A total of 46 ciprofloxacin-resistant (Cip r ) Streptococcus pneumoniae strains were isolated from 1991 to 2001 at the Hospital of Bellvitge. Five of these strains showed unexpectedly high rates of nucleotide variations in the quinolone resistance-determining regions (QRDRs) of their parC, parE, and gyrA genes. The nucleotide sequence of the full-length parC, parE, and gyrA genes of one of these isolates revealed a mosaic structure compatible with an interspecific recombination origin. Southern blot analysis and nucleotide sequence determinations showed the presence of an ant-like gene in the intergenic parE-parC regions of the S. pneumoniae Cip r isolates with high rates of variations in their parE and parC QRDRs. The ant-like gene was absent from typical S. pneumoniae strains, whereas it was present in the intergenic parE-parC regions of the viridans group streptococci (Streptococcus mitis and Streptococcus oralis). These results suggest that the viridans group streptococci are acting as donors in the horizontal transfer of fluoroquinolone resistance genes to S. pneumoniae.

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Four new genotypes appeared in 2006 after childhood vaccination was begun.

The low prevalence of ciprofloxacin-resistant (Cp r ) Streptococcus pneumoniae isolates carrying recombinant topoisomerase IV genes could be attributed to a fitness cost imposed by the horizontal transfer, which often implies the acquisition of larger-than-normal parE-parC intergenic regions. A study of the transcription of these genes and of the fitness cost for 24 isogenic Cp r strains was performed. Six first-level transformants were obtained either with PCR products containing the parC quinolone resistance-determining regions (QRDRs) of S. pneumoniae Cp r mutants with point mutations or with a PCR product that includes parE-QRDR-ant-parC-QRDR from a Cp r Streptococcus mitis isolate. The latter yielded two strains, T6 and T11, carrying parC-QRDR and parE-QRDR-ant-parC-QRDR, respectively. These first-level transformants were used as recipients in further transformations with the gyrA-QRDR PCR products to obtain 18 second-level transformants. In addition, strain Tr7 (which contains the GyrA E85K change) was used. Reverse transcription-PCR experiments showed that parE and parC were cotranscribed in R6, T6, and T11; and a single promoter located upstream of parE was identified in R6 by primer extension. The fitness of the transformants was estimated by pairwise competition with R6 in both one-cycle and two-cycle experiments. In the one-cycle experiments, most strains carrying the GyrA E85K change showed a fitness cost; the exception was recombinant T14. In the two-cycle experiments, a fitness cost was observed in most first-level transformants carrying the ParC changes S79F, S79Y, and D83Y and the GyrA E85K change; the exceptions were recombinants T6 and T11. The results suggest that there is no impediment due to a fitness cost for the spread of recombinant Cp r S. pneumoniae isolates, since some recombinants (T6, T11, and T14) exhibited an ability to compensate for the cost.

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Seven yeasts strains have been isolated from sewage sludge. Also six samples of compost with different sieving, composting times and origins, have been analysed. Apparently, composting processes negatively affect the viability of yeasts, as none could be isolated from the compost samples. The margins of tolerance of the yeasts to Cd, Cu and Zn have been determined. The physiological response to metals was similar in all the species studied, and in general, kinetic parameters (mu and lag) were affected. Metal uptake ability was also studied and inter- and intra-specific heterogeneity was detected, thus indicating that both the tolerance to metals and the capacity of the uptake were dependent on ionic metal and yeast species. The effect of the presence of multi-metal ions on the uptake capacity of each individual metal was assayed for two selected yeasts, Pichia guilliermondii and Torulaspora delbrueckii. The uptake of each individual metal varied with the combination assayed, and when both strains were compared different results were also found.

Two clinical isolates of viridans group streptococci (VS) with different degrees of susceptibility to optochin (OPT), i.e., fully OPT-susceptible (Opt s ) VS strain 1162/99 (for which the MIC was equal to that for Streptococcus pneumoniae, 0.75 g/ml) and intermediate Opt s VS strain 1174/97 (MIC, 6 g/ml) were studied. Besides being OPT susceptible, they showed characteristics typical of VS, such as bile insolubility; lack of reaction with pneumococcal capsular antibodies; and lack of hybridization with rRNA (AccuProbe)-, lytA-, and pnl-specific pneumococcal probes. However, these VS Opt s strains and VS type strains hybridized with ant, a gene not present in S. pneumoniae. A detailed characterization of the genes encoding the 16S rRNA and SodA classified isolates 1162/99 and 1174/97 as Streptococcus mitis. Analysis of the atpCAB region, which encodes the c, a, and b subunits of the F 0 F 1 H ؉ -ATPase, the target of optochin, revealed high degrees of similarity between S. mitis 1162/99 and S. pneumoniae in atpC, atpA, and the N terminus of atpB. Moreover, amino acid identity between S. mitis 1174/97 and S. pneumoniae was found in ␣ helix 5 of the a subunit. The organization of the chromosomal region containing the atp operon of the two Opt s VS and VS type strains was spr1284-atpC, with spr1284 being located 296 to 556 bp from atpC, whereas in S. pneumoniae this distance was longer than 68 kb. In addition, the gene order in S. pneumoniae was IS1239-74 bp-atpC. The results suggest that the full OPT susceptibility of S. mitis 1162/99 is due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae and that the intermediate OPT susceptibility of S. mitis 1174/97 correlates with the amino acid composition of its a subunit.Streptococcus pneumoniae (the pneumococcus) remains a major etiological agent of community-acquired pneumonia, meningitis, and acute otitis media (6). Four phenotypic characteristics are classically used in the diagnostic laboratory for the identification of the pneumococcus: colony morphology on blood agar plates, optochin (OPT) susceptibility, bile solubility, and immunological reaction with type-specific antisera (23). Although their colony morphologies can be very similar, the alpha-hemolytic oral streptococci (known as viridans group streptococci [VS]), notably, Streptococcus mitis and Streptococcus oralis, are classically OPT resistant (Opt r ) and bile insoluble (23).Genetic and biochemical evidence supports that fact that the typical OPT susceptibility of the pneumococcus resides in the characteristics of the F 0 complex of its F 0 F 1 H ϩ -ATPase, an enzyme that is essential for the viability of this organism (10). The primary roles of this enzyme are to create a proton gradient with the energy provided by ATP hydrolysis and to maintain the intracellular pH via proton extrusion (25), as in other related bacteria (18). However, in bacteria with a respiratory chain, the role of the F 0 F 1 H ϩ -ATPase is the synthesis of ATP from the proton gradient of the respiratory chain. Hydr...

Eight optochin-susceptible (Opt s ) alpha-hemolytic (viridans) streptococcus isolates were characterized at the molecular level. These isolates showed phenotypic characteristics typical of both viridans streptococci and Streptococcus pneumoniae. Comparison of the sequence of housekeeping genes from these isolates with those of S. pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae suggested that the Opt s isolates corresponded to streptococci of the mitis group. Besides, the Opt s streptococci were negative by a Gen-Probe AccuProbe pneumococcus test and hybridized with specific pneumococcal probes (lytA and ply) but also with ant, a gene not present in most S. pneumoniae strains. Moreover, the isolates were insoluble in 1% sodium deoxycholate but completely dissolved in 0.1% deoxycholate. Sequence analysis of the lytA gene revealed that the Opt s streptococci carried lytA alleles characteristic of those present in nonpneumococcal streptococci of the mitis group. The determination of the partial nucleotide sequence embracing the atp operon encoding the F o F 1 H ؉ -ATPase indicated that the optochin susceptibility of the isolates was due to the acquisition of atpC, atpA, and part of atpB from S. pneumoniae by horizontal gene transfer.