superfícies de dispositivos médicos introduzidos no organismo. A papaína é mistura complexa de enzimas proteolíticas e peroxidases extraídas do látex de Carica papaya, reconhecida por acelerar os processos de cura de feridas. Este estudo teve como objetivo avaliar a capacidade dos MRSE e MRSHa em produzir biofilmes e analisar a capacidade da papaína em inibir a formação de biofilme ou desintegrar biofilmes já formados por essas bactérias. Observou-se que a papaína tem capacidade de reduzir a formação de biofilme por MRSE (p < 0,06) e MRSHa (p = 0,0005). Além disso, a papaína foi capaz de desintegrar biofilme maduro formado por MRSE (p = 0,014). Nenhuma atividade antibacteriana da papaína foi observada para qualquer das duas espécies de bactérias testadas. A papaína mostrou-se produto potencial para reduzir biofilme.

The antimicrobial susceptibility, biofilm formation and genotypic profiles of Staphylococcus haemolyticus from bloodstream infections.

We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.

Emergence of multiresistant variants of the community-acquired methicillin-resistant Staphylococcus aureus lineage ST1-SCCmecIV in 2 hospitals in Rio de Janeiro, Brazil.

Usually, community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is susceptible to a variety of non-beta-lactam drugs. These isolates commonly display SCCmecIV and are associated with community-acquired infections. More recently, CA-MRSA has been isolated from health-care-associated diseases. We characterized MRSA isolates from 2 hospitals in Rio de Janeiro area to assess the entry of new lineages. The isolates were primary genotyped using a combination of molecular typing methods including SCCmec, restriction modification test, and Panton-Valentine leukocidin (PVL) detection. Pulsed-field gel electrophoresis was carried out for representatives of each lineages found. Disk diffusion test was performed as recommended by the Clinical and Laboratory Standards Institute. SCCmecIV was the predominant cassette mec detected. The most frequent MRSA lineage, a PVL nonproducer, was allocated in the CC1-SCCmecIV. It was found that 56% of these isolates were resistant to 3 or more non-beta-lactam drugs. Multilocus sequence typing of a representative of the CC1 isolates supported our finds that multiresistant variants of a CA-MRSA lineage (ST1-SCCmecIV) emerged in this city.

Comparison of different methods for detecting methicillin resistance in MRSA isolates belonging to international lineages commonly isolated in the American continent.

The aim of the present paper was to compare different methods for detecting methicillin resistance in Staphylococcus aureus. Among the isolates analyzed, 52 belonged to MRSA international lineages commonly detected in the American continent and 14 to sporadic MRSA clones. Both 30 microg-cefoxitin disk and PBP2a had 100% sensibility/specificity when the low-level heterogeneous isolates were tested and, thus, are highly recommended.

Detection and characterization of international community-acquired infections by methicillin-resistant Staphylococcus aureus clones in Rio de Janeiro and Porto Alegre cities causing both community- and hospital-associated diseases.

Community-acquired infections by methicillin-resistant Staphylococcus aureus (CA-MRSA) in the absence of classic risk factors for MRSA diseases have been reported in different continents. In the article presented here, using molecular typing methods as pulsed-field gel electrophoresis, staphylococcal cassette chromosome mec typing, and multilocus sequence typing, we characterized CA-MRSA isolates from Rio Janeiro and Porto Alegre. The results indicated the presence of international CA-MRSA clones in these 2 Brazilian cities. In addition, Panton-Valentine leukocidin and a number of staphylococcal enterotoxin encoding genes were accessed in these MRSA isolates by polymerase chain reaction detection.

Commensal isolates of methicillin-resistant Staphylococcus epidermidis are also well equipped to produce biofilm on polystyrene surfaces.

OBJECTIVES: To study biofilm production and to detect icaAD, atlE and aap genes in 137 isolates of methicillin-resistant Staphylococcus epidermidis (MRSE) obtained from healthy individuals from the community (35 isolates), from hospitalized patients at the Antônio Pedro University Hospital (25 isolates) and from individuals from a home-care system (HCS; 77 isolates). METHODS: Biofilm production was determined in vitro using polystyrene inert surfaces. icaAD, atlE and aap genes were detected using PCR. Hybridization experiments were also carried out to confirm some PCR results. Antimicrobial susceptibility testing was carried out using the NCCLS methods. RESULTS: Although many of the commensal MRSE isolates produced biofilms, the percentage of biofilm producers was significantly higher (P = 0.0107) among hospital isolates (76%) than among isolates from the community (60%) and from the HCS (57%). An association was observed between multiresistance and biofilm production for isolates obtained from healthy individuals from the community and from household contacts from the HCS (P < 0.0001). The concomitant presence of the ica operon and atlE and aap genes was associated with the strong biofilm-producer phenotype (P < 0.0001). CONCLUSION: Because many of the commensal MRSE isolates obtained from nares produced biofilms and carried icaAD, aap and atlE genes, biofilms or such genetic elements should not be used as markers for clinical significance. The biofilm environment seems to increase genetic exchanges and hence may contribute to multiresistance phenotypes.

Isolation of methicillin-resistant coagulase-negative staphylococci from patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and comparison of different molecular techniques for discriminating isolates of Staphylococcus epidermidis.

Coagulase-negative staphylococci (CNS) have emerged as an important pathogen in nosocomial infections. About 80%-90% of CNS isolates associated with hospital infections are methicillin-resistant coagulase-negative staphylococci (MRCNS). The aims of this study were to screen for MRCNS isolates in the flora of a small population of patients undergoing continuous ambulatory peritoneal dialysis (CAPD) and to evaluate the discriminatory power of different molecular methods: pulsed-field gel electrophoresis (PFGE), mecA location, ClaI/mecA polymorphism and arbitrarily primed polymerase chain reaction (AP-PCR) for characterizing isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). Seventy-nine CNS isolates were recovered from the 11 CAPD patients studied. Using a methicillin screening agar and a DNA specific mecA probe we verified that 30 of the 79 (38%) CNS isolates were resistant to methicillin (MRCNS). Twenty-two of the 30 MRCNS (73%) were MRSE, 7 (23%) methicillin-resistant S. haemolyticus (MRSH(ae)) and 1 (3%) methicillin-resistant S. hominis (MRSH(om)). All patients analyzed carried MRCNS in their flora, in one or more sites. Since CAPD patients have high risk for developing peritonitis, the colonization of these patients with MRCNS might represent an additional problem, due to the therapeutic restrictions imposed by these multiresistant isolates. A wide genetic diversity was verified when the PFGE of the MRSE isolates was analyzed. The 22 MRSE isolates displayed a total of 15 PFGE different patterns (11 PFGE types and 4 subtypes). The location of mecA in the SmaI-fragmented genome DNA did not bring any additional advantage for epidemiologic characterization of the isolates. The ClaI/mecA polymorphism was able to correctly discriminate 12 from the 15 PFGE patterns. In addition, the DNA of 20 MRSE isolates were used for AP-PCR typing. These isolates belonged to 14 PFGE patterns (11 types and 3 subtypes) and displayed 15 genotypes (for the association of PFGE, mecA location and ClaI/mecA polymorphism). A total of 17 different amplification patterns was verified using the primer 1. Only for 2 genotypes, strains having identical genetic backgrounds were further discriminated by AP-PCR (2 of 15 genotypes (87%) for AP-PCR and 1 of 15 genotypes for PFGE; (93%). Concluding, our results indicated that the AP-PCR can be an alternative and useful tool for monitoring and genotyping MRSE colonization and also to molecular characterizing MRSE outbreaks in hospitals.