Clinical and microbiological profile of chronic Burkholderia cepacia complex infections in a cystic fibrosis reference hospital in Brazil.

Burkholderia sp. infections are extremely complex in cystic fibrosis (CF) patients, especially considering the lack of knowledge regarding its behavior, its relationship with prognosis, as well as its transmissibility and multidrug resistance features. This study evaluated the frequency of chronic infection by Burkholderia, using microbiological and clinical data. Ninety-eight patients with CF attended from July 2011 to April 2014 in a Brazilian reference hospital were included. Antimicrobial activity, molecular epidemiology, Shwachman score, body mass index, exacerbations, and lung function were analyzed. Nine patients had chronic colonization, and all of them showed preserved pulmonary function levels, body mass index, and Shwachman score. Meropenem was the most effective antibiotic; however, divergent results were shown by other studies. Cross-contamination may have occurred in only two unrelated patients of different ages, who were colonized by B. vietnamiensis, which does not occur frequently. Twelve new sequence types (STs) were identified and three STs have presented intercontinental distribution. None of the patients presented known epidemic strains. In conclusion, a relatively low number of patients with chronic colonization and suspected cross-infection were identified. Different from other studies that have found CF patients chronically colonized with Burkholderia sp. having a greater deterioration of lung function, more frequent antibiotic therapy, and increased mortality, in the current study, the patients showed good clinical outcomes and favorable options for antibiotics therapy. This study also updated the epidemiological database, which facilitates the multicentric collaborative analysis and assists in the control of global infection by these pathogens.

Detection of chromosomal bla(CTX-M-2) in diverse Escherichia coli isolates from healthy broiler chickens.

The rise of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in food-producing animals is a growing concern for public health. We investigated ESBL producers isolated from broiler chickens in Brazil and characterized 19 CTX-M-2-producing E. coli. The ISCR1 was detected upstream of the chromosome-located gene bla(CTX-M-2), associated with sul-1 type integron structure. CTX-M-2-producing E. coli exhibited different PFGE-types and phylogenetic groups, showing a non-clonal dissemination. The sequence types found (ST93, ST155 and ST2309) have been associated with humans and animals worldwide. Herein, we report the chromosomal location of bla(CTX-M-2) on E. coli, highlighting the risks of multidrug-resistant bacteria in food-producing animals.

Changes in vancomycin-resistant Enterococcus faecium causing outbreaks in Brazil.

Enterococci have been implicated in severe human infections as a consequence of associated determinants of virulence and antimicrobial resistance. The majority of vancomycin-resistant Enterococcus faecium (VRE(fm)) connected to outbreaks worldwide pertains to the clonal complex 17 (CC17). In Brazil, the majority of VRE(fm) involved in outbreaks reported so far are not related to CC17. VRE(fm) strains responsible for an outbreak and sporadic cases in hospitals located in the city of Campinas, Brazil, were compared to other VRE(fm) strains in the country. Twenty-two out of 23 E. faecium were vancomycin-resistant and harboured the vanA gene. One vancomycin-susceptible E. faecium (VSE(fm)) strain was included in this study because it was isolated from a patient who one week later harboured a VRE(fm). All strains, except VSE, showed the same alteration in the VanA element characterised by deletion of the left extremity of the transposon and insertion of IS1251 between the vanS and vanH genes. Genes codifying virulence factors such as collagen-adhesin protein, enterococcal surface protein and hyaluronidase were detected in the VRE(fm) and VSE(fm) studied. Both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) revealed that VRE(fm) and VSE(fm) strains have a clonal relationship. New sequence types (STs) were identified by MLST as ST447, ST448, ST478 and ST412 but all belonged to the CC17. The present study revealed that VRE(fm) outbreaks in Brazil were caused by strains that did not share a common evolutionary history, and that VRE(fm) strains belonging to CC17 could be predominant in Brazil as in other countries.

Determinants of beta-lactam resistance in meningitis-causing Enterobacteriaceae in Brazil.

This study analyzed resistance determinants in extended-spectrum beta-lactamase (ESBL)-producing enterobacteria and the epidemiology of 11 Escherichia coli isolates obtained from meningitis patients in a region of Brazil from 2000 to 2005. ESBL-encoding genes and their genetic environment were investigated by PCR and sequencing. The gene blaCTX-M-2 was identified in 3 different enterobacteria (E. coli, Serratia marcescens, and Proteus mirabilis) downstream of the insertion sequence ISCR1 (localized in class 1 integrons), but not as part of the resistance cassettes region. Multilocus sequence typing (MLST) was used to investigate genetic relationships between the 11 E. coli isolates in this study and strains associated with meningitis in the E. coli MLST database. MLST analysis indicated high genetic diversity among isolates, and no significant genetic relationship was identified with meningitis-causing E. coli in the database. The results in this report reinforce the need to be attentive to meningitis suspected to be due to ESBL-producing enterobacterial isolates, especially where ESBL epidemiology is well known.

Virulence factors in vancomycin-resistant and vancomycin - susceptible Enterococcus faecalis from Brazil / Fatores de virulência em Enterococcus faecalis resistentes e suscetível à vancomicina isolados no Brasil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Enterococci são membros da microbiota comensal de animais e insetos, mas também são importantes patógenos oportunistas. Nosso objetivo foi observar se há qualquer diferença na virulência nos diversos grupos de Enterococcus faecalis, principalmente nos E. faecalis resistente à vancomicina (VREFS) isolados de colonização e infecção. VREFS e E. faecalis sensíveis à vancomicina (VSEFS) do Brasil foram pesquisadas quanto a presença de fatores de virulência. Ensaios fenotípicos foram usados para obter a expressão in vivo, entender o potencial patogênico destas amostras e determinar se existe correlação entre virulência e resistência a antibióticos. Diferentes perfis de virulência foram encontrados sugerindo que o clone que está se disseminado pode ter gerado diversas variações. No entanto, nosso estudo mostrou que um conjunto de fatores parece ser mais comum entre as amostras: gelatinase, substância de agregação e esp (GEA). Estes fatores tem sido correlacionados com a agregação de células e formação de biofilmes. A formação de biofilme pode promover a conjugação de plasmídeos contendo genes de resistência entre as espécies. Curiosamente, o perfil GAE não foi exclusivo para VREFS, foi o segundo mais observado em amostras VSEFS provenientes de colonização e infecção em pacientes hospitalizados e também de swabs retais de voluntários saudáveis. Tais linhagens pacerem representar a "porta de entrada" para novos genes de resistência em E. faecalis e podem contribuir para a disseminação de E. faecalis principalmente nos hospitais.

Virulence factors in vancomycin-resistant and vancomycin- susceptible Enterococcus faecalis from Brazil.

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil.

Enterococci are leading causes of hospital-acquired infections that are often difficult to treat because of high-level aminoglycoside and glycopeptide resistance. Vancomycin-resistant enterococci are a global problem, and have been isolated with increasing frequency in hospitals in Brazil. The objective of this study was to determine the genetic relatedness of vancomycin-resistant Enterococcus faecium (VREFM) and vancomycin-sensitive E. faecium (VSEFM) isolated from human infections and faecal sources in Brazil, and to compare these isolates with those from domesticated animals. Isolates (n = 56) were classified by multilocus sequence typing (MLST) and assessed for putative virulence traits. The acm gene was detected in 98% of all isolates. The 56 isolates studied comprised 26 different MLST types. VSEFM isolates from the faeces of pigs were found to be distinct from all human isolates characterised previously by MLST, and were assigned new sequence type (ST) numbers. VREFM isolates were represented by four different STs (ST-114, ST-17, ST-281, ST-50). Among the 26 STs identified in this study, eBURST detected three groups of STs with related allelic profiles, and 19 unrelated STs. Among E. faecium isolates from Brazil, the esp gene was restricted to vancomycin-resistant isolates. Furthermore, isolates classified as ST-17 by MLST, an epidemic strain type isolated internationally with the purK-1 gene, were found among VREFM isolates from Brazil that also harboured the esp and hyl genes.

First report of vancomycin-resistant staphylococci isolated from healthy carriers in Brazil.

Reduced susceptibility or resistance to vancomycin has been reported among clinical isolates of staphylococci in previous studies. In the present study we report on the isolation of four vancomycin-resistant staphylococcal strains from healthy carriers inside and outside the hospital environment. These carriers did not receive treatment with any antibiotic. All coagulase-negative staphylococcal strains showed variable levels of resistance to several antimicrobial agents, including oxacillin, and unstable resistance to vancomycin, with decreased vancomycin MICs (<4 mg/liter) after 10 days of passage in a nonselective medium. However, exposure of these revertants to vancomycin selected staphylococcal strains resistant to vancomycin at very high frequencies (10(-2) and 10(-3)). The vancomycin resistance in these staphylococcal strains was not mediated by the van gene. The cell wall of the staphylococcal strains studied became thickest after culture in medium containing vancomycin, and the differences in cell wall thickness were statistically significant (P < 0.001). Thus, the thickening of the cell wall in these staphylococcal strains may be an important contributor to vancomycin resistance.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil.

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 microg/ml. This isolate was sent to a reference laboratory (Laboratorio Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > ou = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Identification of an unusual VanA element in glycopeptide-resistant Enterococcus faecium in Brazil following international transfer of a bone marrow transplant patient.

A vancomycin-resistant Enterococcus (VRE) was isolated from a blood culture of a patient in a Brazilian hospital who had a treatment history of a bone marrow transplant in the USA. The organism was identified as Enterococcus faecium, which exhibited an MIC (minimum inhibitory concentration) >or= 256 microg/mL for vancomycin. This was confirmed by E-test and the vanA gene was detected by PCR. Overlapping PCR revealed a left IR deletion and an additional 1.5 kb fragment between vanSH genes. DdeI digestion of vanRSHAX genes showed the determinant to be a T type variant, and the element was cloned and sequenced. These results revealed an IS1251 downstream of nucleotide 5820 of the VanA element. Insertions like this have not been reported previously in Brazil, but have been detected in the USA. The genotype and association with a patient previously treated in the USA suggest that this VRE was introduced from abroad, probably through inter-hospital strain spread.

A low molecular weight enterotoxic hemolysin from clinical Enterobacter cloacae.

Seven of 50 Enterobacter cloacae strains from clinical isolates produced small turbid zones of hemolysis in horse and sheep blood agar plates, and the culture supernatants were also positive for hemolytic activity. The hemolysin was partially purified from the culture supernatant of E. cloacae by ultrafiltration (PM-10 membrane) and extraction with acetone. Semipurified hemolysin was stable to heating (100 degrees C, 30 min) and was soluble in organic solvents (acetone, ethanol, and methanol). The toxin showed no loss of biological activity after treatment with trypsin and was stable to acid treatment at pH 2.0 but not at a pH greater than 7.0. In the rat intestinal loop assay, the hemolysin caused hemorrhagic fluid accumulation and severe histological alterations. These findings indicate that this hemolysin may be a putative virulence factor in E. cloacae infections.

Biological activity of Serratia marcescens cytotoxin.

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 g/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli.

Biological activity of Serratia marcescens cytotoxin

Serratia marcescens cytotoxin was purified to homogeneity by ion-exchange chromatography on a DEAE Sepharose Fast Flow column, followed by gel filtration chromatography on a Sephadex G100 column. The molecular mass of the cytotoxin was estimated to be about 50 kDa. Some biological properties of the cytotoxin were analyzed and compared with well-characterized toxins, such as VT1, VT2 and CNF from Escherichia coli and hemolysin produced by S. marcescens. The sensitivity of the cell lines CHO, HeLa, HEp-2, Vero, BHK-21, MA 104 and J774 to the cytotoxin was determined by the cell viability assay using neutral red. CHO and HEp-2 were highly sensitive, with massive cellular death after 1 h of treatment, followed by BHK-21, HeLa, Vero and J774 cells, while MA 104 was insensitive to the toxin. Cytotoxin induced morphological changes such as cell rounding with cytoplasmic retraction and nuclear compactation which were evident 15 min after the addition of cytotoxin. The cytotoxic assays show that 15 min of treatment with the cytotoxin induced irreversible intoxication of the cells, determined by loss of cell viability. Concentrations of 2 CD50 (0.56 æg/ml) of purified cytotoxin did not present any hemolytic activity, showing that the cytotoxin is distinct from S. marcescens hemolysin. Antisera prepared against S. marcescens cytotoxin did not neutralize the cytotoxic activity of VT1, VT2 or CNF toxin, indicating that these toxins do not share antigenic determinants with cytotoxin. Moreover, we did not detect gene sequences for any of these toxins in S. marcescens by PCR assay. These results suggest that S. marcescens cytotoxin is not related to any of these toxins from E. coli

Involvement of NO in the failure of neutrophil migration in sepsis induced by Staphylococcus aureus.

1. Sepsis induced by S. aureus was used to investigate whether neutrophil migration failure to infectious focus correlates with lethality in Gram-positive bacteria-induced sepsis in mice. 2. By contrast with the sub-lethal (SL-group), the lethal (L-group) intraperitoneal inoculum of S. aureus caused failure of neutrophil migration (92% reduction), high CFU in the exudate, bacteremia and impairment of in vitro neutrophil chemotactic activity. 3. Pre-treatments of L-group with adequate doses of aminoguanidine prevented the neutrophil migration failure and improved the survival of the animals (pre-treated: 43%; untreated: 0% survival). Thus, the impairment of neutrophil migration in the L-group appears to be mediated by nitric oxide (NO). 4. The injection of S. aureus SL-inoculum in iNOS deficient (-/-) or aminoguanidine-treated wild-type mice (pre- and post-treatment), which did not present neutrophil migration failure, paradoxically caused severe peritonitis and high mortality. This fact is explainable by the lack of NO dependent microbicidal activity in migrated neutrophils. 5. In conclusion, although the NO microbicidal mechanism is active in neutrophils, the failure of their migration to the infectious focus may be responsible for the severity and outcome of sepsis.

Clinical relevance and virulence factors of pigmented Serratia marcescens.

Pigmented Serratia marcescens isolated in a Brazilian hospital were studied with respect to frequency of isolation, serotyping, antibiotic resistance and virulence factors. The serotype most frequent was O6:K14 (53%) and all isolates were resistant to ampicillin, cephalothin and tetracycline. The majority of the isolates (92%) were resistant to the action of human serum and all produced cytotoxins on Vero, CHO, HEp-2 and HeLa cells. These isolates were virulent for mice (LD(50)=10(7) bacteria ml(-1)) and showed virulence factors, but were isolated with low frequency (3. 4%) and caused infection in only 31% of cases. Analysis of serotyping, phage typing and chromosomal DNA revealed at least 13 unrelated strains among pigmented S. marcescens. In conclusion, this work describes a low frequency of isolation of pigmented S. marcescens from clinical specimens, indicating that non-pigmented strains are clinically more significant.

Effects of the movement of insertion sequences on the structure of VanA glycopeptide resistance elements in Enterococcus faecium.

A Tn1546-related element with IS1216V at position 8839 underwent a structural change after storage of the host strain of Enterococcus faecium at 4 degrees C. The element acquired IS1542 at position 3932, nucleotides 8732 to 8831 were deleted, and the first 3417 nucleotides were lost and replaced by an inverted copy of the IS1216V-vanY-vanZ-inverted-repeat block from the 3' end. Insertion sequence movement is likely to play a key role in the evolution of VanA resistance elements.

Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae

Phenotyping and genotyping methods applied to investigate the relatedness of Brazilian isolates of Enterobacter cloacae.

In order to evaluate the resolving power of several typing methods to identify relatedness among Brazilian strains of Enterobacter cloacae, we selected twenty isolates from different patients on three wards of a University Hospital (Orthopedics, Nephrology, and Hematology). Traditional phenotyping methods applied to isolates included biotyping, antibiotic sensitivity, phage-typing, and O-serotyping. Plasmid profile analysis, ribotyping, and macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) were used as genotyping methods. Sero- and phage-typing were not useful since the majority of isolates could not be subtyped by these methods. Biotyping, antibiogram and plasmid profile permitted us to classify the samples into different groups depending on the method used, and consequently were not reliable. Ribotyping and PFGE were significantly correlated with the clinical epidemiological analysis. PFGE did not type strains containing nonspecific DNase. Ribotyping was the most discriminative method for typing Brazilian isolates of E. cloacae.