Molecular epidemiology of methicillin-resistant Staphylococcus aureus in the Brazilian primary health care system.

OBJECTIVE: To evaluate the molecular epidemiology and to georeference Staphylococcus aureus isolated from wounds and nares of patients seen at Basic Health Units (BHUs) of a Brazilian city. METHODS: Observational, cross-sectional study conducted from 2010 to 2013. A total of 119 S. aureus strains isolated from the wounds and nares of 88 patients were studied. The isolates were characterised by identifying virulence genes encoding enterotoxins A-E, haemolysins α, ß and δ, exfoliatins A, B and D, biofilm production, Panton-Valentine Leukocidin and toxic shock syndrome toxin 1, and by pulsed-field gel electrophoresis (PFGE), multilocus sequence and spa typing. RESULTS: Eighteen methicillin-resistant Staphylococcus aureus (MRSA) (6 SCCmec type II and 12 SCCmec type IV) and 101 (85%) MSSA were identified. PFGE typing resulted in the formation of eight clusters, with STs 1, 5, 8, 30, 188, 1176 and 1635 and spa type t002 being the predominant types among MSSA. The 18 MRSA belonged to STs 5, 8 and 1176 and spa types t002 and t062. CONCLUSION: The results demonstrate widespread dissemination of MSSA and MRSA clones carrying haemolysin, biofilm and toxin genes. Kernel density estimation revealed the highest density of S. aureus in the 4, 5 and 8 BHUs.

OBJECTIF: Evaluer l'épidémiologie moléculaire et géoréférencer le Staphylococcus aureus isolé de plaies et de narines de patients vus dans les unités sanitaires de base (BHU) d'une ville brésilienne. MÉTHODES: Etude observationnelle transversale réalisée de 2010 à 2013. Au total, 119 souches de S. aureus isolées de plaies et de narines de 88 patients ont été étudiées. Les isolats ont été caractérisés par l'identification de gènes de virulence codant pour les entérotoxines AE, les hémolysines α, ß et δ, les exfoliatines A, B et D, la production de biofilm, la leucocidine de Panton-Valentine et la toxine 1 du syndrome de choc toxique, et par typage par électrophorèse sur gel en champ pulsé (PFGE), séquence multilocus et spa. RÉSULTATS: Dix-huit SARM (6 de type II SCCmec et 12 de type IV SCCmec) et 101 (85%) SASM ont été identifiés. Le typage PFGE a résulté à l'obtention de huit grappes, dont STs 1, 5, 8, 30, 188, 1176 et 1635 et le type spa t002 étant les types prédominants parmi les SASM. Les 18 SARM appartenaient aux STs 5, 8 et 1176 et aux types de spa t002 et t062. CONCLUSION: Les résultats démontrent une dissémination étendue des clones de SASM et de SARM portant les gènes de l'hémolysine, de biofilm et de toxine. L'estimation de la densité par noyau a révélé la densité la plus élevée de S. aureus dans les 4, 5 et 8 BHU.

Expression of superantigens and the agr system in Staphylococcus epidermidis.

Infections with coagulase-negative staphylococci (CoNS) can involve the production of toxins such as superantigens, which contribute to tissue degradation and inflammatory immune responses. The accessory gene regulator (agr) quorum sensing system coordinates the expression of most S. aureus virulence factors. Therefore, the aim of this study was to investigate the expression of these superantigens and the presence of the agr locus in CoNS strains isolated from blood cultures. PCR was used to detect enterotoxin and agr genes and expression was analyzed by RT-PCR. Expression of the sea gene was observed in one S. epidermidis isolate andof sec-1 in two, seg and sei were expressed concomitantly in one isolate, and sei was expressed in another isolate. The agr group I was detected in S. epidermidis expressing the sea, seg and sei genes, whereas agr group II was detected in isolates expressing thesec-1 gene. The agr groups were only expressed in strains expressing thesec-1 gene. The results show that enterotoxin genes are highly frequent in CoNS isolated from clinical specimens and confirm the toxin-producing ability of these strains. The agr group II may be associated with enterotoxin C production by S. epidermidis, increasing the virulence of strains isolated from blood cultures and consequently the severity of sepsis caused by these organisms.

Diagnostic accuracy of semi-quantitative and quantitative culture techniques for the diagnosis of catheter-related infections in newborns and molecular typing of isolated microorganisms.

BACKGROUND: Catheter-related bloodstream infections (CR-BSIs) have become the most common cause of healthcare-associated bloodstream infections in neonatal intensive care units (ICUs). Microbiological evidence implicating catheters as the source of bloodstream infection is necessary to establish the diagnosis of CR-BSIs. Semi-quantitative culture is used to determine the presence of microorganisms on the external catheter surface, whereas quantitative culture also isolates microorganisms present inside the catheter. The main objective of this study was to determine the sensitivity and specificity of these two techniques for the diagnosis of CR-BSIs in newborns from a neonatal ICU. In addition, PFGE was used for similarity analysis of the microorganisms isolated from catheters and blood cultures. METHODS: Semi-quantitative and quantitative methods were used for the culture of catheter tips obtained from newborns. Strains isolated from catheter tips and blood cultures which exhibited the same antimicrobial susceptibility profile were included in the study as positive cases of CR-BSI. PFGE of the microorganisms isolated from catheters and blood cultures was performed for similarity analysis and detection of clones in the ICU. RESULTS: A total of 584 catheter tips from 399 patients seen between November 2005 and June 2012 were analyzed. Twenty-nine cases of CR-BSI were confirmed. Coagulase-negative staphylococci (CoNS) were the most frequently isolated microorganisms, including S. epidermidis as the most prevalent species (65.5%), followed by S. haemolyticus (10.3%), yeasts (10.3%), K. pneumoniae (6.9%), S. aureus (3.4%), and E. coli (3.4%). The sensitivity of the semi-quantitative and quantitative techniques was 72.7% and 59.3%, respectively, and specificity was 95.7% and 94.4%. The diagnosis of CR-BSIs based on PFGE analysis of similarity between strains isolated from catheter tips and blood cultures showed 82.6% sensitivity and 100% specificity. CONCLUSION: The semi-quantitative culture method showed higher sensitivity and specificity for the diagnosis of CR-BSIs in newborns when compared to the quantitative technique. In addition, this method is easier to perform and shows better agreement with the gold standard, and should therefore be recommended for routine clinical laboratory use. PFGE may contribute to the control of CR-BSIs by identifying clusters of microorganisms in neonatal ICUs, providing a means of determining potential cross-infection between patients.

Characterization of the clonal profile of MRSA isolated in neonatal and pediatric intensive care units of a University Hospital.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) are important pathogens in neonatal and pediatric intensive care units, which can cause severe infections in hospitalized children. Detection of the mecA gene and classification of the staphylococcal cassette chromosome mec (SCCmec) permit the characterization of MRSA strains isolated from infections caused by these microorganisms. In contrast, pulsed-field gel electrophoresis (PFGE) is used to type MRSA clones. This method is commonly used to analyze the epidemiology of bacteria causing nosocomial infections. The objective of this study was to detect and characterize MRSA isolated from clinical specimens of children hospitalized in the neonatal and pediatric intensive care units of the University Hospital of the Botucatu Medical School. METHODS: A total of 119 S. aureus strains were isolated from clinical specimens and the mecA gene was detected by PCR. SCCmec was detected by multiplex PCR and the clonal profile was analyzed by PFGE. RESULTS: The mecA gene was detected in 17.6% (21/119) of the isolates; 42.9% (9/21) of MRSA were characterized as SCCmec type III and 57.1% (12/21) as type IV. Analysis of the clonal profile of these strains revealed three distinct clones, with SCCmec type III being related to the Brazilian endemic clone and type IV to clones JCSC4469 and USA800. CONCLUSIONS: Replacement of clonal groups occurred in the neonatal and pediatric units over the period studied, a fact highlighting the importance of improving hygiene practices and control measures of nosocomial infections in these units.

Nasopharyngeal carriage of Staphylococcus aureus among imprisoned males from Brazil without exposure to healthcare: risk factors and molecular characterization.

BACKGROUND: Previous studies report high prevalence of Methicillin-resistant Staphylococcus aureus (MRSA) colonization among imprisoned populations. However, there are no data on that prevalence in Brazilian correctional institutions. FINDINGS: We tested 302 male prisoners for nasopharyngeal colonization with Staphylococcus aureus from February 2009 through April 2010. The overall isolation rate of S. aureus was 16.5% (50/302). Men who had sex with men, users of inhalatory drugs and those with previous lung or skin diseases were more likely to be colonized with S. aureus. MRSA was isolated from 0.7% of subjects (2/302). The two Community-associated (CA)-MRSA belonged to ST5 but were unrelated based on the PFGE results. Both harbored SCCmec IV, and did not possess the Panton-Valentine Leukocidin gene. CONCLUSION: We found low prevalence of S. aureus and CA-MRSA among prisoners. MRSA isolates ST5 from two subjects harboured SCCmec IV and presented different PFGE patterns.

Staphylococcus aureus and CA-MRSA Carriage among Brazilian Indians Living in Peri-Urban Areas and Remote Communities.

The emergence of Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections among indigenous populations has been reported. Usually, indigenous communities live in extreme poverty and are at risk of acquiring infections. In Brazil, healthcare inequality is observed in this population. To date, there are no reports of CA-MRSA infections, and no active search for asymptomatic S. aureus carriage has been conducted among Brazilian Indians. The aim of this study was to investigate the prevalence of colonization with S. aureus and CA-MRSA among Brazilian Indians. We screened 400 Indians (from near urban areas and remote hamlets) for S. aureus and CA-MRSA colonization. The isolates were submitted to clonal profiling by pulsed-field gel electrophoresis (PFGE), and selected isolates were submitted to multilocus sequence typing (MLST). Among 931 specimens (nasal and oral) from different indigenous individuals in remote hamlets, S. aureus was cultured in 190 (47.6%). Furthermore, CA-MRSA was found in three isolates (0.7%), all SCCmec type IV. PFGE analysis identified 21 clusters among the S. aureus isolates, and MLST analysis showed a predominance of sequence type 5 among these isolates. Our study revealed a higher prevalence of S. aureus carriage among Shanenawa ethnicity individuals (41.1%). Therefore, ethnicity appears to be associated with the prevalence of S. aureus in these populations.

Phenotypic and Molecular Characterization of Nonfermenting Gram-Negative Bacilli Causing Peritonitis in Peritoneal Dialysis Patients.

(1) Background: Peritonitis due to nonfermenting Gram-negative bacilli (NF-GNB) is a dramatic complication of peritoneal dialysis (PD) with bad outcomes. Previous studies of PD-related peritonitis due to Pseudomonas species have shown a low-resolution rate, without a high resistance rate to antipseudomonal antibiotics. This suggests that bacterial virulence factors can act and influence peritonitis evolution. This study aimed to describe the microbiological characteristics of NF-GNB causing PD-related peritonitis and analyze their influence on the outcome. (2) Methods: We analyze the 48 isolates from NF-GNB peritonitis, which were stored in our culture collection regarding bacterial resistance, biofilm, and other virulence factors' production, and clonal profile. Additionally, we collected data on treatment and outcomes from patients' clinical registers. (3) Results: The etiologies were species of Pseudomonas (50%), Acinetobacter (36%), and other NF-GNB (14%). There was a high (75%) proportion of biofilm producer lineages. The in vitro susceptibility rate of Pseudomonas spp. to amikacin, ciprofloxacin, and ceftazidime was significantly greater than that of Acinetobacter spp. and other species; however, there was a similar low-resolution rate (<45%) among the episodes attributable to them. Pseudomonas species have a polyclonal profile, while we found a clone of five multiresistant Acinetobacter baumannii over an 8-year interval (2000-2008), which suggest an origin from the healthcare environment. (4) Conclusions: We are not able to identify any predictor of outcome, but it is possible that biofilm and others virulence factors can act in concert and contribute to the bad outcome.

Staphylococcal Biofilm on the Surface of Catheters: Electron Microscopy Evaluation of the Inhibition of Biofilm Growth by RNAIII Inhibiting Peptide.

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) have become the main causative agents of medical device-related infections due to their biofilm-forming capability, which protects them from the host's immune system and from the action of antimicrobials. This study evaluated the ability of RNA III inhibiting peptide (RIP) to inhibit biofilm formation in 10 strains isolated from clinical materials, including one S. aureus strain, two S. epidermidis, two S. haemolyticus, two S. lugdunensis, and one isolate each of the following species: S. warneri, S. hominis, and S. saprophyticus. The isolates were selected from a total of 200 strains evaluated regarding phenotypic biofilm production and the presence and expression of the ica operon. The isolates were cultured in trypticase soy broth with 2% glucose in 96-well polystyrene plates containing catheter segments in the presence and absence of RIP. The catheter segments were observed by scanning electron microscopy. The results showed inhibition of biofilm formation in the presence of RIP in all CoNS isolates; however, RIP did not interfere with biofilm formation by S. aureus. RIP is a promising tool that might be used in the future for the prevention of biofilm-related infections caused by CoNS.

Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR.

Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains-including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states-by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon-mainly responsible for biofilm formation-as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from a Brazilian university hospital.

The aim of this study was to perform SCCmec typing in Staphylococcus aureus isolates and to characterize the clonal profile of these isolates. Forty-six mecA gene-positive strains isolated between 2002 and 2006 were submitted to antimicrobial resistance testing by the E-test, SCCmec typing by multiplex PCR, and clonal profile analysis by pulsed-field gel electrophoresis. Forty-one (89.1%) isolates were typed as SCCmec III and five (10.9%) as SCCmec IV. Four circulating clones were detected, one of them comprising isolates related to the Brazilian epidemic clone. This clone was detected throughout the study period. The SCCmec III isolates were associated with a high rate of multidrug resistance and clonal dissemination of methicillin-resistant S. aureus in the wards of the University Hospital of the Botucatu School of Medicine, Universidade Estadual Paulista.

Molecular epidemiology of methicillin-resistant Staphylococcus aureus in a burn unit from Brazil.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a threat for patients in burn units. Studies that mix epidemiological designs with molecular typing may contribute to the development of strategies for MRSA control. We conducted a study including: molecular characterization of Staphylococcal Chromosome Cassette mecA (SCCmec), strain typing with pulsed field gel electrophoresis (PFGE) and detection of virulence genes, altogether with a case-case-control study that assessed risk factors for MRSA and for methicillin-susceptible S. aureus (MSSA), using S. aureus negative patients as controls. Strains were collected from clinical and surveillance cultures from October 2006 through March 2009. MRSA was isolated from 96 patients. Most isolates (94.8%) harbored SCCmec type III. SCCmec type IV was identified in isolates from four patients. In only one case it could be epidemiologically characterized as "community-associated". PFGE typing identified 36 coexisting MRSA clones. When compared to MSSA (38 isolates), MRSA isolates were more likely to harbor two virulence genes: tst and lukPV. Previous stay in other hospital and admission to Intensive Care Unit were independent risk factors for both MRSA and MSSA, while the number of burn wound excisions was significantly related with the former (OR=6.80, 95%CI=3.54-13.07). In conclusion, our study found polyclonal endemicity of MRSA in a burn unit, possibly related to importing of strains from other hospitals. Also, it pointed out to a role of surgical procedures in the dissemination of MRSA strains.

Antimicrobial resistance and persistence of Staphylococcus epidermidis clones in a Brazilian university hospital.

Oxacillin is an alternative for the treatment of Staphylococcus spp. infections; however, resistance to this drug has become a major problem over recent decades. The main objective of this study was to epidemiologically characterize coagulase-negative staphylococci (CoNS) strains recovered from blood of patients hospitalized in a Brazilian teaching hospital. Oxacillin resistance was analyzed in 160 strains isolated from blood culture samples by phenotypic methods, detection of the mecA gene, and determination of intermediate sensitivity to vancomycin on brain heart infusion agar supplemented with 4 and 6 µg/mL vancomycin. In addition, characterization of the epidemiological profile by staphylococcal cassette chromosome mec (SCCmec) typing and clonal analysis by pulsed-field gel electrophoresis (PFGE) were performed. The mecA gene was detected in 72.5% of the isolates. Methicillin-resistant CoNS isolates exhibited the highest minimum inhibitory concentrations and multiresistance when compared to methicillin-susceptible CoNS strains. Typing classified 32.8% of the isolates as SCCmec I and 50% as SCCmec III. PFGE typing of the SCCmec III Staphylococcus epidermidis isolates identified 6 clones disseminated in different wards that persisted from 2002 to 2009. The high oxacillin resistance rates found in this study and clonal dissemination in different wards highlight the importance of good practices in nosocomial infection control and of the rational use of antibiotic therapy in order to prevent the dissemination of these clones.

Enterotoxin genes, multidrug resistance, and molecular typing of Staphylococcus spp. isolated from organic bovine milk / Genes de enterotoxinas, multirresistência a antimicrobianos e caracterização molecular de espécies de Staphylococcus spp. isoladas de leite bovino orgânico

The multidrug resistant and the emergence of methicillin-resistant staphylococci isolated from animals, food, and humans are public health concern. These microorganisms produce different toxins related to food poisoning in humans. This study aimed to characterize Staphylococcus spp. isolated from two organic milk farms in Brazil. A total of 259 milk samples were collected, from which 58 (22.4%) Staphylococcus spp. were isolated. The highest sensibility to ceftiofur and sulfamethoxazole/trimethoprim was observed in 96.6% of Staphylococcus spp., and whereas 89% were resistant to penicillin G. The mecA gene was detected in 13.8% of the isolates. SEA and SEC were the most common enterotoxins detected. PFGE revealed genetic heterogeneity from S. intermedius and S. warneri analyzed, while S. aureus presented similar profiles among isolates from the two studied herds. To the best of our knowledge, the current study describes for the first time presence of enterotoxins, mecA gene, and genetic diversity of staphylococci isolated from organic dairy farms in Brazil.(AU)

A emergência de estafilococos multirresistentes e resistentes à meticilina, isolados de animais, alimentos e humanos é uma preocupação em saúde pública. Esses micro-organismos produzem diferentes toxinas relacionadas à intoxicação alimentar em humanos. Este estudo caracterizou Staphylococcus spp. isolados em duas fazendas orgânicas no Brasil. Foram coletadas 259 amostras de leite em duas propriedades leiteiras orgânicas, nas quais 58 (22,4%) estirpes de Staphylococcus spp. foram isoladas. A maior sensibilidade dos isolados foi observada para ceftiofur e sulfametoxazol/trimetoprim em 96,6%. Em contraste, acima de 89% de resistência dos estafilicocos foi encontrada para penicilina G. O gene mecA foi identificado em 13,8% dos isolados. SEA e SEC foram as enterotoxinas mais comumente detectadas. PFGE revelou heterogeneidade genética entre S. intermedius e S. warneri, enquanto S. aureus demonstraram perfis semelhantes entre isolados dos dois rebanhos estudados. Relata-se pela primeira vez no Brasil a detecção de enterotoxinas, o gene mecA e diversidade genética em estafilococos isolados de vacas em produção orgânica.(AU)

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.