Chromosomally and Extrachromosomally Mediated High-Level Gentamicin Resistance in Streptococcus agalactiae.

Streptococcus 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).

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.

Evolutionary dynamics of ompA, the gene encoding the Chlamydia trachomatis key antigen.

Chlamydia trachomatis is the trachoma agent and causes most bacterial sexually transmitted infections worldwide. Its major outer membrane protein (MOMP) is a well-known porin and adhesin and is the dominant antigen. So far, investigation of MOMP variability has been focused mainly on molecular epidemiological surveys. In contrast, we aimed to evaluate the impact of the host pressure on this key antigen by analyzing its evolutionary dynamics in 795 isolates from urogenital infections, taking into account the MOMP secondary structure and the sizes/positions of antigenic regions. One-third of the specimens showed a mutational drift from the corresponding genotype, where approximately 42% of the mutations had never been described. Amino acid alterations were sixfold more frequent within B-cell epitopes than in the remaining protein (P = 0.027), and some mutations were also found within or close to T-cell antigenic clusters. Interestingly, the two most ecologically successful genotypes, E and F, showed a mutation rate 60.3-fold lower than that of the other genotypes (P < 10(-8)), suggesting that their efficacy may be the result of a better fitness in dealing with the host immune system rather than of specific virulence factors. Furthermore, the variability exhibited by some genetic variants involved residues that are known to play a critical role during the membrane mechanical movements, contributing to a more stable and flexible porin conformation, which suggests some plasticity to deal with environmental pressure. Globally, these MOMP mutational trends yielded no mosaic structures or important phylogenetic changes, but instead yielded point mutations on specific protein domains, which may enhance pathogen's infectivity, persistence, and transmission.

Lymphogranuloma venereum in Portugal: unusual events and new variants during 2007.

BACKGROUND: Several European countries identified an ongoing LGV outbreak, particularly among men who have sex with men (MSM). In Portugal, no particular surveillance measures were launched. Nonetheless, circulating LGV strains could eventually be detected through the routine Chlamydia trachomatis ompA genotyping procedure held in the Portuguese National Institute of Health (NIH). METHODS: During 2007, 178 Chlamydia trachomatis specimens were genotyped through amplification and automated-sequencing of ompA. Sequences of 891bp (nt142-nt1032) were aligned with currently available chlamydial sequences from GenBank to identify the corresponding genotype. RESULTS: Eight Chlamydia trachomatis specimens matched LGV-genotypes (7 "L2" and 1 mixed E+L2 undetermined variant). These specimens were identified in samples collected from 4 women and 4 men. One HIV(+) MSM presented LGV related symptoms, while the other infected persons were either asymptomatic or presented no clear LGV symptoms. All samples revealed ompA sequences different from the L2/434 reference strain and from the L2b/144276, which is the most frequently described genotype during the recent LGV outbreak. CONCLUSIONS: The detection of 7 LGV specimens during 2007 in contrast with their absence over the previous 5 years. The LGV infected individuals do not seem to be related to any sexual networks of MSM, contrarily to those described in other European countries. Moreover, all Lisbon LGV specimens revealed unusual ompA sequences that differentiate them from the currently reported LGV infections in Europe. The results of the current study further justify an attentive surveillance of LGV strains infecting different populations and the study of their relation with clinical aspects and disease patterns.

Capsular Type, Sequence Type and Microbial Resistance Factors Impact on DNase Activity of Streptococcus agalactiae Strains from Human and Bovine Origin.

Extracellular 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.

Natural Mutations in Streptococcus agalactiae Resulting in Abrogation of ß Antigen Production.

Streptococcus agalactiae genome encodes 21 two-component systems (TCS) and a variety of regulatory proteins in order to control gene expression. One of the TCS, BgrRS, comprising the BgrR DNA-binding regulatory protein and BgrS sensor histidine kinase, was discovered within a putative virulence island. BgrRS influences cell metabolism and positively control the expression of bac gene, coding for ß antigen at transcriptional level. Inactivation of bgrR abrogated bac gene expression and increased virulence properties of S. agalactiae. In this study, a total of 140 strains were screened for the presence of bac gene, and the TCS bgrR and bgrS genes. A total of 53 strains carried the bac, bgrR and bgrS genes. Most of them (48 strains) expressed ß antigen, while five strains did not express ß antigen. Three strains, in which bac gene sequence was intact, while bgrR and/or bgrS genes had mutations, and expression of ß antigen was absent, were complemented with a constructed plasmid pBgrRS(P) encoding functionally active bgrR and bgrS gene alleles. This procedure restored expression of ß antigen indicating the crucial regulatory role of TCS BgrRS. The complemented strain A49V/BgrRS demonstrated attenuated virulence in intraperitoneal mice model of S. agalactiae infection compared to parental strain A49V. In conclusion we showed that disruption of ß antigen expression is associated with: i) insertion of ISSa4 upstream the bac gene just after the ribosomal binding site; ii) point mutation G342A resulting a stop codon TGA within the bac gene and a truncated form of ß antigen; iii) single deletion (G) in position 439 of the bgrR gene resulting in a frameshift and the loss of DNA-binding domain of the BgrR protein, and iv) single base substitutions in bgrR and bgrS genes causing single amino acid substitutions in BgrR (Arg187Lys) and BgrS (Arg252Gln). The fact that BgrRS negatively controls virulent properties of S. agalactiae gives a novel clue for understanding of S. agalactiae adaptation to the human.

Accuracy of prenatal culture in predicting intrapartum group B streptococcus colonization status.

OBJECTIVE: To evaluate the positive predictive value (PPV) of group B Streptococcus (GBS) cultures at 35-37 weeks of gestation relative to GBS colonization status at delivery. METHODS: Rectovaginal swabs from 221 women at labor in four Lisbon hospitals were collected for GBS screening according to the CDC guidelines. RESULTS: The PPV was 24.4%. IAP was administered to 100% of prenatally GBS positive women. There was no case of early onset GBS disease (EOD). CONCLUSIONS: Poor accuracy of prenatal cultures in identifying true candidates for IAP highlights the need for Portuguese clinical and laboratory guidelines to prevent EOD and antibiotic overtreatment of pregnant women.

Antimicrobial resistance and molecular epidemiology of streptococci from bovine mastitis.

Streptococcus agalactiae (Group B Streptococcus, GBS), Streptococcus dysgalactiae subsp. dysgalactiae (Group C Streptococcus, GCS) and Streptococcus uberis are relevant mastitis pathogens, a highly prevalent and costly disease in dairy industry due to antibiotherapy and loss in milk production. The aims of this study were the evaluation of antimicrobial drug resistance patterns, particularly important for streptococcal mastitis control and the identification of strain molecular features. Antimicrobial resistance was assessed by disk diffusion against amoxicillin-clavulanic acid, cefazolin, cefoperazone, pirlimycin-PRL, rifaximin, streptomycin, chloramphenicol, erythromycin-ERY, gentamicin, tetracycline-TET and vancomycin. Genotypic relationships were identified using pulsed-field gel electrophoresis (PFGE), macrolide and/or tetracycline resistance gene profiling, GBS capsular typing, GBS virulence gene profiling and GBS and S. uberis multi locus sequence typing (MLST). The majority of the isolates were susceptible to all drugs except to aminoglycoside, macrolide, lincosamide and tetracycline. Close to half of the TET resistant isolates have tetO and tetK and almost all ERY-PRL resistant isolates have ermB. A high degree of intra-species polymorphism was found for GCS. The GBS belonged to ST-2, -554, -61, -23 lineages and five new molecular serotypes and human GBS insertion sequences in the cpsE gene were found. Also, GBS of serotype V with scpB and lmb seem to be related with GBS isolates of human origin (same ST-2 and similar PFGE). Overall our results suggested that different therapeutic programs may have been implemented in the different farms and that in most cases clones were herd-specific.

Molecular epidemiology of group B streptococcal meningitis in children beyond the neonatal period from Angola.

Streptococcus agalactiae is a major pathogen of neonates and immunocompromised adults. Prior studies have demonstrated that, beyond the neonatal period, S. agalactiae rarely causes invasive infections in children. However, during 2004-2005, S. agalactiae was the causative agent of 60 meningitis episodes in children aged 3 months to 12 years from Angola. To identify and study the specific causative genetic lineages of S. agalactiae childhood meningitis, which lack characterization to date, we conducted an extensive molecular analysis of the recovered isolates (n = 21). This constitutes what we believe to be the first molecular study of the population structure of invasive S. agalactiae isolates from Africa. A low genetic diversity was observed among the isolates, where the majority belonged to clonal complex (CC) 17 presenting the capsular subtype III-2 (86 % of cases) and marked by the intron group II GBSi1, which has previously been observed to be associated with neonatal hosts. The predominance of single-locus variants of sequence type (ST) 17 suggested the local diversification of this hypervirulent clone, which displayed novel alleles of the fbsB and sip virulence genes. The absence of the scpB-lmb region in two S. agalactiae isolates with the Ia/ST23 genotype is more typical of cattle than human isolates. Globally, these data provide novel information about the enhanced invasiveness of the CC17 genetic lineage in older children and suggest the local diversification of this clone, which may be related to the future emergence of a novel epidemic clone in Angola.

Comparative expression profiling of the Chlamydia trachomatis pmp gene family for clinical and reference strains.

BACKGROUND: Chlamydia trachomatis, an obligate intracellular pathogen, is a leading worldwide cause of ocular and urogenital diseases. Advances have been made in our understanding of the nine-member polymorphic membrane protein (Pmp) gene (pmp) family of C. trachomatis. However, there is only limited information on their biologic role, especially for biological variants (biovar) and clinical strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated expression for pmps throughout development for reference strains E/Bour and L2/434, representing different biovars, and for clinical E and L2 strains. Immunoreactivity of patient sera to recombinant (r)Pmps was also determined. All pmps were expressed at two hours. pmpA had the lowest expression but was up-regulated at 12 h for all strains, indicating involvement in reticulate body development. For pmpD, expression peaked at 36 h. Additionally, 57.7% of sera from infected and 0% from uninfected adolescents were reactive to rPmpD (p = 0.001), suggesting a role in immunogenicity. pmpF had the highest expression levels for all clinical strains and L2/434 with differential expression of the pmpFE operon for the same strains. Sera were nonreactive to rPmpF despite immunoreactivity to rMOMP and rPmpD, suggesting that PmpF is not associated with humoral immune responses. pmpFE sequences for clinical strains were identical to those of the respective reference strains. We identified the putative pmpFE promoter, which was, surprisingly, 100% conserved for all strains. Analyses of ribosomal binding sites, RNase E, and hairpin structures suggested complex regulatory mechanism(s) for this >6 Kb operon. CONCLUSIONS/SIGNIFICANCE: The dissimilar expression of the same pmp for different C. trachomatis strains may explain different strain-specific needs and phenotypic distinctions. This is further supported by the differential immunoreactivity to rPmpD and rPmpF of sera from patients infected with different strains. Furthermore, clinical E strains did not correlate with the E reference strain at the gene expression level, reinforcing the need for expansive studies of clinical strains.

Evolution of Chlamydia trachomatis diversity occurs by widespread interstrain recombination involving hotspots.

Chlamydia trachomatis is an obligate intracellular bacterium of major public health significance, infecting over one-tenth of the world's population and causing blindness and infertility in millions. Mounting evidence supports recombination as a key source of genetic diversity among free-living bacteria. Previous research shows that intracellular bacteria such as Chlamydiaceae may also undergo recombination but whether this plays a significant evolutionary role has not been determined. Here, we examine multiple loci dispersed throughout the chromosome to determine the extent and significance of recombination among 19 laboratory reference strains and 10 present-day ocular and urogenital clinical isolates using phylogenetic reconstructions, compatibility matrices, and statistically based recombination programs. Recombination is widespread; all clinical isolates are recombinant at multiple loci with no two belonging to the same clonal lineage. Several reference strains show nonconcordant phylogenies across loci; one strain is unambiguously identified as recombinantly derived from other reference strain lineages. Frequent recombination contrasts with a low level of point substitution; novel substitutions relative to reference strains occur less than one per kilobase. Hotspots for recombination are identified downstream from ompA, which encodes the major outer membrane protein. This widespread recombination, unexpected for an intracellular bacterium, explains why strain-typing using one or two genes, such as ompA, does not correlate with clinical phenotypes. Our results do not point to specific events that are responsible for different pathogenicities but, instead, suggest a new approach to dissect the genetic basis for clinical strain pathology with implications for evolution, host cell adaptation, and emergence of new chlamydial diseases.

Polymorphisms in the nine polymorphic membrane proteins of Chlamydia trachomatis across all serovars: evidence for serovar Da recombination and correlation with tissue tropism.

Chlamydia trachomatis is an intracellular bacterium responsible for ocular, respiratory, and sexually transmitted diseases. The genome contains a nine-member polymorphic membrane protein (Pmp) family unique to members of the order Chlamydiales. Genomic and molecular analyses were performed for the entire pmp gene family for the 18 reference serological variants (serovars) and genovariant Ja to identify specific gene and protein regions that differentiate chlamydial disease groups. The mean genetic distance among all serovars varied from 0.1% for pmpA to 7.0% for pmpF. Lymphogranuloma venereum (LGV) serovars were the most closely related for the pmp genes and were also the most divergent, compared to ocular and non-LGV urogenital disease groups. Phylogenetic reconstructions showed that for six of nine pmp genes (not pmpA, pmpD, or pmpE), the serovars clustered based on tissue tropism. The most globally successful serovars, E and F, clustered distantly from the urogenital group for five pmp genes. These pmp genes may confer a biologic advantage that may facilitate infection and transmission for E and F. Surprisingly, serovar Da clustered with the ocular group from pmpE to pmpI, which are located together in the chromosome, providing statistically significant evidence for intergenomic recombination and acquisition of a genetic composition that could hypothetically expand the host cell range of serovar Da. We also identified distinct domains for pmpE, pmpF, and pmpH where substitutions were concentrated and associated with a specific disease group. Thus, our data suggest a possible structural or functional role that may vary among pmp genes in promoting antigenic polymorphisms and/or diverse adhesions-receptors that may be involved in immune evasion and differential tissue tropism.