Browsing by Author "Florindo, Carlos"
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- Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine OriginPublication . Florindo, Carlos; Barroco, Cinthia Alves; Silvestre, Inês; Damião, Vera; Gomes, João Paulo; Spellerberg, Barbara; Santos-Sanches, Ilda; Borrego, Maria JoséExtracellular deoxyribonucleases (DNases) contribute to the spread of pathogenic bacteria through the evasion from host innate immunity. Our main objective was to evaluate the production of extracellular DNases by human and bovine Streptococcus agalactiae clinical strains and perform a correlation of genetic lineages and DNase activity with capsular type, genetic determinants, clinical origin (colonization and infection), and host (human or bovine). DNase activity was evaluated by qualitative and quantitative assays for a collection of 406 human (n = 285) and bovine (n = 121) strains. All (121/121) bovine were isolated from mastitis and revealed to be DNase (+), indicating a putative pathogenic role in this clinical scenario. From the human S. agalactiae strains, 86% (245/285) showed DNase activity, among which all strains belonging to capsular types, namely, Ia, Ib, III-2, and IV. All CC17 strains (n = 58) and 56/96 (58.3%) of the CC19 displayed DNase activity. DNase (-) strains belonged to the CC19 group. However, the subcharacterization of CC19 S. agalactiae strains through multiple-locus variable number tandem repeat analysis (MLVA), antibiotic resistance, mobile elements, and surface proteins did not provide any distinction among DNase producers and non-producers. The production of DNases by all human CC17 strains, about two-fifths of human CC19, and all bovine strains, suggest an important contribution of DNases to hypervirulence.
- Chromosomally and Extrachromosomally Mediated High-Level Gentamicin Resistance in Streptococcus agalactiaePublication . Sendi, Parham; Furitsch, Martina; Mauerer, Stefanie; Florindo, Carlos; Kahl, Barbara C.; Shabayek, Sarah; Berner, Reinhard; Spellerberg, BarbaraStreptococcus agalactiae (group B Streptococcus [GBS]) is a leading cause of sepsis in neonates. The rate of invasive GBS disease in nonpregnant adults also continues to climb. Aminoglycosides alone have little or no effect on GBS, but synergistic killing with penicillin has been shown in vitro. High-level gentamicin resistance (HLGR) in GBS isolates, however, leads to the loss of a synergistic effect. We therefore performed a multicenter study to determine the frequency of HLGR GBS isolates and to elucidate the molecular mechanisms leading to gentamicin resistance. From eight centers in four countries, 1,128 invasive and colonizing GBS isolates were pooled and investigated for the presence of HLGR. We identified two strains that displayed HLGR (BSU1203 and BSU452), both of which carried the aacA-aphD gene, typically conferring HLGR. However, only one strain (BSU1203) also carried the previously described chromosomal gentamicin resistance transposon designated Tn3706. For the other strain (BSU452), plasmid purification and subsequent DNA sequencing resulted in the detection of plasmid pIP501 carrying a remnant of a Tn3 family transposon. Its ability to confer HLGR was proven by transfer into an Enterococcus faecalis isolate. Conversely, loss of HLGR was documented after curing both GBS BSU452 and the transformed E. faecalis strain from the plasmid. This is the first report showing plasmid-mediated HLGR in GBS. Thus, in our clinical GBS isolates, HLGR is mediated both chromosomally and extrachromosomally.
- Genotypes and antimicrobial resistant phenotypes of Neisseria gonorrhoeae in Portugal (2004-2009)Publication . Florindo, Carlos; Pereira, Rui; Boura, Márcia; Nunes, Baltazar; Paulino, Albertina; Gomes, João Paulo; Borrego, Maria J.OBJECTIVES: To determine the antibiotic phenotype and MAST-genotype distribution of Neisseria gonorrhoeae isolates in Portugal between 2004 and 2009, and to evaluate specific associations between MAST-genotypes and sexual orientation, age and antibiotic resistance. METHODS: A total of 236 N gonorrhoeae isolates were typed through N gonorrhoeae multiantigen sequence typing (NG-MAST). The degree of polymorphism and the phylogenetic relatedness among NG-MAST sequence types (STs) were evaluated with MEGA4 software on concatenated sequences of por and tbpb alleles. Etest was used to determine the susceptibility to ceftriaxone, ciprofloxacin, penicillin and spectinomycin. RESULTS: No isolates displayed resistance to spectinomycin and ceftriaxone, whereas 79.1% and 37.4% were resistant to penicillin and ciprofloxacin, respectively. A total of 104 different STs (one per 2.3 isolates) were found; the most common were ST210 (8.1%) and ST225 (7.6%). STs formed two major groups separated by 159.8 (SE 8.9) nucleotide differences, yielding several subgroups, one of them including the worldwide-prevalent ST225. The probability of ciprofloxacin resistance among isolates within this subgroup was 73.5-fold higher than for the remaining isolates. Indeed, for the genetically closest subgroup, which includes the most prevalent ST (ST210), only 8.0% of isolates were resistant to ciprofloxacin. There was a non-homogeneous distribution per year for ST225 (p<0.001), ST210 (p=0.011) and ST2 (p=0.007). CONCLUSIONS: The heterogeneous ST scenario may represent the 'tip of the iceberg', reflecting a high number of undiagnosed and unreported gonorrhoea cases. A laboratory-based national surveillance of N gonorrhoeae infections is necessary to provide a broader spectrum of isolates that will allow the sexual network situation in Portugal to be established.
- Selection of reference genes for real-time expression studies in Streptococcus agalactiaePublication . Florindo, Carlos; Ferreira, Rita; Borges, Vitor; Spellerberg, Barbara; Gomes, João Paulo; Borrego, Maria JoséStreptococcus agalactiae, group B streptococci (GBS) is the leading cause of severe bacterial infections in newborns. GBS expression studies allowed the identification and characterization of virulence factors and a better understanding of the host-pathogen-environment interactions. The measurement of transcript levels by quantitative real-time PCR (qRT-PCR) is a widely used technique in GBS; however, a systematic evaluation and validation of reference gene stability for normalization purposes in GBS expression studies is currently lacking. Therefore, we analyzed the stability of 10 candidate reference genes (16SrRNA, glcK, glnA, groEL, gyrA, recA, rpoB, rpsL, sdhA and tkt) in three GBS prototype strains (O90R, NEM316 and 2603V/R) grown at different temperature conditions (37°C and 40°C). Our approach was based on the calibration of transcript levels from each gene against the number of bacteria from the same sample (ratio messenger RNA/genomic DNA). As a complementary analysis, reference gene stability was also investigated through the bioinformatic applications, geNorm and NormFinder. Considering the whole GBS development cycle, only a minority of genes were stable under both growth conditions, but this number increased when restricting the analysis to the logarithmic time-points. The range of stable genes was higher at 37°C, where recA and sdhA were stable simultaneously for the three strains, and six out of 10 genes were stable for at least two strains. At 40°C, recA showed up again as one of the best options, suggesting its potential use as reference gene in future qRT-PCR studies. The results generated with geNorm and NormFinder were consistent with those obtained experimentally and evidenced minor variations either among strains or temperature conditions. In conclusion, the fluctuation of expression of reference genes observed among different GBS strains and growth conditions highlights the importance of carefully validating, for each experimental scenario, the use of reference genes for qRT-PCR normalization purposes. Nevertheless, recA seems to be a good candidate for such optimizations.