Comparing ranibizumab monotherapy and combination with single photodynamic therapy in wet AMD: retreatment and morphologic results.

PURPOSE: To evaluate retreatment indications/morphologic responses to ranibizumab monotherapy and combination with verteporfin photodynamic therapy (PDT). METHODS: A total of 40 patients received 3 monthly intravitreal ranibizumab 0.3 mg injections combined with either PDT or sham PDT at baseline (1:1) followed by as-needed ranibizumab based on predetermined vision/anatomical criteria. RESULTS: Retreatment criteria were visual acuity (VA) loss (59%/58%), central retinal thickness (CRT) increase (27%/26%), or both (14%/16%). One month before retreatment, intraretinal cysts (IRC) were present in 84%/74%, subretinal fluid (SRF) in 70%/63%, and at least one of them in 84%/89% of eyes. A significant decrease in mean leakage area, IRC, and SRF as well as a reduction in presence of hemorrhages and hard exudates occurred in both treatment groups at 12 months (compared to baseline). CONCLUSIONS: Retreatment indications were mostly based on VA loss, probably due to the quantitative optical coherence tomography criterion. Intraretinal cysts and SRF were earlier predictors for recurring choroidal neovascularization (CNV) activity than CRT/VA changes. Both treatment strategies were equally potent in reducing CNV activity.

Altered lipid composition in Streptococcus pneumoniae cpoA mutants.

BACKGROUND: Penicillin-resistance in Streptococcus pneumoniae is mainly due to alterations in genes encoding the target enzymes for beta-lactams, the penicillin-binding proteins (PBPs). However, non-PBP genes are altered in beta-lactam-resistant laboratory mutants and confer decreased susceptibility to beta-lactam antibiotics. Two piperacillin resistant laboratory mutants of Streptococcus pneumoniae R6 contain mutations in the putative glycosyltransferase gene cpoA. The CpoA gene is part of an operon including another putative glycosyltransferase gene spr0982, both of which being homologous to glycolipid synthases present in other Gram-positive bacteria. RESULTS: We now show that the cpoA mutants as well as a cpoA deletion mutant are defective in the synthesis of galactosyl-glucosyl-diacylglycerol (GalGlcDAG) in vivo consistent with the in vitro function of CpoA as α-GalGlcDAG synthase as shown previously. In addition, the proportion of phosphatidylglycerol increased relative to cardiolipin in cpoA mutants. Moreover, cpoA mutants are more susceptible to acidic stress, have an increased requirement for Mg(2+) at low pH, reveal a higher resistance to lysis inducing conditions and are hypersensitive to bacitracin. CONCLUSIONS: The data show that deficiency of the major glycolipid GalGlcDAG causes a pleitotropic phenotype of cpoA mutant cells consistent with severe membrane alterations. We suggest that the cpoA mutations selected with piperacillin are directed against the lytic response induced by the beta-lactam antibiotic.

Genome of Streptococcus oralis strain Uo5.

Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.

The genome of Streptococcus mitis B6--what is a commensal?

Streptococcus mitis is the closest relative of the major human pathogen S. pneumoniae. The 2,15 Mb sequence of the Streptococcus mitis B6 chromosome, an unusually high-level beta-lactam resistant and multiple antibiotic resistant strain, has now been determined to encode 2100 genes. The accessory genome is estimated to represent over 40%, including 75 mostly novel transposases and IS, the prophage phiB6 and another seven phage related regions. Tetracycline resistance mediated by Tn5801, and an unusual and large gene cluster containing three aminoglycoside resistance determinants have not been described in other Streptococcus spp. Comparative genomic analyses including hybridization experiments on a S. mitis B6 specific microarray reveal that individual S. mitis strains are almost as distantly related to the B6 strain as S. pneumoniae. Both species share a core of over 900 genes. Most proteins described as pneumococcal virulence factors are present in S. mitis B6, but the three choline binding proteins PcpA, PspA and PspC, and three gene clusters containing the hyaluronidase gene, ply and lytA, and the capsular genes are absent in S. mitis B6 and other S. mitis as well and confirm their importance for the pathogenetic potential of S. pneumoniae. Despite the close relatedness between the two species, the S. mitis B6 genome reveals a striking X-alignment when compared with S. pneumoniae.

An ABC transporter of Streptococcus pneumoniae involved in susceptibility to vancoresmycin and bacitracin.

Vancoresmycin is a novel tetramic acid antibiotic, probably interfering with functions of the cytoplasmic membrane. To investigate its mode of action, mutants of Streptococcus pneumoniae exhibiting reduced susceptibility to vancoresmycin were isolated at a low frequency. Four of them were further examined and showed similar pleiotropic phenotypes, including reduced growth rate, early autolysis, and chain formation. In one mutant, the level of transcripts from a single locus encoding the potential ABC transporter Spr0812-Spr0813 was increased sixfold. The corresponding DNA sequence revealed a nonsense mutation (C1744T) in spr0813, leading to the formation of a truncated permease lacking 2 of the 10 predicted transmembrane helices. As demonstrated by deletion and transformation analysis and reconstructing the spr0813(C1744T) mutation in the wild-type background, this nucleotide exchange was sufficient to cause reduced susceptibility to vancoresmycin and higher amounts of spr0812-spr0813 mRNA. Mapping and reporter assays of the cognate promoter P(abc) showed that spr0812 and spr0813 are cotranscribed with a preceding small gene and that the spr0813(C1744T) mutation does not affect the activity of P(abc). Due to the similarity of Spr0812-Spr0813 to bacitracin transporters of Streptococcus mutans and Bacillus spp., the bacitracin susceptibility of spr0813 mutants was examined. Both the spr0813(C1744T) nonsense mutation and the deletion of the transporter genes led to a clearly increased sensitivity to bacitracin. Thus, the intact transporter is required for intrinsic resistance to bacitracin, whereas reduced vancoresmycin susceptibility is mediated by the truncated permease.

Transcript map of the temperate Lactobacillus gasseri bacteriophage phiadh.

Temporal transcription of phage phiadh was analysed during lytic reproduction. Based on Northern hybridizations the phage genome was divided into regions of early, middle and late transcription. Eight groups of overlapping transcripts, probably originating from common precursors, were distinguished. Early transcription of a 10.9 kb region adjacent to the lytic/lysogenic switch started within the first 10 min of infection and produced three groups of mRNAs mostly related to DNA replication. Four middle transcripts were observed 30 min after infection, corresponding to an 8.5 kb genomic region, which started at the replication origin (ori) and encompassed a DNA packaging function and the cos site. Three groups of late transcripts were first observed 50 min after infection, corresponding to a 21.1 kb region between the middle region and the attachment site (attP), encoding functions for capsid morphogenesis and host cell lysis. A fourth group of late-appearing mRNAs was divergently transcribed from the 3.2 kb section between attP and the lytic/lysogenic switch, including the repressor and integrase genes. Except for one set of early mRNAs, all the transcripts persisted until the end of the reproduction cycle. Two confirmed and two predicted promoters were assigned to transcript 5' ends in the early region.

Crystal structure of the dinuclear zinc aminopeptidase PepV from Lactobacillus delbrueckii unravels its preference for dipeptides.

PepV from Lactobacillus delbrueckii, a dinuclear zinc peptidase, has been characterized as an unspecific amino dipeptidase. The crystal structure of PepV in complex with the phosphinic inhibitor AspPsi[PO(2)CH(2)]AlaOH, a dipeptide substrate mimetic, reveals a "catalytic domain" and a "lid domain," which together form an internal active site cavity that traps the inhibitor. The catalytic domain is topologically similar to catalytic domains from amino- and carboxypeptidases. However, the lid domain is unique among the related enzymes. In contrast to the other related exopeptidases, PepV recognizes and fixes the dipeptide backbone, while the side chains are not specifically probed and can vary, rendering it a nonspecific dipeptidase. The cocrystallized inhibitor illustrates the two roles of the two catalytic zinc ions, namely stabilization of the tetrahedral intermediate and activation of the catalytic water molecule.

Inducible promoter-repressor system from the Lactobacillus casei phage phiFSW.

With the aim to extend the presently available inducible gene expression systems for lactobacilli, we have isolated a thermoinducible promoter-repressor cassette from the temperate Lactobacillus casei phage phiFSW-TI in Escherichia coli. The phiFSW-TI promoter fragment was abutted to the plasmid-borne promoterless beta-glucuronidase (gusA) reporter gene and shown to direct its transcription in L. casei. In addition, the functionality of the promoter-repressor system was verified in the L. casei phiFSW-TI lysogen by showing that the gusA reporter gene, controlled by the isolated phiFSW-TI promoter, was repressed at 28 degrees C and expressed at 42 degrees C. Moreover, a homology search revealed that the C terminus of the isolated phiFSW repressor shows a high similarity to the small mutS-related domain of the MutS2 protein family that is unprecedented for phage-encoded repressor proteins.

Expression of the phospho-beta-glycosidase ArbZ from Lactobacillus delbrueckii subsp. lactis in Lactobacillus helveticus: substrate induction and catabolite repression.

ArbZ from Lactobacillus delbrueckii subsp. lactis was previously shown to enable utilization of the beta-glucoside arbutin by Escherichia coli. The arbZ gene was cloned and expressed in the industrially used beta-glucoside-negative strain Lactobacillus helveticus 3036(62). The transformants were able to ferment not only arbutin, but also cellobiose, salicin and methyl-beta-glucoside (MbetaGlc). Cleavage of beta-glucosides by the transformants depended on the integrity of the cytoplasmic membrane, whereas in cell-free extracts only C(6)-phosphorylated substrates were hydrolysed. This suggested that ArbZ is a phospho-beta-glycosidase. ArbZ activity in transformants of Lb. helveticus was subject to substrate induction mediated by the beta-glucosides arbutin, salicin and MbetaGlc, whereas cellobiose or the beta-galactoside lactose had no inducing effect. Northern blot analysis proved that induction by MbetaGlc was due to enhanced transcription of arbZ. Catabolite repression of arbZ induction was observed with glucose, mannose, fructose and galactose. The induction kinetics observed in the presence of these sugars indicated that at least two different mechanisms are operative in catabolite repression of arbZ in Lb. helveticus.

PepR1, a CcpA-like transcription regulator of Lactobacillus delbrueckii subsp. lactis.

The PepR1 protein from Lactobacillus delbrueckii subsp. lactis DSM 7290 shares extensive homology with catabolite-control proteins from various Gram-positive bacteria. Expression of the subcloned pepR1 gene allowed for partial complementation of a ccpA defect in Staphylococcus xylosus. The influence of PepR1 on transcription of the prolidase gene pepQ, which is located adjacent to pepR1, was examined by use of lacZ reporter gene fusions in Escherichia coli. PepR1 stimulated transcription initiation at the pepQ promoter about twofold, and this effect required the integrity of a 14 bp palindromic cre-like sequence located 74 nt upstream of pepQ. In gel-mobility-shift assays, PepR1 specifically interacted with the pepQ promoter region and also with DNA fragments covering the promoters of the pepX, pepl and brnQ genes of Lb. delbrueckii subsp. lactis, which encode two additional peptidases and a branched-chain amino acid transporter, respectively. cre-like elements were identified in each of these DNA fragments. Catabolite control of PepQ was demonstrated in Lb. delbrueckii subsp. lactis. During growth with lactose the enzyme activity was twofold higher than in the presence of glucose, and corresponding differences were also detected in the level of pepQ transcription.