Human IgMhiCD300a+ B Cells Are Circulating Marginal Zone Memory B Cells That Respond to Pneumococcal Polysaccharides and Their Frequency Is Decreased in People Living with HIV.

CD300a is differentially expressed among B cell subsets, although its expression in immunoglobulin (Ig)M+ B cells is not well known. We identified a B cell subset expressing CD300a and high levels of IgM (IgMhiCD300a+). The results showed that IgMhiCD300a+ B cells were CD10-CD27+CD25+IgDloCD21hiCD23-CD38loCD1chi, suggesting that they are circulating marginal zone (MZ) IgM memory B cells. Regarding the immunoglobulin repertoire, IgMhiCD300a+ B cells exhibited a higher mutation rate and usage of the IgH-VDJ genes than the IgM+CD300a- counterpart. Moreover, the shorter complementarity-determining region 3 (CDR3) amino acid (AA) length from IgMhiCD300a+ B cells together with the predicted antigen experience repertoire indicates that this B cell subset has a memory phenotype. IgM memory B cells are important in T cell-independent responses. Accordingly, we demonstrate that this particular subset secretes higher amounts of IgM after stimulation with pneumococcal polysaccharides or a toll-like receptor 9 (TLR9) agonist than IgM+CD300a- cells. Finally, the frequency of IgMhiCD300a+ B cells was lower in people living with HIV-1 (PLWH) and it was inversely correlated with the years with HIV infection. Altogether, these data help to identify a memory B cell subset that contributes to T cell-independent responses to pneumococcal infections and may explain the increase in severe pneumococcal infections and the impaired responses to pneumococcal vaccination in PLWH.

Characterization of recombinant fluoroquinolone-resistant pneumococcus-like isolates.

Fourteen fluoroquinolone-resistant streptococcal isolates with recombinant DNA topoisomerase genes, preliminarily identified as pneumococci, were further characterized using phenotypic and genotypic approaches. Phenotypic tests classified them as atypical pneumococci. Phylogenetic relationships were analyzed by using the sequences of seven housekeeping alleles from these isolates and from isolates of Streptococcus pneumoniae, Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. Four isolates grouped with S. pneumoniae, seven grouped with S. pseudopneumoniae, and three grouped with S. mitis. These results generally agreed with those obtained with an optochin susceptibility test and with the organization of the atp operon chromosomal region, encoding the F(o)F(1) H(+)-ATPase (the target of optochin). All seven isolates grouping with S. pseudopneumoniae share the same spr1368-atpC-atpA gene order; all four grouping with S. pneumoniae share the spr1368-IS1239-atpC-atpA order, and two out of the three grouping with S. mitis share the spr1284-atpC-atpA order. In addition, evidence for recombination within the seven housekeeping alleles of the S. pseudopneumoniae population was provided by several methods: the index of association (0.4598, P < 0.001), the pairwise homoplasy index, and the split-decomposition method. This study confirms the existence of pneumococci among the alpha-hemolytic streptococci with DNA topoisomerase genes showing a mosaic structure and reveals a close relationship between atypical pneumococci and S. pseudopneumoniae.

Nonoptimal DNA topoisomerases allow maintenance of supercoiling levels and improve fitness of Streptococcus pneumoniae.

Fluoroquinolones, which target gyrase and topoisomerase IV, are used for treating Streptococcus pneumoniae infections. Fluoroquinolone resistance in this bacterium can arise via point mutation or interspecific recombination with genetically related streptococci. Our previous study on the fitness cost of resistance mutations and recombinant topoisomerases identified GyrAE85K as a high-cost change. However, this cost was compensated for by the presence of a recombinant topoisomerase IV (parC and parE recombinant genes) in strain T14. In this study, we purified wild-type and mutant topoisomerases and compared their enzymatic activities. In strain T14, both gyrase carrying GyrAE85K and recombinant topoisomerase IV showed lower activities (from 2.0- to 3.7-fold) than the wild-type enzymes. These variations of in vitro activity corresponded to changes of in vivo supercoiling levels that were analyzed by two-dimensional electrophoresis of an internal plasmid. Strains carrying GyrAE85K and nonrecombinant topoisomerases had lower (11.1% to 14.3%) supercoiling density (σ) values than the wild type. Those carrying GyrAE85K and recombinant topoisomerases showed either partial or total supercoiling level restoration, with σ values being 7.9% (recombinant ParC) and 1.6% (recombinant ParC and recombinant ParE) lower than those for the wild type. These data suggested that changes acquired by interspecific recombination might be selected because they reduce the fitness cost associated with fluoroquinolone resistance mutations. An increase in the incidence of fluoroquinolone resistance, even in the absence of further antibiotic exposure, is envisaged.

New species genetic approach to identify strains of mitis group streptococci that are donors of rifampin resistance to Streptococcus pneumoniae.

Eight rifampin-resistant streptococci of the mitis group were identified at the species level by using a concatenated 16S rRNA gene-sodA-rpoB-hlpA sequence. Characterization of their rpoB alleles showed single amino acid changes involved in rifampin resistance. Comparison of RpoB sequences from pneumococcal recombinant isolates, viridans isolates, and type strains revealed a species-specific amino acid signature, which allowed it to be ascertained that recombinant RpoBs were originated in genetic interchanges with Streptococcus mitis and Streptococcus oralis.

Changes in fluoroquinolone-resistant Streptococcus pneumoniae after 7-valent conjugate vaccination, Spain.

Among 4,215 Streptococcus pneumoniae isolates obtained in Spain during 2006, 98 (2.3%) were ciprofloxacin resistant (3.6% from adults and 0.14% from children). In comparison with findings from a 2002 study, global resistance remained stable. Low-level resistance (30 isolates with MIC 4-8 microg/mL) was caused by a reserpine-sensitive efflux phenotype (n = 4) or single topoisomerase IV (parC [n = 24] or parE [n = 1]) changes. One isolate did not show reserpine-sensitive efflux or mutations. High-level resistance (68 isolates with MIC >or=16 microg/mL) was caused by changes in gyrase (gyrA) and parC or parE. New changes in parC (S80P) and gyrA (S81V, E85G) were shown to be involved in resistance by genetic transformation. Although 49 genotypes were observed, clones Spain9V-ST156 and Sweden15A-ST63 accounted for 34.7% of drug-resistant isolates. In comparison with findings from the 2002 study, clones Spain14-ST17, Spain23F-ST81, and ST8819F decreased and 4 new genotypes (ST9710A, ST57016, ST43322, and ST71733) appeared in 2006.

Feasibility of copper uptake by the yeast Pichia guilliermondii isolated from sewage sludge.

Copper is one of the most abundant toxic heavy metals in municipal wastewaters and, in consequence, in sewage sludge and compost. The ability of a strain of the yeast Pichia guilliermondii, which was isolated from sewage sludge, to eliminate copper has been evaluated, using both viable and nonviable biomass. It has been found that raising concentrations of copper affected both morphology and physiological parameters of the viable yeast, and it is thought that a process of bioaccumulation may be involved in its copper uptake. The growth rate of nonadapted cells decreased with increasing concentrations of copper, mainly due to a decrease in the biomass yield. The cells could be adapted by training with increasing copper concentrations up to 317.7 mg/l. This adaptation was an all-or-nothing process: once cells had adapted, the biomass yield, metabolic flux and consequent growth rate were constant and independent of the external copper concentration. Also, it was determined that up to 20 mg of copper per gram of viable adapted biomass could accumulate from the medium (i.e., double the amount when using nonadapted viable biomass). Finally, adsorption data on nonviable cells were found to be well modeled by the Langmuir isotherm (qmax = 9.09 mg/g of biomass).

Fitness of Streptococcus pneumoniae fluoroquinolone-resistant strains with topoisomerase IV recombinant genes.

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.

Molecular characterization of disease-associated streptococci of the mitis group that are optochin susceptible.

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.

Genetic characterization of optochin-susceptible viridans group streptococci.

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 micro g/ml) and intermediate Opt(s) VS strain 1174/97 (MIC, 6 micro 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 alpha 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.

Viridans group streptococci are donors in horizontal transfer of topoisomerase IV genes to Streptococcus pneumoniae.

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.

Fluoroquinolone resistance in penicillin-resistant Streptococcus pneumoniae clones, Spain.

Among 2,882 Streptococcus pneumoniae sent to the Spanish Reference Laboratory during 2002, 75 (2.6%) were ciprofloxacin-resistant. Resistance was associated with older patients (3.9% in adults and 7.2% in patients > or =65 years of age), with isolation from noninvasive sites (4.3% vs. 1.0%), and with penicillin and macrolide resistance. Among 14 low-level resistant (MIC 4-8 microg/mL) strains, 1 had a fluoroquinolone efflux phenotype, and 13 showed single ParC changes. The 61 high-level ciprofloxacin-resistant (MIC > or =16 microg/mL) strains showed either two or three changes at ParC, ParE, and GyrA. Resistance was acquired either by point mutation (70 strains) or by recombination with viridans streptococci (4 strains) at the topoisomerase II genes. Although 36 pulsed-field gel electrophoresis patterns were observed, 5 international multiresistant clones (Spain23F-1, Spain6B-2, Spain9V-3, Spain14-5 and Sweden15A-25) accounted for 35 (46.7%) of the ciprofloxacin-resistant strains. Continuous surveillance is needed to prevent the dissemination of these clones.

Occurrence of yeasts in municipal wastes and their behaviour in presence of cadmium, copper and zinc.

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.