Angela C. D. Castro

Identification of Clinical Isolates of Indole-Positive and Indole-Negative Klebsiella spp.

ABSTRACT Biochemical methods employed to classify bacterial species have limitations and may have contributed to the taxonomic complexity recently reported for the genus Klebsiella. The objective of the present study was to apply a simple biochemical test panel to classify a collection of human Klebsiella isolates. We found that with only three additional tests, it is possible to place most isolates in a defined species. Analysis of a 512-bp sequence of the rpoB gene was used as the reference. A total of 16 conventional and 4 supplementary tests were used to evaluate 122 recent isolates identified as Klebsiella from 120 patients, isolated at the clinical laboratory of a university hospital in Minas Gerais, Brazil. Of these, 102 (84%) isolates were identified as Klebsiella pneumoniae or Klebsiella variicola, 19 (15%) as Klebsiella oxytoca, and 1 (1%) as Raoultella planticola. Enterobacterial repetitive intergenic consensus-PCR typing revealed a diversity of genotypes. rpoB gene sequencing confirmed the phenotypic identification and detected five K. variicola isolates among the K. pneumoniae/K. variicola group. Three additional tests that include growth at 10°C and histamine and d-melezitose assimilation should be considered essential tests for the typing of Klebsiella isolates.

Phenotypic and genotypic characterization of clinical and intestinal enterococci isolated from inpatients and outpatients in two Brazilian hospitals.

The phenotypic and genotypic characteristics of clinical and intestinal enterococcal isolates recovered from inpatients and outpatients of two Brazilian hospitals, located in Niterói city, Rio de Janeiro, Brazil, were compared. A total of 601 strains were studied, including 253 isolated from different clinical sources and 348 intestinal strains (205 isolated from inpatients and 143 from outpatients) recovered from fecal specimens. Isolates were identified by using conventional physiological tests and evaluated for high-level resistance to aminoglycosides (HLR-A) and resistance to vancomycin and ampicillin by the agar screening technique. Susceptibility to several antimicrobial agents was evaluated by the disk diffusion method. The genetic diversity of Enterococcus faecalis strains presenting HLR-A was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. E. faecalis was the most frequent species among clinical isolates (90.1%) and intestinal strains from inpatients (53.6%). E. casseliflavus was the prevalent species among intestinal isolates from outpatients (35.0%). Clinical isolates were shown to be resistant to erythromycin (53.0%), tetracycline (52.2%), ciprofloxacin (36.4%), gentamicin (36.4%), streptomycin (30.4%), chloramphenicol (34.4%), norfloxacin (32.0%), imipenem (3.2%), and ampicillin (2.8%). Vancomycin resistance was only detected in intrinsic vancomycin-resistant enterococcal species. The overall prevalence of HLR-A was 52.2% among clinical isolates and 40.5% among intestinal strains. However, HLR-A was significantly more frequent among intestinal strains obtained from inpatients (56.6%) than among strains from outpatients (17.5%). Three major clonal groups were found among E. faecalis strains exhibiting HLR-GE or HLR-GE/ST (clonal groups GE-A and GE-B), and strains exhibiting HLR-ST (clonal group ST-A). HLR-A, particularly HLR-GE, was most frequently associated with enterococcal strains of nosocomial origin. Isolates included in the major clonal groups were recovered from clinical and intestinal sources from patients in both hospitals, indicating both intrahospital and interhospital spread of strains.

Genetic and Phenotypic Features of Streptococcus pyogenes Strains Isolated in Brazil That Harbor New emm Sequences

ABSTRACT In the present study, 37 group A Streptococcus (GAS) strains belonging to 13 new emm sequence types identified among GAS strains randomly isolated in Brazil were characterized by using phenotypic and genotypic methods. The new types were designated st204, st211,st213, st809, st833,st854, st2904, st2911,st2917, st2926, st3757,st3765, and st6735. All isolates were susceptible to the antimicrobial agents tested, except to tetracycline. They all carried the speB gene, and 94.6% produced detectable SpeB. Most strains belonging to a given emmtype had similar or highly related pulsed-field gel electrophoresis profiles that were distinct from profiles of strains of another type. The other characteristics were variable from isolate to isolate, although some associations were consistently found within some emm types. Unlike the other isolates, all type st213 isolates were speA positive and produced SpeA. Strains belonging to st3765 were T6 and opacity factor (OF) negative. Individual isolates within OF-positive emm types were associated with uniquesof gene sequence types, while OF-negative isolates weresof negative by PCR. This report provides information on new emm sequence types first detected in GAS isolates from a geographic area not extensively surveyed. Such data can contribute to a better understanding of the local and global dynamics of GAS populations and of the epidemiological aspects of GAS infections occurring in tropical regions.

Expression of the Major Heat Shock Proteins DnaK and GroEL in Streptococcus pyogenes: A Comparison to Enterococcus faecalis and Staphylococcus aureus

One of the outstanding problems in the field of heat shock response has been to elucidate the mechanism underlying the induction of heat shock proteins (HSPs). In this work, we initiate an analysis of the expression of heat shock groEL and dnaK genes and their promoters in S. pyogenes. The synthesis of total cellular proteins was studied upon transfer of a log-phase culture from 37°C to 42°C by performing 5-min pulse-labeling experiments with 35S-Met. The heat shock responses in the pathogenic Gram-positive cocci, Enterococcus faecalis and Staphylococcus aureus, were also analyzed.

Expression of heat-shock proteins in Streptococcus pyogenes and their immunoreactivity with sera from patients with streptococcal diseases

The heat-shock response of Streptococcus pyogenes following exposure to elevated growth temperatures, and the immunological reactivity of heat-shock proteins (HSPs) in streptococcal infections were studied. Two major proteins of 65 and 75 kDa were expressed when a S. pyogenes strain was shifted from 37 degrees C to heat-shock temperatures of 40, 42 and 45 degrees C. Such proteins are members of the GroEL and DnaK families recognised in a Western blot assay with polyclonal antibodies against Escherichia coli GroEL and E. coli DnaK, respectively. Two-dimensional autoradiograms of polypeptides labelled at 37 or 42 degrees C showed an increased intensity of three spots at 42 degrees C. A monoclonal antibody (MAb) against HSP 63 of Bordetella pertussis also recognised the 65-kDa inducible protein, although MAbs against Mycobacterium tuberculosis HSP 65 failed to recognise this protein. Immunoblot analysis of sera from individuals with rheumatic fever or uncomplicated streptococcal diseases revealed seven major immunogenic protein bands, two of which also reacted with anti-E. coli GroEL and DnaK polyclonal antibodies. Furthermore, antibodies to the GroEL and DnaK proteins were also detected in sera from patients with either rheumatoid arthritis or systemic lupus erythematosus. These results demonstrated a heat-shock response of S. pyogenes, and indicated the presence of an immune response against HSPs in streptococcal diseases.

Streptococcus agalactiae em gestantes: prevalência de colonização e avaliação da suscetibilidade aos antimicrobianos

PURPOSE: to verify the occurrence of colonization by Streptococcus agalactiae in pregnant women attended at the prenatal outpatient clinic of the Teaching Maternity Hospital of Rio de Janeiro University (UFRJ) and to evaluate the susceptibility of the isolates to antimicrobial agents. METHODS: a total of 167 pregnant women between the 32nd and 41st week of gestation, regardless of risk factors, attended at the antenatal clinic between February 2003 and February 2004, were evaluated. The vaginal/anal material, collected by the same swab, was inoculated in Todd-Hewitt broth to which nalidixic acid (15 µg/mL) and gentamicin (8 µg/mL) were added, with following subcultures onto sheep blood-agar. Identification was carried out observing colony morphology and beta-hemolysis type on blood-agar, catalase, cAMP, and serological tests. The antimicrobial susceptibility testing used agar diffusion and agar dilution methods. Statistical analysis was performed by the c2 test with the level of significance set at p 0.05). All 32 isolated strains were susceptible to penicillin, cefotaxime, ofloxacin, chloramphenicol, vancomycin and meropenem. Resistance to erythromycin and clindamycin was detected in 9.4 and 6.2% of the isolates, respectively. CONCLUSIONS: the relatively high incidence (19.2%) of colonization by S. agalactiae among the evaluated pregnant women and the recovery of antimicrobial resistant strains, especially those recommended in cases of penicillin allergy, emphasize the importance, for a correct prevention of neonatal infections, of detecting colonization at the end of pregnancy and evaluating antimicrobial susceptibility.

Antimicrobial Susceptibility and Survey of Macrolide Resistance Mechanisms among Streptococcus pyogenes Isolated in Rio de Janeiro, Brazil

A total of 357 clinical Streptococcus pyogenes isolates collected between 1994 and 1999 in Rio de Janeiro city were tested for susceptibility to 10 antimicrobial drugs by agar-diffusion tests. All isolates were susceptible to penicillin, cephems, and vancomycin. High resistance rates were observed for tetracycline (43.1%) and trimethoprim/sulfamethoxazole (77.9%). Three isolates (0.8%) were resistant to erythromycin, and three exhibited intermediate susceptibility. Determination of the erythromycin MICs by the agar dilution method, showed 1.6% of erythromycin resistant isolates (the three erythromycin-resistant and the three erythromycin-intermediate isolates found by agar-diffusion test). Of the erythromycin-resistant isolates subjected to the double-disc diffusion test for erythromycin and clindamycin, three isolates expressed the iMLSB and three the M phenotype. The resistance phenotypes were confirmed by comparing the clindamycin MICs determined under normal testing conditions and those determined after induction by pre-growth in 0.06 microg/ml of erythromycin. Three ermTR and three mefA-containing isolates were detected by PCR. In strains belonging to the iMLSB phenotype, two clones were identified by PFGE following restriction with SmaI. M phenotype isolates could not be restricted with SmaI. Our results indicate a low rate of erythromycin resistance among S. pyogenes isolated in Rio de Janeiro, Brazil, and pointed to the presence of both resistance mechanisms found in streptococci.

Extracellular deoxyribonucleases of streptococci: a comparison of their occurrence and levels of production Smong beta-hemolytic strains of various serological groups

Production of extracellular deoxyribonuclease by 394 strains of beta hemolytic streptococci was examined employing a deoxyribonucleic acid-methyl green assay. Enzymatic activities were measured in supernatants of bacterial cultures. Of the strains tested, 316 (80%) produced the enzyme. Nuclease production was demonstrated in 100% of group A strains and in 85, 74 and 58% of groups B, C and G, respectively. Levels of nuclease activity were then evaluated statistically. The analysis of variance showed that group A strains produced more enzyme than did streptococci of groups B, C or G. Group B strains produced less nuclease than did isolates of groups C or G. There was no significant difference in the levels of nuclease produced by groups C and G or by the various serological types of group B streptococci. Human group C strains produced more enzyme than animal strains.

Correlation between growth in antibiotic-medium and hemolytic activity of group C and G streptococci

The effect of a subminimal inhibitory concentration of penicillin on the production of bound and free hemolysins by streptococci was examined using sheep red blood cells. A marked decrease of a group C cell-free and bound activities was observed with penicillin at a concentration of 1/3 of the MIC whereas an increase was observed with those of a group G strain. Potassium ferricyanide and anti-streptolysin O (group A streptococcus) were strongly inhibitory for the free activities of both strains. The cell-bound activities were stimulated by addition of RNA during bacterial growth in control cultures and also in drug-containing media.

Efeito de concentrações subinibitórias de penicilina sobre antígenos de estreptococos do grupo G

The effect of subinhibitory concetrations of penicillin on a strain of streptococcus belonging to Lancefield group G was studied. Production of the group-specific antigen and extra- cellular hyaluronidase by drug-exposed cells were examined. At all concentrations levels a higher amount of group-specific antigen was extracted from cells and increases of up to 1400% in the specific activity of hyaluronidase were determined in culture supernates. The higher increase in the expression of both antigens was observed at 1/2 MIC.