José Augusto Adler Pereira

Staphylococcus haemolyticus disseminated among neonates with bacteremia in a neonatal intensive care unit in Rio de Janeiro, Brazil

Oxacillin-resistant Staphylococcus haemolyticus (ORSH) was found as the most prevalent (77.5%) species of coagulase-negative staphylococci associated with bacteremia in neonates making use of intravenous catheters in an intensive care unit of a Brazilian teaching hospital. Thirty-one blood isolates were confirmed as S. haemolyticus by sequencing of the 16S and clustered in 6 pulsed-field gel electrophoresis types (with 58% of the strains belonging to 2 predominant types B and D). S. haemolyticus was mostly oxacillin-resistant (90.3%) displaying multiresistance profiles (70.4%). However, the mecA gene was undetected in 22.6% strains. ORSH exhibited slime production on Congo-Red agar (67.7%), adherence to polystyrene (96.7%), and glass (87%) surfaces. Interestingly, ica-operon was detected in 58% strains, mostly belonging to the B, D, and F genotypes, which is a significantly higher percentage when compared to other studies conducted at different parts of the globe. Data indicated that ica operon and biofilm-forming ORSH are endemic in Brazilian nosocomial environment.

β-lactamase producing enterobacteria isolated from surveillance swabs of patients in a Cardiac Intensive Care Unit in Rio de Janeiro, Brazil

There is a high incidence of infections caused by betalactamase-producing Gram-negative microorganisms in Brazil. These organisms are of clinical and epidemiological importance, since their mobile genetic elements facilitate cross-infection. The present study was conducted in sentinel rectal swabs from patients admitted to a cardiac surgery hospital in Rio de Janeiro, from January through December 2007, in a consecutive manner. The aim of the study was to characterize the genotype and phenotype of these isolates from colonized patients. Biochemical tests, antimicrobial susceptibility tests, a confirmatory test for the expression of extended spectrum betalactamase (ESBL) production and polymerase chain reaction for the blaTEM, blaSHV, CTX-M1, Toho-1 and AmpC genes were performed at the University Hospital of Universidade do Estado do Rio de Janeiro (UERJ). The most frequently isolated bacteria were Escherichia coli 9/41 (21.95%) and Klebsiella pneumoniae 14/41 (34.1%). In 24/41 (58%), the ESBL genotype was confirmed. The most prevalent genes in samples that expressed ESBL were blaTEM 13/24 (54%), AmpC 12/24 (50%), blaSHV 6/24 (25%), CTX-M1 7/24 (29%), and Toho-1 6/24 (25%). Of these, 14/24 (58%) presented more than one genotype for the tested primers. In nine (37%) samples other than E. coli, K. pneumoniae or Proteus spp., the phenotype for ESBL was found and confirmed by PCR. The most sensitive substrate in the approximation test in ESBL positive samples was ceftriaxone (83%). Fifty percent of the samples expressed AmpC were associated with other genes. Intermediate susceptibility to ertapenem was found in 2/41 (5%).

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

Newborn intestinal colonization by multidrug resistant enterobacteria in a neonatal unit

OBJECTIVE: To evaluate the occurrence of intestinal colonization in newborns by multidrug-resistant enterobacteria strains (MDRES) during hospital stay after birth. We used selective media in an attempt to determine the relationship between isolation of these strains and some of the presumed colonization risk factors. METHOD: A sequencial inclusion study of 30 newborns was carried out in the neonatal unit of the HUPE, State University Hospital, a general 600-bed tertiary care hospital. We obtained clinical and epidemiological information from medical records and collected a fecal sample from each newborn, which was plated in gentamicin (8mg/ml) medium and potassium tellurite (25mg/ml) medium. The isolated strains were biochemically identified and also submitted to tests of antimicrobial susceptibility. Nine MDRES were submitted to an assay for plasmid conjugational transfer. RESULTS: We isolated 56 distinct MDRES from 14 among 30 newborns (46.7%). Klebsiella pneumoniae was the most common bacterial species (38/56 (68%). We found statistical association between individual MDRES isolation and presence of 3 or 4 of the following colonization risk factors considered: antimicrobial use, low weight (<2.500g), more than 6 days of hospitalization and artificial milk feeding (p< 0.02). We could detect plasmid resistance transfer by bacterial conjugation for 8 among 9 MDRES. CONCLUSION: The seletive cultura media were useful to detect the high frequence of newborns colonized by MDRES in association with well established infection risk factors. We emphasize the importance of reinforcing control rules aiming at preventing intestinal colonization viewed as a risk of nosocomial infection.

Trimethoprim-sulfamethoxazole and fluoroquinolones Resistant Escherichia Coli in Community-Acquired and Nosocomial Urinary Tract Infections in Rio De Janeiro, Brazil

To investigate the multidrug resistance (MDR) patterns of Escherichia coli causative of urinary tract infections (UTI) in patients attending a tertiary university hospital of Rio de Janeiro, Brazil. Antibiotic susceptibility testing was performed by the disk diffusion method. MDR, extensively-resistance (XDR) and pan-resistance (PDR) were defined by using recently described criteria. Retrospective analyses of clinical, microbiological and demographic features of outpatients and inpatients with UTI (n=416) were also performed. High antibiotic resistance rates for trimethoprimsulfamethoxazole - SXT-TMP (n=177; 46.7%) and fluoroquinolones - FQ [n=117; norfloxacin (27%) and ciprofloxacin (26.8%) – (FQ) were demonstrated for E. coli strains isolated from community and healthcare-onsets. Risk factors associated with UTIs due to MDR E. coli isolates included prior three-month hospitalization (OR: 2.4; CI 95%: 1.3-4.4; p<0.005), presence of neurogenic bladder (OR: 3.7; CI 95% :1.7-8.3; p<0.01 ) and kidney transplantation (OR: 3.1; CI 95%:1-0.5; p<0.04). A high prevalence of community-acquired and nosocomial urinary tract infections due SXT-TMP/ FQ resistant E. coli strains was observed in Rio de Janeiro metropolitan area, Brazil. According to IDSA Guidelines, initial empirical therapy for community-associated UTI with SXT-TMP and FQ should be avoided in Rio de Janeiro. Nitrofurantoin, amoxicillin/clavulanic, piperacillin/tazobactam or gentamicin associations were effective for the empiric therapy for community-acquired and healthcare-associated UTIs, respectively.

Evidences of gentamicin resistance amplification in Klebsiella pneumoniae isolated from faeces of hospitalized newborns

The intestinal microbiota, a barrier to the establishment of pathogenic bacteria, is also an important reservoir of opportunistic pathogens. It plays a key role in the process of resistance-genes dissemination, commonly carried by specialized genetic elements, like plasmids, phages, and conjugative transposons. We obtained from strains of enterobacteria, isolated from faeces of newborns in a university hospital nursery, indication of phenotypical gentamicin resistance amplification (frequencies of 10(-3) to 10(-5), compatible with transposition frequencies). Southern blotting assays showed strong hybridization signals for both plasmidial and chromosomal regions in DNA extracted from variants selected at high gentamicin concentrations, using as a probe a labeled cloned insert containing aminoglycoside modifying enzyme (AME) gene sequence originated from a plasmid of a Klebsiella pneumoniae strain previously isolated in the same hospital. Further, we found indications of inactivation to other resistance genes in variants selected under similar conditions, as well as, indications of co-amplification of other AME markers (amikacin). Since the intestinal environment is a scenario of selective processes due to the therapeutic and prophylactic use of antimicrobial agents, the processes of amplification of low level antimicrobial resistance (not usually detected or sought by common methods used for antibiotic resistance surveillance) might compromise the effectiveness of antibiotic chemotherapy.

Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis?

Corynebacterium diphtheriae strains continue to circulate worldwide causing diphtheria and invasive diseases, such as endocarditis, osteomyelitis, pneumonia and catheter-related infections. Presumptive C. diphtheriae infections diagnosis in a clinical microbiology laboratory requires a primary isolation consisting of a bacterial culture on blood agar and agar containing tellurite (TeO32−). In this study, nine genome sequenced and four unsequenced strains of C. diphtheriae from different sources, including three samples from a recent outbreak in Brazil, were characterized with respect to their growth properties on tellurite-containing agar. Levels of tellurite-resistance (TeR) were evaluated by determining the minimum inhibitory concentrations of potassium tellurite (K2TeO3) and by a viability reduction test in solid culture medium with K2TeO3. Significant differences in TeR levels of C. diphtheriae strains were observed independent of origin, biovar or presence of the tox gene. Data indicated that the standard initial screening with TeO32−-selective medium for diphtheria bacilli identification may lead to false-negative results in C. diphtheriae diagnosis laboratories.

Sphygmomanometers and thermometers as potential fomites of Staphylococcus haemolyticus: biofilm formation in the presence of antibiotics

BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

An epidemiological study of hospital infection and its resistance both to antimicrobials and to tellurite in Enterobacteriaceae with special reference to Klebsiella pneumoniae

As prevalências de Klebsiella pneumoniae em fezes de pacientes hospitalizados e ambulatoriais foram similares. A média de marcadores de resistência não determinou diferença significativa para os dois ambientes. Em outra abordagem verificamos, entretanto, que a prevalência de enterobactérias fecais resistentes ao íon telurito foi maior entre pacientes internados há mais de uma semana do que entre aqueles com menos de uma semana. Na segunda etapa de internação o número de marcadores de resistência por cepa foi maior, além de detectarmos uma modificação no espectro de espécies resistentes ao telurito. Sugerimos que tais fenômenos representem a pressão de colonização de germes hospitalares sobre a flora fecal de pacientes hospitalizados. A u tiliz ação de m eios se le tiv o s , con tendo antimicrobianos, no estudo dos processos de colonização pode trazer contribuições para o conhecimento dos mesmos processos. As modificações da microbiota intestinal determinam alterações nas espécies microbianas associadas a infecções hospitalares assim como nos seus perfis de resistência. Pudemos mostrar que cepas de K. pneumoniae isoladas de infecções urinárias adquiridas no am biente hospitalar apresentavam codificação plasmidial para a multirresistência a antimicrobianos e a íons telurito e mercúrico. Detectamos plasmídios únicos de alto peso molecular transferíveis por conjugação. Entre cepas de K. pneumoniae isoladas de surto de in fecção nosocom ial encontram os uma heterogeneidade detectada pela biotipagem e uma diversidade de plasmídeos codificando a multirresistência. O não achado de plasmídio único lev o u -n o s a su g e rir que o prob lem a da

Use of a selective medium with potassium tellurite to follow intestinal colonization of hospitalized patients by drug-resistant Enterobacteriaceae

Nosocomial infections are a relevant factor in complicating the recovery of patients interned for even minor causes. In a attempt to determine their origin it is crucial to consider that their origin is of an endogenous nature. Looking for an accessible expression of intestinal colonization we analyzed fecal samples from 3 separate groups of hospital patients collected after different lengths of time. For practical reasons one group was studied prospectively and two other groups (patients hospitalized for up to 7 days and patients hospitalized for more than 7 days) were compared to one another. We looked for the emergence of tellurite resistance among Enterobacteriaceae using a selective medium, MacConkey potassium tellurite (MCPT). The frequency of prospectively studied patients with tellurite resistant strains was significantly greater after 7 days of hospitalization. For the two other groups, patients with more than 7 days of hospitalization showed a significant increase of bacterial species and of strains with new antimicrobial resistance markers. High molecular weight plasmids were detected in some of these strains. These data show that the MCPT medium is a useful tool for the investigation of bowel colonization in hospitalized patients by drug-resistant Enterobacteriaceae.