In vitro compared activity of telithromycin and azithromycin against northwest Italian isolates of Streptococcus pyogenes and Streptococcus pneumoniae with different erythromycin susceptibility

Aims: This study compared the in vitro activity of telithromycin with that of azithromycin against 438 Streptococcus pyogenes and 198 Streptococcus pneumoniae, isolated over the period 2005–2007 from specimens of different human origin obtained in three Piemonte Region’s hospitals. Methods and Results: The determination of antimicrobial activity was evaluated by the microdilution broth method and the erythromycin‐resistant (Ery‐R) phenotypes by the triple‐disc test. Exactly 78·8% of S. pyogenes and 69·2% of S. pneumoniae were erythromycin‐susceptible (Ery‐S). Concerning S. pyogenes, telithromycin was active against M and inducible MLS_B, subtype‐C, phenotypes but not against constitutive MLS_B strains. Telithromycin acted well against all S. pneumoniae, irrespective of their mechanism of macrolide‐resistance. On the contrary, the Ery‐R isolates, both S. pyogenes and S. pneumoniae, were resistant to azithromycin. Conclusions: Our results indicate that macrolide resistance in streptococci still persist in northwest Italy (21·2% of S. pyogenes and 30·8% of S. pneumoniae) and that telithromycin is confirmed as being extremely active even against recent clinical Ery‐R streptococcal isolates. Significance and Impact of the Study: The present study emphasizes that an active surveillance of the phenotype distribution and antibacterial resistance in streptococci is essential in guiding the effective use of empirical treatment option for streptococcal infections, also at regional level.     

Ecological and biotechnological importance of secondary metabolites produced by coral-associated bacteria

Symbiotic relationships between corals and their associated micro-organisms are essential to maintain host homeostasis. Coral-associated bacteria (CAB) can have different beneficial roles in the coral metaorganism, such as metabolizing essential nutrients for the coral host and protecting the coral from pathogens. Many CAB exert these functions via secondary metabolites, which include antibacterial, antifouling, antitumour, antiparasitic and antiviral compounds. This review describes how analysis of CAB has led to the discovery of secondary metabolites with potential biotechnological applications. The most commonly found types of secondary metabolites, antimicrobial and antibiofilm compounds, are emphasized and described. Recently developed methods that can be applied to enhance the culturing of CAB from shallow-water reefs and the less-studied deep-sea coral reefs are also discussed. Last, we suggest how the combined use of meta-omics and innovative growth-diffusion techniques can vastly improve the discovery of novel compounds in coral environments.     

Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. as antibiofilm producing and antiquorum sensing in Streptococcus pyogenes

Biofilm formation has been demonstrated as a potentially important mechanism contributing to antibiotic treatment failure on Streptococcus pyogenes. It could play a significant role in recurrent and chronic infections. Boesenbergia pandurata (Roxb.) Schltr., Eleutherine americana Merr. and Rhodomyrtus tomentosa (Aiton) Hassk. have been previously reported from our laboratory as effective agents against S. pyogenes. Therefore, in the present study, we observed the effect of these plants on biofilm formation. The bacterial biofilms were quantified by safranin staining and absorbance at 492 nm. The results clearly demonstrated that all subinhibitory concentrations [1/32-1/2 minimal inhibitory concentration (MIC)] of E. americana (7.81-125 microg mL(-1)) and R. tomentosa (0.24-7.81 microg mL(-1)) extracts significantly prevented biofilm formation while 1/2MIC (7.81 microg mL(-1)) of B. pandurata extract produced this effect. The issue of antiquorum sensing of this pathogenic bacterium has been further explored. A correlation between antiquorum-sensing and antibiofilm-producing activities was demonstrated. Strong inhibition on quorum sensing was displayed with the extract of R. tomentosa. Eleutherine americana extract showed partial inhibition, while B. pandurata did not show this activity. By contrast, an assay of microbial adhesion to hydrocarbon revealed no changes in the cell-surface hydrophobicity of the treated organisms. Active organisms with the ability to inhibit quorum sensing and biofilm formation are worth studying as they may provide complimentary medicine for biofilm-associated infections.     

Antimicrobial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria

Aims: The aim of this study is to assess the antibacterial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria. Methods and Results: The antibacterial activity was determined by broth microdilution method. The results showed that although Enterocuccus faecium OB7084 and Klebsiella pneumoniae OB7088 had high tolerance to sodium citrate, several oral bacteria including Fusobacterium nucleatum JCM8532^T, Streptococcus mutans JCM5705^T and Strep. pneumoniae NBRC102642^T were susceptible. Furthermore, the bactericidal activity of sodium citrate against Strep. pneumoniae NBRC102642^T was not influenced by pH in the range of 5·0–8·0, whereas that of sodium lactate was weakened at neutral or weak alkaline pH. When Strep. pneumoniae NBRC102642^T was treated with sodium citrate for 2 h, many burst cells were observed. However, addition of MgCl₂ or CaCl₂ to an assay medium weakened the antimicrobial activity although ZnCl₂ or MnCl₂ did not influence. Conclusions: Independent of pH, sodium citrate inhibited the growth of oral bacteria, which suggests that the mechanism is different from that of sodium lactate. Significance and Impact of the Study: The results presented in this study would be available for understanding the antimicrobial property of sodium citrate.     

Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes

We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PCR). Genomic DNA from 22 isolates representing 14 M serotypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and ennX were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which correspond to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein.     

Prediction of surface exposed proteins in Streptococcus pyogenes,with a potential application to other Gram‐positive bacteria

The in silico prediction of bacterial surface exposed proteins is of growing interest for the rational development of vaccines and in the study of bacteria–host relationships, whether pathogenic or host beneficial. This interest is driven by the increase in the use of DNA sequencing as a major tool in the early characterization of pathogenic bacteria and, more recently, even of complex ecosystems at the host–environment interface in metagenomics approaches. Current protein localization protocols are not suited to this prediction task as they ignore the potential surface exposition of many membrane‐associated proteins. Therefore, we developed a new flow scheme, SurfG+, for the processing of protein sequence data with the particular aim of identification of potentially surface exposed (PSE) proteins from Gram‐positive bacteria, which was validated for Streptococcus pyogenes. The results of an exploratory case study on closely related lactobacilli of the acidophilus group suggest that the yogurt bacterium Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) dedicates a relatively important fraction of its coding capacity to secreted proteins, while the probiotic gastrointestinal (GI) tract bacteria L. johnsonii and L. gasseri appear to encode a larger variety of PSE proteins, that may play a role in the interaction with the host.     

Hyaluronan Breakdown Contributes to Immune Defense against Group A Streptococcus

Group A Streptococcus (GAS) commonly infects human skin and occasionally causes severe and life-threatening invasive diseases. The hyaluronan (HA) capsule of GAS has been proposed to protect GAS from host defense by mimicking endogenous HA, a large and abundant glycosaminoglycan in the skin. However, HA is degraded during tissue injury, and the functions of short-chain HA that is generated during infection have not been studied. To examine the impact of the molecular mass of HA on GAS infection, we established infection models in vitro and in vivo in which the size of HA was defined by enzymatic digestion or custom synthesis. We discovered that conversion of high molecular mass HA to low molecular mass HA facilitated GAS phagocytosis by macrophages and limited the severity of infection in mice. In contrast, native high molecular mass HA significantly impaired internalization by macrophages and increased GAS survival in murine blood. Thus, our data demonstrate that GAS virulence can be influenced by the size of HA derived from both the bacterium and host and suggest that high molecular mass HA facilitates GAS deep tissue infections, whereas the generation of short-chain HA can be protective.     

Insertional inactivation of virR in Streptococcus pyogenes M49 demonstrates that VirR functions as a positive regulator of ScpA, FcRA, OF, and M protein

Abstract Several reports have shown that Streptococcus pyogenes strains which produce opacity factor (OF+) have diverged significantly from OF⁻ serotypes. This study questions whether several surface proteins of an OF+ culture are regulated by the positive regulatory protein VirR, in a manner similar to OF~ strains. Interruption of the virR region of an OF⁺ S. pyogenes (strain CS101, M type 49) was performed using a temperature-sensitive plasmid containing a fragment of virR . Interruption of the virR region produced cultures with (indétectable amounts of M49 and ScpA proteins, and reduced the yield of FcRA protein. In addition, mutants had a significant reduction in detectable opacity factor. These results suggest that virR functions as a positive regulator of a variety of surface proteins in OF⁺ strains.     

Consideration of cysteine protease activity for serological M-typing of clinical Streptococcus pyogenes isolates

Clinical isolates of Streptococcus pyogenes were classified by serological typing of their surface M protein. Non-M typeable strains with the emm1 gene were characterized as the degradation of M protein caused by overproduction of the extracellular cysteine protease, SpeB. These events are dependent on the growth phase. M protein produced prior to expression of SpeB is degraded in the stationary phase when the active form of SpeB is detected. The proteolytic degradation of M protein should be considered for precise M typing analysis.     

Binding of complement subcomponent C1q to Streptococcus pyogenes: evidence for interactions with the M5 and FcRA76 proteins

Binding of C1q, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of C1q to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between C1q and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound C1q; alkaline and SDS extracts yielded the 52 kDa component as the main C1q-binding substance. Trypsin treatment of the SDS extracts of two GAS strains suggested the C1q-binding component(s) to be of protein nature. C1q-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human C1q and fibrinogen, conjugated to horse radish peroxidase (HRP) as well as rabbit IgG antibodies complexed to HRP (PAP system). The 52 kDa component was non-reactive with fibrinogen or rabbit IgG. However, C1q-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either fibrinogen (type M5) or both fibrinogen and rabbit IgG (type M76); the molecular mass of these components, 55 kDa and 43–40 kDa, respectively, was in agreement with the reported molecular mass of the M5 and FcRA76 proteins. Our findings suggest that C1q may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with C1q as biologically important.     

Isolation of a new superantigen with potent mitogenic activity to murine T cells from Streptococcus pyogenes

Abstract A mitogenic substance on murine lymphocytes was detected in the culture supernate of Streptococcus pyogenes type 12 strain. This substance had a molecular weight of 28 000 and p I 9.2, and was designated as S. pyogenes mitogen (SPM). The proliferative response of C3H/HeN spleen cells began at 1 ng ml⁻¹ and reached a maximal response at 100 ng ml⁻¹ of SPM for 4 days culture. Anti-Thy 1.2 mAb and complement-treated spleen cells abrogated the proliferative response to any dose of SPM. Although the anti-major histocompatibility complex class I mAbs had no blocking effect on proliferation by SPM, this proliferation was substantially inhibited by the addition of either anti-I-A or anti-I-E mAb, and complete inhibition was produced by the addition of both mAbs. Fixed antigen-presenting cells still induced T cell proliferation by SPM. A significant expansion of T cells bearing Vβ13 T-cell receptor was observed up to 73% among the Thy1.2⁺ cells in cultures stimulated with SPM, indicating expansion in a Vβ-specific manner. Immunoblotting of IEF-separated proteins showed that anti-streptococcal pyrogenic exotoxin (SPE) C reacted with a protein of p I 6.9 and anti-SPEB did not show any reactivity. SPEA was reported to expand Vβ8.1 and 8.2 bearing murine T cells, and SPM did not. SPM also exhibited potent mitogenic activity on human T cells and Vβ21⁺ T cells were selectively expanded. These results lead to the conclusion that SPM was neither SPEA, B nor C, but a new protein belonging to a group of streptococcal superantigens with activity on not only human but also murine lymphocytes.     

A single emm gene-specific oligonucleotide probe does not recognise all members of the Streptococcus pyogenes M type 1

Abstract Serological typing of the streptococcal M protein has recently been challenged by a number of unique molecular methodologies based on oligonucleotide recognition of allelic variations within the M protein ( emm ) gene. In these methods, stringent hybridization of an oligonucleotide probe to a polymerase chain reaction amplified emm gene is used as confirmation of specific M type identity. A sample of 17 isolates from 7 previously defined distinct genotypes were tested using a single M1 oligonucleotide probe. Isolates from only three of the genotypes hybridized with the probe. The results demonstrate that a single emm -specific oligonucleotide probe can not identify all members of M type 1, as defined by conventional serotyping using polyclonal antisera.     

Assembly mechanism of FCT region type 1 pili in serotype M6 Streptococcus pyogenes

The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner.     

In vitro activity of xanthorrhizol against Streptococcus mutans biofilms

AIMS: We determined the effect of xanthorrhizol (XTZ) purified from the rhizome of Curcuma xanthorrhiza Roxb. on the Streptococcus mutans biofilms in vitro. METHODS AND RESULTS: The biofilms of S. mutans at different phases of growth were exposed to XTZ at different concentrations (5, 10 and 50 micromol l(-1)) and for different time exposures (1, 10, 30 and 60 min). The results demonstrated that the activity of XTZ in removing S. mutans biofilm was dependent on the concentration, exposure time and the phase growth of biofilm. A concentration of 5 micromol l(-1) of XTZ completely inhibited biofilm formation by S. mutans at adherent phases of growth, whereas 50 micromol l(-1) of XTZ removed 76% of biofilm at plateau accumulated phase when exposed to S. mutans biofilm for 60 min. CONCLUSIONS: Xanthorrhizol isolated from an edible plant (C. xanthorrhiza Roxb.) shows promise as an antibacterial agent for inhibiting and removing S. mutans biofilms in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: XTZ could be used as a potential antibacterial agent against biofilm formation by S. mutans.     

不同培养基对放线菌TRM10325抑制群感效应活性的影响

检测不同培养基条件下,放线菌TRM10325发酵液抑制群感效应的活性,初步了解其活性稳定性,并为筛选最优发酵培养基提供实验依据。选取17种合成培养基、26种天然培养基发酵放线菌TRM10325,采用微孔板半定量法检测其对紫色素杆菌群感效应以及表皮葡萄球菌生物膜形成的抑制作用。不同配方的培养基对放线菌10325抑制群感效应活性的影响也不相同,其中Am6培养基抑制群感效应效果最佳。成分不同的培养基,明显影响微生物不同次级代谢产物的产生。同一微生物在不同培养基中发酵,其次生代谢产物的种类和含量变化很大。最终选定Am6培养基为最适发酵培养基。