A Counterselectable Sucrose Sensitivity Marker Permits Efficient and Flexible Mutagenesis in Streptococcus agalactiae.

Streptococcus agalactiae (group B Streptococcus [GBS]) is a cause of severe infections, particularly during the newborn period. While methods exist for generating chromosomal mutations in GBS, they are cumbersome and inefficient and present significant challenges if the goal is to study subtle mutations, such as single-base-pair polymorphisms. To address this problem, we have developed an efficient and flexible GBS mutagenesis protocol based on sucrose counterselection against levansucrase (SacB) expressed from a temperature-selective shuttle vector. GBS containing the SacB expression cassette demonstrates lethal sensitivity to supplemental sucrose whether the plasmid DNA is replicating outside of the chromosome or has been integrated during a crossover event. Transmission electron microscopy shows that SacB-mediated lethal sucrose sensitivity results from the accumulation of inclusion bodies that eventually lead to complete degradation of normal cellular architecture and subsequent lysis. We used this new mutagenesis technique to generate an in-frame, allelic exchange knockout of the GBS sortase gene srtA, demonstrating that >99% of colonies that emerge from our protocol had the expected knockout phenotype and that among a subset tested by sequencing, 100% had the correct genotype. We also generated barcoded nonsense mutations in the cylE gene in two GBS strains, showing that the approach can be used to make small, precise chromosomal mutations.IMPORTANCE The ability to generate chromosomal mutations is fundamental to microbiology. Historically, however, GBS pathogenesis research has been made challenging by the relative genetic intractability of the organism. Generating a single knockout in GBS using traditional techniques can take many months, with highly variable success rates. Furthermore, traditional methods do not offer a straightforward way to generate single-base-pair polymorphisms or other subtle changes, especially to noncoding regions of the chromosome. We have developed a new sucrose counterselection-based method that permits rapid, efficient, and flexible GBS mutagenesis. Our technique requires no additional equipment beyond what is needed for traditional approaches. We believe that it will catalyze rapid advances in GBS genetics research by significantly easing the path to generating mutants.

Diminished Capsule Exacerbates Virulence, Blood-Brain Barrier Penetration, Intracellular Persistence, and Antibiotic Evasion of Hyperhemolytic Group B Streptococci.

Group B streptococci (GBS) are encapsulated, ß-hemolytic bacteria that are a common cause of infections in human newborns and certain adults. Two factors important for GBS virulence are the sialic acid capsular polysaccharide that promotes immune evasion and the hemolytic pigment that induces host cell cytotoxcity. These virulence factors are often oppositely regulated by the CovR/CovS two-component system. Clinical GBS strains exhibiting hyperhemolysis and low capsule due to pathoadaptive covR/S mutations have been isolated from patients. Given the importance of capsule to GBS virulence, we predicted that a decrease or loss of capsule would attenuate the virulence of covR/S mutants. Surprisingly, hyperhemolytic GBS with low or no capsule exhibit increased virulence, intracellular persistence, and blood-brain barrier penetration, which was independent of a Trojan horse mechanism of barrier penetration. Additionally, intracellular persistence enabled both hemolytic and hyperhemolytic GBS to evade antibiotics routinely used to treat these infections. The finding that diminished capsule expression promotes GBS virulence, intracellular persistence, and antibiotic evasion has important implications for sustained antibiotic therapy and efficacy of capsule-based vaccines.

Modification of the CpsA protein reveals a role in alteration of the Streptococcus agalactiae cell envelope.

The bacterial cell envelope is a crucial first line of defense for a systemic pathogen, with production of capsular polysaccharides and maintenance of the peptidoglycan cell wall serving essential roles in survival in the host environment. The LytR-CpsA-Psr proteins are important for cell envelope maintenance in many Gram-positive species. In this study, we examined the role of the extracellular domain of the CpsA protein of the zoonotic pathogen group B Streptococcus in capsule production and cell wall integrity. CpsA has multiple functional domains, including a DNA-binding/transcriptional activation domain and a large extracellular domain. We demonstrated that episomal expression of extracellularly truncated CpsA causes a dominant-negative effect on capsule production when expressed in the wild-type strain. Regions of the extracellular domain essential to this phenotype were identified. The dominant-negative effect could be recapitulated by addition of purified CpsA protein or a short CpsA peptide to cultures of wild-type bacteria. Changes in cell wall morphology were also observed when the dominant-negative peptide was added to wild-type cultures. Fluorescently labeled CpsA peptide could be visualized bound at the mid-cell region near the division septae, suggesting a novel role for CpsA in cell division. Finally, expression of truncated CpsA also led to attenuation of virulence in zebrafish models of infection, to levels below that of a cpsA deletion strain, demonstrating the key role of the extracellular domain in virulence of GBS.

RovS and its associated signaling peptide form a cell-to-cell communication system required for Streptococcus agalactiae pathogenesis.

UNLABELLED: Bacteria can communicate with each other to coordinate their biological functions at the population level. In a previous study, we described a cell-to-cell communication system in streptococci that involves a transcriptional regulator belonging to the Rgg family and short hydrophobic peptides (SHPs) that act as signaling molecules. Streptococcus agalactiae, an opportunistic pathogenic bacterium responsible for fatal infections in neonates and immunocompromised adults, has one copy of the shp/rgg locus. The SHP-associated Rgg is called RovS in S. agalactiae. In this study, we found that the SHP/RovS cell-to-cell communication system is active in the strain NEM316 of S. agalactiae, and we identified different partners that are involved in this system, such as the Eep peptidase, the PptAB, and the OppA1-F oligopeptide transporters. We also identified a new target gene controlled by this system and reexamined the regulation of a previously proposed target gene, fbsA, in the context of the SHP-associated RovS system. Furthermore, our results are the first to indicate the SHP/RovS system specificity to host liver and spleen using a murine model, which demonstrates its implication in streptococci virulence. Finally, we observed that SHP/RovS regulation influences S. agalactiae's ability to adhere to and invade HepG2 hepatic cells. Hence, the SHP/RovS cell-to-cell communication system appears to be an essential mechanism that regulates pathogenicity in S. agalactiae and represents an attractive target for the development of new therapeutic strategies. IMPORTANCE: Rgg regulators and their cognate pheromones, called small hydrophobic peptides (SHPs), are present in nearly all streptococcal species. The general pathways of the cell-to-cell communication system in which Rgg and SHP take part are well understood. However, many other players remain unidentified, and the direct targets of the system, as well as its link to virulence, remain unclear. Here, we identified the different players involved in the SHP/Rgg system in S. agalactiae, which is the leading agent of severe infections in human newborns. We have identified a direct target of the Rgg regulator in S. agalactiae (called RovS) and examined a previously proposed target, all in the context of associated SHP. For the first time, we have also demonstrated the implication of the SHP/RovS mechanism in virulence, as well as its host organ specificity. Thus, this cell-to-cell communication system may represent a future target for S. agalactiae disease treatment.

SecA localization and SecA-dependent secretion occurs at new division septa in group B Streptococcus.

Exported proteins of Streptococcus agalactiae (GBS), which include proteins localized to the bacterial surface or secreted into the extracellular environment, are key players for commensal and pathogenic interactions in the mammalian host. These proteins are transported across the cytoplasmic membrane via the general SecA secretory pathway and those containing the so-called LPXTG sorting motif are covalently attached to the peptidoglycan by sortase A. How SecA, sortase A, and LPXTG proteins are spatially distributed in GBS is not known. In the close relative Streptococcus pyogenes, it was shown that presence of the YSIRKG/S motif (literally YSIRKX3Gx2S) in the signal peptide (SP) constitutes the targeting information for secretion at the septum. Here, using conventional and deconvolution immunofluorescence analyses, we have studied in GBS strain NEM316 the localization of SecA, SrtA, and the secreted protein Bsp whose signal peptide contains a canonical YSIRKG/S motif (YSLRKykfGlaS). Replacing the SP of Bsp with four other SPs containing or not the YSIRKG/S motif did not alter the localized secretion of Bsp at the equatorial ring. Our results indicate that secretion and cell wall-anchoring machineries are localized at the division septum. Cell wall- anchored proteins displayed polar (PilB, Gbs0791), punctuate (CspA) or uniform distribution (Alp2) on the bacterial surface. De novo secretion of Gbs0791 following trypsin treatment indicates that it is secreted at the septum, then redistributed along the lateral sides, and finally accumulated to the poles. We conclude that the ±YSIRK SP rule driving compartimentalized secretion is not true in S. agalactiae.

Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.

Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.

Structural differences between the Streptococcus agalactiae housekeeping and pilus-specific sortases: SrtA and SrtC1.

The assembly of pili on the cell wall of Gram-positive bacteria requires transpeptidase enzymes called sortases. In Streptococcus agalactiae, the PI-1 pilus island of strain 2603V/R encodes two pilus-specific sortases (SrtC1 and SrtC2) and three pilins (GBS80, GBS52 and GBS104). Although either pilus-specific sortase is sufficient for the polymerization of the major pilin, GBS80, incorporation of the minor pilins GBS52 and GBS104 into the pilus structure requires SrtC1 and SrtC2, respectively. The S. agalactiae housekeeping sortase, SrtA, whose gene is present at a different location and does not catalyze pilus polymerization, was shown to be involved in cell wall anchoring of pilus polymers. To understand the structural basis of sortases involved in such diverse functions, we determined the crystal structures of S. agalactiae SrtC1 and SrtA. Both enzymes are made of an eight-stranded beta-barrel core with variations in their active site architecture. SrtA exhibits a catalytic triad arrangement similar to that in Streptococcus pyogenes SrtA but different from that in Staphylococcus aureus SrtA. In contrast, the SrtC1 enzyme contains an N-terminal helical domain and a 'lid' in its putative active site, which is similar to that seen in Streptococcus pneumoniae pilus-specific sortases, although with subtle differences in positioning and composition. To understand the effect of such differences on substrate recognition, we have also determined the crystal structure of a SrtC1 mutant, in which the conserved DP(W/F/Y) motif was replaced with the sorting signal motif of GBS80, IPNTG. By comparing the structures of WT wild type SrtA and SrtC1 and the 'lid' mutant of SrtC1, we propose that structural elements within the active site and the lid may be important for defining the role of specific sortase in pili biogenesis.

The GBS PI-2a pilus is required for virulence in mice neonates.

BACKGROUND: Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis and meningitis in newborns. Most bacterial pathogens, including gram-positive bacteria, have long filamentous structures known as pili extending from their surface. Although pili are described as adhesive organelles, they have been also implicated in many other functions including thwarting the host immune responses. We previously characterized the pilus-encoding operon PI-2a (gbs1479-1474) in strain NEM316. This pilus is composed of three structural subunit proteins: PilA (Gbs1478), PilB (Gbs1477), and PilC (Gbs1474), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component whereas PilA, the pilus associated adhesin, and PilC the pilus anchor are both accessory proteins incorporated into the pilus backbone. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the role of the major pilin subunit PilB was tested in systemic virulence using 6-weeks old and newborn mice. Notably, the non-piliated ΔpilB mutant was less virulent than its wild-type counterpart in the newborn mice model. Next, we investigated the possible role(s) of PilB in resistance to innate immune host defenses, i.e. resistance to macrophage killing and to antimicrobial peptides. Phagocytosis and survival of wild-type NEM316 and its isogenic ΔpilB mutant in immortalized RAW 264.7 murine macrophages were not significantly different whereas the isogenic ΔsodA mutant was more susceptible to killing. These results were confirmed using primary peritoneal macrophages. We also tested the activities of five cationic antimicrobial peptides (AMP-1D, LL-37, colistin, polymyxin B, and mCRAMP) and found no significant difference between WT and ΔpilB strains whereas the isogenic dltA mutant showed increased sensitivity. CONCLUSIONS/SIGNIFICANCE: These results question the previously described role of PilB pilus in resistance to the host immune defenses. Interestingly, PilB was found to be important for virulence in the neonatal context.

Metallopeptidases produced by group B Streptococcus: influence of proteolytic inhibitors on growth and on interaction with human cell lineages.

Group B Streptococcus (GBS) is a major etiologic agent of neonatal bacterial infections and is the most common cause of sepsis and pneumonia in newborns. Surface and secreted molecules of GBS are often essential virulence factors which are involved in the adherence of the bacteria to host cells or are required to suppress the defense mechanisms of hosts. We analyzed the peptidase profiles of GBS by detection of proteolytic activities on SDS-PAGE containing copolymerized gelatin as substrate. Based on the inhibition by o-phenathroline and EGTA, three distinct peptidases of 220, 200 and 180 kDa were identified in the culture medium, besides one major cell-associated proteolytic activity, a 200-kDa metallopeptidase, suggesting that all were zinc-metallopeptidases. GBS culture supernatants, rich in metallotype peptidases, also cleaved fibronectin, laminin, type IV collagen, fibrinogen and albumin. Cleavage of the host extracellular matrix by GBS may be a relevant factor in the process of bacterial dissemination and/or invasion. Notably, metallopeptidase inhibitors strongly blocked GBS growth as well as its interaction with human cell lineages. Understanding the contribution of peptidases to the pathogenesis of GBS disease may broaden our perception of how this significant pathogen causes severe infections in newborn infants.

Cytochemical study of Streptococcus agalactiae and macrophage interaction.

Light and electron microscopy were used to analyse the process of interaction of Streptococcus agalactiae (serotypes Ia, III, and V) with resident and activated mouse peritoneal macrophages. Transmission electron microscopy showed that adherence of the S. agalactiae serotype Ia, but not III and V serotypes, to the surface of activated macrophages triggers the respiratory oxidative burst as revealed by the presence of reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase in the phagocytic vacuoles. Fusion of macrophage lysosomes with bacteria-containing phagocytic vacuoles was observed in macrophages treated with Lucifer yellow as well as by localization of acid phosphatase for all serotypes.

Effects of tamoxifen, melatonin, coenzyme Q10, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins.

The involvement of toxic oxygen intermediates in the bacteriostatic effects of mycotoxins (T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1, and fumonisin B1) was investigated by producing bacterial growth curves using turbidimetry assays in the presence and absence of oxygen radical-scavenging substances. The strains used in this study included Escherichia coli (FT 101), Streptococcus agalactiae (FT 311, FT 313, FT 315), Staphylococcus aureus (FT 192), Yersinia enterocolitica (FT 430), Salmonella infantis (FT 431), Erysipelothrix rhusiopathiae (FT 432), Lactobacillus plantarum (FT234) and Lactobacillus casei (FT 232). Tamoxifen, melatonin, l-carnitine and coenzyme Q10 were used as radical scavengers against oxygen toxicity to the strains studied. Tamoxifen was the most effective in inhibiting bacterial growth when used at a high concentration, whereas melatonin and l-carnitine were less effective. A combination of l-carnitine and coenzyme Q10 provided better protection against oxygen toxicity caused by the mycotoxins growth than they did individually. It was concluded that oxygen radicals are involved in the killing of bacteria and that there is endogenous formation of toxic oxygen products by mycotoxins. The objective of this study was to determine whether the antioxidants were able to counteract the toxic effects of the mycotoxins. The data obtained indicate that bacterial growth can be inhibited especially by T-2 toxin, aflatoxin B1 and ochratoxin A and that this effect can be partially counteracted by antioxidants such as coenzyme Q10 plus l-carnitine.

Regulation of cultured human chorion cell chemokine production by group B streptococci and purified bacterial products.

PROBLEM: To determine if different strains of group B streptococci (GBS) and purified bacterial products regulate chemokine production by cultured human chorion cells. METHOD OF STUDY: Primary cultures of human chorion cells were established from placentae isolated from normal women at term gestation having repeat cesarean section. Five different strains of heat-killed GBS were incubated with confluent chorion cells at 10(7) bacteria/ml for 16 hours at 37 degrees C. In separate experiments, lipoteichoic acid and sialic acid at various concentrations were incubated with chorion cells for 16 hours at 37 degrees C. Culture supernatants were collected and then assayed to determine concentrations of interleukin-8 (IL-8) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) by ELISA. RESULTS: We found that GBS stimulated chorion cell production of MIP-1 alpha in a strain-specific fashion. We also found that both lipoteichoic acid and sialic acid stimulated concentration-dependent increases in chorion cell IL-8 production. Chorion cells, however, did not increase MIP-1 alpha production in response to either lipoteichoic acid or sialic acid. Two strains of GBS tested induced concentration-dependent increases in both IL-8 and MIP-1 alpha, but both stimulated IL-8 production to a greater extent. Similarly, IL-1 beta also caused chorion cells to produce more IL-8 than MIP-1 alpha. CONCLUSIONS: Our data are the first to show that GBS and purified bacterial products can stimulate chemokine production by fetal gestational tissues. We suggest that chorion cells may produce specific types of chemokines to attract different types of inflammatory cells and thus may participate in the pathophysiology of infection-mediated preterm labor by directing specific inflammatory responses.

Effect of different surfactants on pulmonary group B streptococcal infection in premature rabbits.

OBJECTIVES: To evaluate the effects of different surfactants on pulmonary infection with group B streptococci in premature rabbits and to examine the effects of different surfactants on pulmonary alveolar macrophage function of newborn rabbits. MODEL: Preterm and term rabbit pups. METHODS: Rabbit pups were infected with GBS aerosols followed by intratracheal administration of either calf lung surfactant extract, minced porcine lung surfactant (Curosurf), synthetic surfactant (Exosurf Neonatal), minced bovine lung surfactant (Survanta), human amniotic fluid-derived surfactant, rabbit surfactant, saline vehicle, or no treatment. Intrapulmonary clearance of GBS was determined by comparing bacterial counts in left lungs cultured immediately after aerosol infection with similarly infected lungs analyzed 4 hours after surfactant therapy. Phagocytosis of streptococci was ascertained by microscopic examination of the right lungs fixed in situ at 4 hours. For comparison, an in vitro method was used to measure growth of GBS in the different surfactants. RESULTS: Preterm animals had a sixfold increase in pulmonary bacterial growth compared with a slight decrease in intrapulmonary GBS in term animals when all were delivered by cesarean section (p < 0.05). In premature rabbits, GBS proliferation was lowest in animals treated with Exosurf Neonatal and highest in animals receiving Curosurf and human amniotic fluid-derived surfactant (p < 0.05). None of the surfactants promoted accelerated growth of GBS in comparison with control animals. Similar growth of GBS was seen in in vitro cultures. Intrapulmonary phagocytosis of GBS in premature pups was not altered by any of the surfactants. In term rabbit pups, the following measures of macrophage population kinetics remained normal at 1 and 24 hours after surfactant administration: viability, cell numbers based on lung lavage, and in vivo incorporation of thymidine. CONCLUSIONS: Surfactants used in clinical practice do not accelerate the in vivo growth of group B streptococci in the lungs of preterm rabbits. Some surfactants inhibit streptococcal proliferation. The effects of different surfactants are not explained by changes in macrophage function.

Experimental neonatal group B streptococcal pneumonia: effect of a modified porcine surfactant on bacterial proliferation in ventilated near-term rabbits.

We studied bacterial proliferation in relation to surfactant treatment in a model of neonatal group B streptococcal (GBS) pneumonia. Surfactant (Curosurf) was isolated from pig lungs with a method preserving only polar lipids and hydrophobic proteins. Near-term rabbit fetuses were ventilated in a body plethysmograph system. At 15 min, a suspension of GBS strain 090 Ia LD (5 mL/kg, concentration approximately 10(9)/mL) was instilled intratracheally. At 30 min, surfactant (n = 12) or sterile saline (n = 13) was administered via the airways (2.5 mL/kg). A control group (n = 12) received the same volumes of saline. After 5 h the animals were killed, and samples for blood cultures and blood gases were taken from the heart. The left lung was aseptically removed, weighed, homogenized, serially diluted, and cultured on blood agar plates. The results were expressed as mean log10 colony forming units/g lung +/- SD. Compared with animals (n = 12) killed immediately after GBS instillation (8.13 +/- 0.54), there was a significant increase in bacterial numbers in both groups ventilated for 5 h, but values for surfactant-treated animals (8.96 +/- 0.38) were lower than those for animals receiving saline (9.46 +/- 0.50; p < 0.05). After 5 h, 96% of GBS-infected animals had positive blood cultures. Light microscopic examination of the right lung of GBS-infected animals revealed inflammatory changes that tended to be less prominent in surfactant-treated rabbits. We conclude that intratracheal inoculation of near-term rabbits with GBS resulted in a significant bacterial proliferation during 5 h of ventilation and that bacterial growth was mitigated by treatment with surfactant.

Properties of group B streptococci with protein surface antigens X and R.

A total of 128 bovine and 134 human group B streptococci were serotyped by conventional methods. Among the bovine cultures, 60 (47%) had type antigen X, and among the human cultures, 53 (39%) had type antigen R. The occurrence of type antigens X and R was significantly related to the growth pattern of the bacteria in fluid media. Type X- and R-positive cultures and most of the nontypeable cultures predominantly formed long chains and grew as granular sediment with clear supernatant. In addition, group B streptococci with surface antigen X or R showed compact colony formation in soft agar and reacted positively in the salt aggregation test. These properties, possibly caused by the surface charges of the X- and R-positive cultures, might be related to bacterial pathogenicity.

Comparison of a rapid enzyme-linked immunosorbent assay test and the Gram stain for detection of group B streptococcus in high-risk antepartum patients.

Early-onset neonatal sepsis with group B streptococci is a major problem in the management of high-risk obstetrics. Intrapartum treatment of the colonized mother reduces neonatal acquisition; however, many high-risk patients are delivered before culture results are available. This study prospectively evaluated a new enzyme-linked immunosorbent assay and the Gram stain for their accuracy in rapid detection of group B streptococci in 131 high-risk patients. Twenty positive cultures for group B streptococci were identified in the study population and were used as the control for test comparisons. The enzyme-linked immunosorbent assay test was 60% sensitive, whereas the Gram stain was 45% sensitive. The enzyme-linked immunosorbent assay showed an increase in sensitivity as the colony count increased; however, two cases of severe neonatal sepsis occurred in patients with low colony counts and both had enzyme-linked immunosorbent assay negative results. In conclusion, the need for a rapid sensitive test for group B streptococci detection still exists.

[The morphology of microcolonies as a criterion for recognizing bacteria in the differentiation of Streptococcus pyogenes and Streptococcus group B]. / Morfologiia mikrokolonii kak kriterii raspoznavaniia bakterii pri differentsiatsii piogennogo streptokokka i streptokokkov gruppy B.

The authors studied the diagnostic importance of the morphology of microcolonies of S. pyogenes and group B streptococci in comparison with the currently used tests for the differentiation of these two species: the bacitracin test, the hydrolysis of sodium hippurate and the CAMP test. The standard tests proved to be positive in 94-97% and microcolonies had typical morphology in 86-95.7%. The statistical indices of the diagnostic effectiveness of differential tests varied within 93.8-98.9%. The diagnostic value of the study of the morphology of colonies was characterized by the following data: the sensitivity and prognostic negative value of the study were 95% for S. pyogenes and 86-89% for group B streptococci, while its specificity and prognostic positive value were 100% due to the absence of false positive results.

Serological differentiation of strains of group B streptococci by the soft-agar technique.

Soft-agar technique was applied for serological determination of strains of group B streptococci. With representative type strains, their colonial morphology was converted from diffuse- to compact-type growth only by the addition of homologous rabbit anti-serum in the medium and no conversion of colonial morphology was observed by heterologous rabbit antisera. Twenty seven out of 30 fresh isolates obtained from human clinical specimens showed diffuse-type growth in soft-agar medium and were subjected to this examination. Twenty-one strains reacted with a single antiserum, 5 strains showed reactivities to two different antisera although reaction to an antiserum were significantly higher than those of the other antiserum and no reaction was observed with a strain. Twenty single and 5 major serotypes determined by this technique were coincided with those differentiated by Lancefield's precipitin method. From these experimental results, soft-agar technique was regarded being available for serological typing of strains of group B streptococci.

Lysis and protoplast formation of group B streptococci by mutanolysin.

Group B streptococci, refractory to previously tested muralysins under physiological conditions, were successfully converted to protoplasts by use of a recently describede N-acetyl muramidase, mutanolysin, derived from a streptomycete. Purified enzyme was effective, but crude preparations, although degrading cell walls, simultaneously produced peculiar effects of cytoplasmic coagulation, retention of cell shape, loss of some intracellular enzymes, and a rise in optical density. Addition of purified mutanolysin to the array of muralysins (group C streptococcal phage-associated lysin, lysozyme), previously successful in preparing protoplasts of different streptococci, now makes possible enzymatic preparation of protoplasts of streptococci of groups A, B, C. D. G, and H.