High-Virulence Clone of Group B Streptococci Unable to Grow at High Temperatures Is Present in Serotypes Other Than Type III

Highly virulent clonotypes of serotype III seem to cause much of the perinatal morbidity and mortality attributed to Streptococcus agalactiae (group B streptococci, GBS), One of these clonal types, designated the “high-virulence clone” (HVC), was identified by its inability to grow at 40°C in a chemically defined medium. In the present study, this inability to grow at high temperatures was used as a marker to identify HVC in a sample of 286 Mexican GBS isolates. Forty-three isolates (15%) were identified as belonging to this clone: 15 were invasive isolates, 33 were serotype III (77%), and 10 were of serotypes other than type III (23%). These results demonstrate that HVC is more prevalent in Mexico than previously reported and that this clone is not restricted to serotype III isolates.     

Identification of the High-Virulence Clone of Group B Streptococci in Mexican Isolates by Growth Characteristics at 40°C

Group B streptococci (GBS) colonizing the vagina and rectum of pregnant women cause invasive disease of the offspring in a small number of cases. The immune status of the host and differences in virulence among strains appear to be the main determinants for neonatal infection. A high-virulence clone (HVC) was proposed to cause much of the morbidity and mortality when a collection of GBS isolates was examined by multilocus enzyme electrophoresis. HVC isolates could be further distinguished by their inability to grow at 40°C. This characteristic was used in the present study to examine a collection of 57 GBS isolates from Mexico City for the HVC. Three serotype III invasive strains were classified in the HVC. The other eleven invasive strains and all carrier isolates had growth curves unaffected at 40°C. These results demonstrate the presence of the HVC in Mexico. Such a low prevalence could explain in part the low rate of GBS invasive neonatal disease in Mexico. Received: 21 May 1998 / Accepted: 14 September 1998     

Temperature sensitivity of fructose-1,6-biphosphate aldolase accounts for the inability of the high-virulence clone ofStreptococcus agalactiae to grow at 40°C

A high-virulence clone of serotype IIIStreptoccus agalactiae causing invasive neonatal disease has recently been identified by multilocus enzyme electrophoresis and can be further distinguished by its inability to grow at 40°C in a chemically defined medium. The basis for the unusual growth inhibition at 40°C was examined in the present study and shown to be owing to a temperature-sensitive fructose-1,6-bisphosphate aldolase (fba). Crude enzyme preparations (75% saturated ammonium sulfate precipitates) of fba obtained from a high-virulence clone demonstrated a 75% reduction in aldolase activity when preincubated at 40°C for 30 min compared with 37°C. In contrast, fba from a serotype III isolate obtained from an asymptomatically colonized infant demonstrated <10% decrease in activity at 40°C. Comparison of another enzyme, lactate dehydrogenase (ldh), from both organisms indicated no loss in activity at 40°C compared with 37°C. Glyceraldehyde-3-phosphate, one of the end-products of fba activity, relieved growth inhibition at 40°C.     

A practical method for subtyping of Streptococcus agalactiae serotype III, of human origin, using rolling circle amplification

Group B streptococcus (GBS; Streptococcus agalactiae) serotype III is one of the most common and virulent serotypes of the species. It can be divided into several subtypes, which vary in their distribution among invasive isolates from different patient groups. In this study, we used 91 well-characterized GBS serotype III isolates to compare three subtyping methods, and developed a novel padlock probe and rolling circle amplification (RCA) method to identify informative single nucleotide polymorphisms (SNPs) that define the major subtypes. There was good agreement between partial sequencing of the capsule polysaccharide synthesis (cps) gene cluster, a 3-set genotyping system and multilocus sequence typing (MLST). Serosubtype III-2/multilocus sequence type (ST)-17 represents a virulent clone which is significantly associated with late onset GBS neonatal infections. RCA provides a simple, reproducible method for rapid identification of the two most common GBS serotype III subtypes (III-1/ST-19 and III-2/ST-17).     

Serotype distribution and invasive potential of group B streptococcus isolates causing disease in infants and colonizing maternal-newborn dyads

Background

Serotype-specific polysaccharide based group B streptococcus (GBS) vaccines are being developed. An understanding of the serotype epidemiology associated with maternal colonization and invasive disease in infants is necessary to determine the potential coverage of serotype-specific GBS vaccines.

Methods

Colonizing GBS isolates were identified by vaginal swabbing of mothers during active labor and from skin of their newborns post-delivery. Invasive GBS isolates from infants were identified through laboratory-based surveillance. GBS serotyping was done by latex agglutination. Serologically non-typeable isolates were typed by a serotype-specific PCR method. The invasive potential of GBS serotypes associated with sepsis within seven days of birth was evaluated in association to maternal colonizing serotypes.

Results

GBS was identified in 289 (52.4%) newborns born to 551 women with GBS-vaginal colonization and from 113 (5.6%) newborns born to 2,010 mothers in whom GBS was not cultured from vaginal swabs. The serotype distribution among vaginal-colonizing isolates was as follows: III (37.3%), Ia (30.1%), and II (11.3%), V (10.2%), Ib (6.7%) and IV (3.7%). There were no significant differences in serotype distribution between vaginal and newborn colonizing isolates (P = 0.77). Serotype distribution of invasive GBS isolates were significantly different to that of colonizing isolates (P<0.0001). Serotype III was the most common invasive serotype in newborns less than 7 days (57.7%) and in infants 7 to 90 days of age (84.3%; P<0.001). Relative to serotype III, other serotypes showed reduced invasive potential: Ia (0.49; 95%CI 0.31–0.77), II (0.30; 95%CI 0.13–0.67) and V (0.38; 95%CI 0.17–0.83).

Conclusion

In South Africa, an anti-GBS vaccine including serotypes Ia, Ib and III has the potential of preventing 74.1%, 85.4% and 98.2% of GBS associated with maternal vaginal-colonization, invasive disease in neonates less than 7 days and invasive disease in infants between 7–90 days of age, respectively.     

Acidic pH Strongly Enhances In Vitro Biofilm Formation by a Subset of Hypervirulent ST-17 Streptococcus agalactiae Strains

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a primary colonizer of the anogenital mucosa of up to 40% of healthy women and an important cause of invasive neonatal infections worldwide. Among the 10 known capsular serotypes, GBS type III accounts for 30 to 76% of the cases of neonatal meningitis. In recent years, the ability of GBS to form biofilm attracted attention for its possible role in fitness and virulence. Here, a new in vitro biofilm formation protocol was developed to guarantee more stringent conditions, to better discriminate between strong-, low-, and non-biofilm-forming strains, and to facilitate interpretation of data. This protocol was used to screen the biofilm-forming abilities of 366 GBS clinical isolates from pregnant women and from neonatal infections of different serotypes in relation to medium composition and pH. The results identified a subset of isolates of serotypes III and V that formed strong biofilms under acidic conditions. Importantly, the best biofilm formers belonged to serotype III hypervirulent clone ST-17. Moreover, the abilities of proteinase K to strongly inhibit biofilm formation and to disaggregate mature biofilms suggested that proteins play an essential role in promoting GBS biofilm initiation and contribute to biofilm structural stability.     

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.     

Genetic islands of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains NEM316 and 2603VR and their presence in other Group B Streptococcal strains

Background  

Streptococcus agalactiae (Group B Streptococcus; GBS) is a major contributor to obstetric and neonatal bacterial sepsis. Serotype III strains cause the majority of late-onset sepsis and meningitis in babies, and thus appear to have an enhanced invasive capacity compared with the other serotypes that cause disease predominantly in immunocompromised pregnant women. We compared the serotype III and V whole genome sequences, strains NEM316 and 2603VR respectively, in an attempt to identify genetic attributes of strain NEM316 that might explain the propensity of strain NEM316 to cause late-onset disease in babies. Fourteen putative pathogenicity islands were described in the strain NEM316 whole genome sequence. Using PCR- and targeted microarray- strategies, the presence of these islands were assessed in a diverse strain collection including 18 colonizing isolates from healthy pregnant women, and 13 and 8 invasive isolates from infants with early- and late-onset sepsis, respectively.     

Restriction pattern of the major outer-membrane protein gene provides evidence for a homogeneous invasive group among ruminant isolates of Chlamydia psittaci.

Thirty-six ruminant isolates of Chlamydia psittaci, previously classified as invasive or non-invasive in a mouse model of virulence, were compared by analysing AluI restriction patterns of the major outer-membrane protein (MOMP) gene after DNA amplification by the polymerase chain reaction. The 24 invasive isolates, although from various origins, all belonged to serotype 1 and represented a strictly homogeneous group sharing a specific MOMP-gene restriction pattern that was not observed in the non-invasive strains. On the other hand, the 12 non-invasive strains, although all belonging to serotype 2, constituted a heterogeneous group with eight distinct MOMP-gene restriction patterns. However, all eight patterns shared a 180 bp fragment or the corresponding restricted fragments of 110 and 70 bp. MOMP-gene restriction patterns also clearly distinguished the ruminant strains from an avian C. psittaci isolate, a C. pneumoniae isolate and two C. trachomatis isolates which were studied for comparison. The homogeneous character of the invasive C. psittaci strains argues strongly for their genetic relatedness. Our results illustrate the usefulness of the MOMP-gene restriction mapping in typing chlamydiae.     

Restriction fragment polymorphism in mitochondrial DNA of Cryptococcus neoformans

The restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support the current taxonomic classification of C. neoformans into two varieties.     

Diversity of prophage DNA regions of Streptococcus agalactiae clonal lineages from adults and neonates with invasive infectious disease

The phylogenetic position and prophage DNA content of the genomes of 142 S. agalactiae (group-B streptococcus, GBS) isolates responsible for bacteremia and meningitis in adults and neonates were studied and compared. The distribution of the invasive isolates between the various serotypes, sequence types (STs) and clonal complexes (CCs) differed significantly between adult and neonatal isolates. Use of the neighbor-net algorithm with the PHI test revealed evidence for recombination in the population studied (PHI, P = 2.01 × 10(-6)), and the recombination-mutation ratio (R/M) was 6:7. Nevertheless, the estimated R/M ratio differed between CCs. Analysis of the prophage DNA regions of the genomes of the isolates assigned 90% of the isolates to five major prophage DNA groups: A to E. The mean number of prophage DNA fragments amplified per isolate varied from 2.6 for the isolates of prophage DNA group E to 4.0 for the isolates of prophage DNA group C. The isolates from adults and neonates with invasive diseases were distributed differently between the various prophage DNA groups (P < 0.00001). Group C prophage DNA fragments were found in 52% of adult invasive isolates, whereas 74% of neonatal invasive isolates had prophage DNA fragments of groups A and B. Differences in prophage DNA content were also found between serotypes, STs and CCs (P < 0.00001). All the ST-1 and CC1 isolates, mostly of serotype V, belonged to the prophage DNA group C, whereas 84% of the ST-17 and CC17 isolates, all of serotype III, belonged to prophage DNA groups A and B. These data indicate that the transduction mechanisms, i.e., gene transfer from one bacterium to another by a bacteriophage, underlying genetic recombination in S. agalactiae species, are specific to each intraspecies lineage and population of strains responsible for invasive diseases in adults and neonates.     

Properties of extracellular proteases from three psychrotolerant Stenotrophomonas maltophilia isolated from Antarctic soil

Three Antarctic psychrotolerant Stenotrophomonas maltophilia were isolated and the characteristics of their extracellular serine proteases were described. The isolates were able to grow at 14 and 34°C, but grew better between 20 and 28°C. The highest protease secretion was reached at 20–24°C. The purified enzyme preparations had maximal activity at 55–60°C and alkaline pH. They showed high pH stability, retaining more than 60% of residual activity after 3 h of incubation at a pH range of 4–12. The thermal stability was slightly lower compared with a commercial mesophilic protease, with 74–79% residual activity after 90 min at 40°C and 50% inactivation at 50°C between 43 and 69 min. These properties suggest that the Antarctic isolates could be adapted to cold by means of synthesising more enzymes with high activity but that the proteases they produce are not truly cold-active, being more similar to mesophilic enzymes.     

A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain variation in virulence

The bacterial factors responsible for the variation in invasive potential between different clones and serotypes of Streptococcus pneumoniae are largely unknown. Therefore, the isolation of rare serotype 1 carriage strains in Indigenous Australian communities provided a unique opportunity to compare the genomes of non-invasive and invasive isolates of the same serotype in order to identify such factors. The human virulence status of non-invasive, intermediately virulent and highly virulent serotype 1 isolates was reflected in mice and showed that whilst both human non-invasive and highly virulent isolates were able to colonize the murine nasopharynx equally, only the human highly virulent isolates were able to invade and survive in the murine lungs and blood. Genomic sequencing comparisons between these isolates identified 8 regions >1 kb in size that were specific to only the highly virulent isolates, and included a version of the pneumococcal pathogenicity island 1 variable region (PPI-1v), phage-associated adherence factors, transporters and metabolic enzymes. In particular, a phage-associated endolysin, a putative iron/lead permease and an operon within PPI-1v exhibited niche-specific changes in expression that suggest important roles for these genes in the lungs and blood. Moreover, in vivo competition between pneumococci carrying PPI-1v derivatives representing the two identified versions of the region showed that the version of PPI-1v in the highly virulent isolates was more competitive than the version from the less virulent isolates in the nasopharyngeal tissue, blood and lungs. This study is the first to perform genomic comparisons between serotype 1 isolates with distinct virulence profiles that correlate between mice and humans, and has highlighted the important role that hypervariable genomic loci, such as PPI-1v, play in pneumococcal disease. The findings of this study have important implications for understanding the processes that drive progression from colonization to invasive disease and will help direct the development of novel therapeutic strategies.     

The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library

Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms.     

Comparative genomics inferred two distinct populations of piscine pathogenic Streptococcus agalactiae,serotype Ia ST7 and serotype III ST283, in Thailand and Vietnam

The genomes of Streptococcus agalactiae (group B streptococcus; GBS) collected from diseased fish in Thailand and Vietnam over a nine-year period (2008–2016) were sequenced and compared (n = 21). Based on capsular serotype and multilocus sequence typing (MLST), GBS isolates are divided into 2 groups comprised of i) serotype Ia; sequence type (ST)7 and ii) serotype III; ST283. Population structure inferred by core genome (cg)MLST and Bayesian clustering analysis also strongly indicated distribution of two GBS populations in both Thailand and Vietnam. Deep phylogenetic analysis implied by CRISPR array's spacer diversity was able to cluster GBS isolates according to their temporal and geographic origins, though ST7 has varying CRISPR1-spacer profiles when compared to ST283 strains. Based on overall genotypic features, Thai ST283 strains were closely related to the Singaporean ST283 strain causing foodborne illness in humans in 2015, thus, signifying zoonotic potential of this GBS population in the country.     

Characterization of DNA restriction-modification systems in Spirulina platensis strain pacifica

Four unique restriction enzymes were identified in the soluble protein fraction of Spirulina platensis strain pacifica, a commercially important strain of marine cyanobacterium that is used as a supplement in a human diets. These are SpaI, SpaII, SpaIII and SpaIV, which are isoschizomers of Tth111I, Pvul, PvuII and HindIII, respectively. The recognition sites of each of these four enzymes were identified by restriction digests of different plasmid DNAs of known sequence and determining the cleavage sites by sequencing. SpaI is the most predominant restriction enzyme present in S. platensis strain pacifica. It shows high activity at 37 °C compared to 65 °C for its isoschizomer Tth111I.Department of Plant Molecular Physiology     

Analysis of cytomegalovirus genomes with restriction endonucleases Hin D III and EcoR-1.

Cleavage of genomes of eleven human, one simian, and one simian-related cytomegalovirus (CMV) isolate by the restriction endonucleases HinD III and EcoR-1 generated reproducible DNA fragments. The size range of CMV DNA fragments as estimated by contour length measurements in comparison with simian virus 40 form II DNA and by coelectrophoresis with EcoR-1 fragments of herpes simplex virus DNA varied between 15 X 10(6) and 0.5 X 10(6) daltons. Comparison of the cleavage products of each isolate in 1% agarose slab gels showed extensive comigration of fragments among the human CMV isolates. In the HinD III digests, three fragment bands comigrated among all human CMV isolates, and six fragments comigrated among most, but not all, human CMV isolates. In the EcoR-1 digests, nine fragment bands comigrated among all human CMV isolates, and five bands comigrated among most, but not all human isolates. Each isolate had a distinctive electrophoretic profile with either HinD III or EcoR-1 digests. No two isolates had identical HinD III or EcoR-1 patterns although some isolates did share more general pattern similarities than others. No clear-cut subgrouping of isolates based on cleavage pattern characteristics could be discerned. Comparison of HinD III and EcoR-1 patterns showed that human isolates differ greatly from nonhuman CMV isolates. HinD III and EcoR-1 digests of each isolate contained both major and minor molar classes of DNA fragments that ranged from about 1 and multiples of 1 M down to about 0.25 M; however, the summed molecular weights for major molar fragments resulting from HinD III or EcoR-1 digests of several isolates closely approximated the molecular weight of 10(8) of the intact genome.     

PCR-based restriction fragment length polymorphism (RFLP) analysis and serotyping of Campylobacter jejuni isolates from diarrheic patients in China and Japan

Abstract A molecular typing approach for Campylobacter jejuni was applied with restriction fragment length polymorphism (RFLP) analysis of a 702-bp PCR-amplified portion of the flagellin-A ( flaA ) gene. We analyzed a total of 179 strains, including 69 independent clinical isolates from diarrheic patients in Japan, 85 isolates in China, and 25 heat-stable (HS) serotype strains by Penner and Hennessy ((1980) J. Clin. Microbiol. 12, 732–737). Six Afa I, seven Mbo I, and five Hae III RFLPs were found in the 702-bp flaA segment from the 179 strains. Using a combination of these three enzymes, 25 separate RFLP groups were recognized. While 59 of 154 (38.3%) strains obtained in Japan and China were nontypeable by the HS antigenic scheme, all but two of 154 (98.7%) could be typed by RFLP typing. All 11 isolates of HS-19 strains, which are frequently isolated from Guillain-Barré syndrome (GBS) patients, showed an identical RFLP pattern (Cj-1), and Cj-1 consisted only of HS-19 strains. This suggests that the HS-19:Cj-l strain is distinct among C. jejuni strains. This molecular typing method provides a rapid and reliable typing scheme for epidemiological studies of C. jejuni , and may also be useful for the analysis of C. jejuni subtypes from GBS patients.     

Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene.

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P.aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.