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Background and Aims Intestinal microbiota dysbiosis is implicated in Crohn’s disease [CD] and may play an important role in triggering postoperative disease recurrence [POR]. We prospectively studied faecal and mucosal microbial recolonisation following ileocaecal resection to identify the predictive value of recurrence-related microbiota. Methods Mucosal and/or faecal samples from 121 CD patients undergoing ileocaecal resection were collected at predefined time points before and after surgery. Ileal biopsies were collected from 39 healthy controls. POR was defined by a Rutgeerts score ≥i2b. The microbiota was evaluated by 16S rRNA sequencing. Prediction analysis was performed using C5.0 and Random Forest algorithms. Results The mucosa-associated microbiota in CD patients was characterised by a depletion of butyrate-producing species (false discovery rate [FDR] Conclusions Microbiota recolonisation after ileocaecal resection is different between recurrence and non-recurrence patients, with Fusobacteria as the most prominent player driving early POR. These bacteria involved in the early recolonisation and POR represent a promising therapeutic strategy in the prevention of disease recurrence.

Background Therapeutic aims in IBDs are to induce remission through rapidly effective treatment. Conventional therapeutic methods including immunosuppression have been available for more than half a century with biological therapies the treatment of choice in patients who fail immunosuppressive drugs. There has been little attempt to longitudinally examine the differences in biopsychological factors and their associations with treatment modalities in IBD patients. Methods 50 IBD participants (24 UC, 26 CD) in clinical remission were followed for 12 months. Complete longitudinal datasets and monthly stool and blood samples for inflammatory biomarkers were collected for analysis of association. Microbiome analysis was performed using V4 16SrRNA for identification of microbial phylogenetic relationships. Patients were grouped on whether or not they were in remission and on biological agents. Results At the baseline there were similar ecological indices between the two treatment options in both disease classes; (Shannon, p = 0.380; Pielou’s, p = 0.246; Chao1, p = 0.934). At family level, the CD group showed significant differences (Wilcoxon, p ≤ 0.05) between the treatment received (biologics vs non-biologic) in the abundance of Barnesiellaceae, Bifidobacteriaceae, unclassified Clostridia, and Clostridiaceae (Figure 1A). Results showed similar ecological index values in samples from the two treatment options. Samples in UC groups on biological and non-biologic treatment modes were found to have significantly different microbial communities based on Bray-Curtis dissimilarity (PERMANOVA, p = 0.0031). Microbial family composition in UC group and on two treatment modalities showed a total of 12 families that were significantly different (Wilcoxon, p ≤ 0.05): Acidaminococcaceae, unclassified Bacteroidales, Barnesiellaceae, Christensenellaceae, Clostridiales vadinBB60 group, Coriobacteriaceae, Defluvitaleaceae, Eggerthelaceaem Fusobacteriaceae, Muribaculaceae, Prevotellaceae, and Streptococcaceae (Figure1B). Results identified significant differences in microbial diversity (Shannon index, p = 0.041), and evenness (Pielou’s evenness, p = 0.045) between the two treatment modes. No differences were identified in richness (Chao1) in UC participants. Conclusion Conclusion: Baseline biological indices were mostly similarly distributed between the two treatment options in remissive IBD patients, however biological therapy significantly influenced longitudinal trends of some microbiome dynamics, especially in UC. This suggests an underlying interrelationship between mode of treatment and biopsychological trajectories which might overshadow response to the treatment, requiring further assessment.

BACKGROUND: Myelosuppression-related infections remain important causes of morbidity and mortality in children with acute lymphoblastic leukemia (ALL). METHODS: By analyzing fecal samples collected at diagnosis and after each of the initial 3 phases of chemotherapy, we evaluated the role of gut microbiota in predicting infections in 199 children with newly diagnosed ALL. The bacterial 16S rRNA gene was analyzed by high-depth sequencing to determine the diversity and composition of the microbiome. RESULTS: After the induction and reinduction I phases of chemotherapy, microbial diversity decreased significantly relative to the prechemotherapy value. After chemotherapy, the relative abundance of certain bacterial taxa (eg, Bacteroidetes) decreased significantly, whereas that of other taxa (eg, Clostridiaceae and Streptococcaceae) increased. A baseline gut microbiome characterized by Proteobacteria predicted febrile neutropenia. Adjusting for the chemotherapy phase and ALL risk level, Enterococcaceae dominance (relative abundance ≥30%) predicted significantly greater risk of subsequent febrile neutropenia and diarrheal illness, whereas Streptococcaceae dominance predicted significantly greater risk of subsequent diarrheal illness. CONCLUSIONS: In children undergoing therapy for newly diagnosed ALL, the relative abundance of Proteobacteria before chemotherapy initiation predicts development of febrile neutropenia, and domination of the gut microbiota by Enterococcaceae or Streptococcaceae at any time during chemotherapy predicts infection in subsequent phases of chemotherapy. CLINICAL TRIAL REGISTRATION: NCT00549848.

The effect of the flavonol morin on Streptococcus pyogenes biofilm growth was determined using a static biofilm model, in which reduced biofilm biomass was observed in the presence of morin, suggesting that morin inhibited biofilm development. Morin at concentrations exceeding 225 μM had the greatest impact on biofilm biomass causing reductions of up to 65%, which was found to be statistically significant. Morin was also shown to induce rapid bacterial aggregation. Approximately 55% of S. pyogenes in liquid suspension aggregated when incubated with morin at concentrations of 275 and 300 μM for 120 min, compared to the control group in which only 10% of the cells aggregated, this was also shown to be statistically significant.

Lacticin 3147 is a two-peptide broad spectrum lantibiotic produced by Lactococcus lactis DPC3147 shown to inhibit a number of clinically relevant Gram-positive pathogens. Initially isolated from an Irish kefir grain, lacticin 3147 is one of the most extensively studied lantibiotics to date. In this study, the bacterial diversity of the Irish kefir grain from which L. lactis DPC3147 was originally isolated was for the first time investigated using a high-throughput parallel sequencing strategy. A total of 17 416 unique V4 variable regions of the 16S rRNA gene were analysed from both the kefir starter grain and its derivative kefir-fermented milk. Firmicutes (which includes the lactic acid bacteria) was the dominant phylum accounting for > 92% of sequences. Within the Firmicutes, dramatic differences in abundance were observed when the starter grain and kefir milk fermentate were compared. The kefir grain-associated bacterial community was largely composed of the Lactobacillaceae family while Streptococcaceae (primarily Lactococcus spp.) was the dominant family within the kefir milk fermentate. Sequencing data confirmed previous findings that the microbiota of kefir milk and the starter grain are quite different while at the same time, establishing that the microbial diversity of the starter grain is not uniform with a greater level of diversity associated with the interior kefir starter grain compared with the exterior.

Streptococcus pyogenes causes a broad spectrum of acute infections and is the bacterium most frequently isolated from patients with pharyngitis. A number of antibiotics including penicillin have been shown to be effective, although antibiotic treatment failure in cases of streptococcal pharyngitis have been reported. Herein, we aimed to elucidate the features of recurrent strains using clinical isolates. Ninety-three S. pyogenes organisms were obtained from Japanese patients with recurrent pharyngitis. Following genetic characterization, M-type isolates from patients with recurrent pharyngitis differed from those obtained at initial onset in 11 of 49 episodes, and pulsed field gel electrophoresis analysis showed different patterns in those cases. Additionally, spe genotyping revealed that the Spe type of the strains obtained at secondary onset corresponded with those from the initial onset in 22 cases. Furthermore, antibiotic susceptibility testing revealed that more than half of the strains were resistant to macrolides and lincosamides, which was a much greater ratio as compared with the strains obtained from initial onsets in previous studies. Our results suggest that recurrence and reinfection are often confused during the diagnosis of repetitive and persistent streptococcal pharyngitis. Moreover, the present S. pyogenes organisms were less susceptible to antibiotics, which raises caution about their appropriate use in clinical practice.

Gastrointestinal survival of the bacteriocin-producing strain, Lactococcus lactis DPC6520, was evaluated systematically in vitro and in vivo with a view to using this strain to deliver biologically active lacticin 3147, a broad-spectrum bacteriocin, to the gut. The activity of the lacticin 3147 producer was also evaluated against two clinically relevant pathogens: Clostridium difficile and Listeria monocytogenes. When suspended in an appropriate matrix, the lactococcal strain is capable of surviving simulated gastrointestinal juices similar to the porcine probiotic, Lactobacillus salivarius DPC6005. Upon administration of L. lactis DPC6520 to pigs (n=4), excretion rates of ∼10(2) -10(5) CFU g(-1) faeces were observed by day 5. Although passage through the gastrointestinal tract (GIT) did not affect lacticin 3147 production by L. lactis DPC6520 isolates, activity was undetectable in faecal samples by an agar well diffusion assay. Furthermore, L. lactis DPC6520 had no inhibitory effect on C. difficile or other bacterial populations in a human distal colon model, while lactococcal counts declined 10,000-fold over 24 h. The lacticin 3147 producer failed to prevent L. monocytogenes infection in a mouse model, even though a mean L. lactis DPC6520 count of 4.7 × 10(4) CFU g(-1) faeces was obtained over the 5-day administration period. These data demonstrate that L. lactis DPC6520 is capable of surviving transit through the GIT, and yet lacks antimicrobial efficacy in the models of infection used.

In this study, we used a previously described method of controlling gene expression with computer-based gene design and de novo DNA synthesis to attenuate the virulence of Streptococcus pneumoniae. We produced 2 S. pneumoniae serotype 3 (SP3) strains in which the pneumolysin gene (ply) was recoded with underrepresented codon pairs while retaining its amino acid sequence and determined their ply expression and pneumolysin production in vitro and their virulence in a mouse pulmonary infection model. Expression of ply and production of pneumolysin of the recoded SP3 strains were decreased, and the recoded SP3 strains were less virulent in mice than the wild-type SP3 strain or a Δply SP3 strain. Further studies showed that the least virulent recoded strain induced a markedly reduced inflammatory response in the lungs compared with the wild-type or Δply strain. These findings suggest that reducing pneumococcal virulence gene expression by altering codon-pair bias could hold promise for rational design of live-attenuated pneumococcal vaccines.

Aims: Enterococcus faecalis is associated with a significant number of refractory endodontic infections. Previous studies report a prevalence of Ent. faecalis ranging from 24% up to 77% in teeth with failed endodontic treatment. The origin of the micro-organism remains unclear, as enterococci do not belong to the normal oral microflora. The aim of this study was to determine whether these enterococci were of endogenous or exogenous origin. Methods and Results: Fifty consecutive patients with apical periodontitis in need of endodontic orthograde re-treatment were included. Samples were collected from root canals, saliva and faeces and subjected to microbiological culturing. The genetic relationship between Ent. faecalis from root canals and isolates from the different host sources was determined using pulsed-field gel electrophoresis. In 16% (8 ⁄50) of the patients, enterococci were collected from the root canal samples. The genetic analysis showed that the isolates from the root canals were not related to those from the normal gastrointestinal microflora. None of these patients had enterococci in their saliva samples. Conclusions: Endodontic infections with Ent. faecalis are probably not derived from the patient’s own normal microflora, which indicates that these infections ent. faecalis are of exogenous origin. Significance and Impact of the Study: This is the first study to genetically compare endodontic infectious Ent. faecalis isolates with isolates from the hosts’ own normal microflora.

Streptococcal histidine triad protein was identified recently as a cell surface-associated protein family. Five members of this family (PhtA, PhtB, PhtD, PhtE and HtpA), derived from Streptococcus pneumoniae and Streptococcus pyogenes, have been shown as antigens that confer protection to the host on infection. In this report, a gene sequence highly homologous to htpA and phtD (designated htpS, the histidine triad protein of Streptococcus suis) was identified from S. suis 2 Chinese strain 05ZYH33. Our data revealed that htpS is extremely conserved in S. suis 2 and widely distributed in 83% (29/35) of 35 S. suis serotypes. It was also demonstrated by Western blot and flow cytometry that HtpS is a cell surface-associated protein that was expressed during S. suis 2 infection. An antibody against HtpS could increase the deposition of human complement 3 on S. suis 2 and also enhance the clearance of S. suis 2 in whole blood. In addition, mice could be immunized against S. suis 2 infection and were well protected after immunization with recombinant HtpS.

The taxonomic characteristics of β-hemolytic streptococcal strains that reacted with Lancefield group M antisera were investigated. Group M streptococci have not been proposed as a species to date. Four strains of the group M streptococci isolated from dog were located within the pyogenic group of the genus Streptococcus on 16S rRNA gene-based phylogenetic analysis; the group M strains were located a short distance away from all other members of the group. The homology values of 16S rRNA gene sequences between group M strains and all other streptococci were

Aim: The objective of this study was to isolate and characterize the active compound from Trachyspermum ammi seeds, exhibiting antibiofilm activity against Streptococcus mutans, a major causal organism of dental caries. Methods and Results: Purification of the active compound from the seeds was performed by silica gel chromatography, and spectroscopic methods (FTIR, NMR and MS) were employed for its identification and structure determination. Antibiofilm and antiadherence activities of the active compound against S. mutans were analysed. Confocal microscopy was performed to visualize the effect of the compound on biofilm structure of S. mutans. Around 50% reduction was observed in adherence at 39·06 μg ml−1 and in biofilm at 78·13 μg ml−1. It was found effective against adherent cells of S. mutans, reduced water-insoluble glucan synthesis and inhibited the reduction in pH. Confocal microscopy revealed scattered cells at sub-MIC concentration of the compound, resulting in distorted biofilm architecture in contrast to clustered cells seen in control. Conclusion: This study revealed a novel compound, a naphthalene derivative, isolated first time from T. ammi seeds with antibiofilm activity against S. mutans. Significance and Impact of the Study: Trachyspermum ammi represents an interesting source of a novel compound, (4aS, 5R, 8aS) 5, 8a-di-1-propyl-octahydronaphthalen-1-(2H)-one, with a great potential to be used as a therapeutic agent against dental caries.

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 JCM8532T, Streptococcus mutans JCM5705T and Strep. pneumoniae NBRC102642T were susceptible. Furthermore, the bactericidal activity of sodium citrate against Strep. pneumoniae NBRC102642T 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 NBRC102642T 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.

Streptococcus suis is a worldwide cause of various swine infections and is also an important agent of zoonosis. Strains of S. suis are classified according to their serotype, and currently, 35 serotypes are recognized. The aim of this study was to characterize nontypeable isolates of S. suis with regard to their cell surface properties and compare them with serotype 2 strains, the most frequently associated with infections. The seven nontypeable strains of S. suis isolated from infected animals demonstrated a stronger capacity to adhere to a fibronectin-coated polystyrene surface than the serotype 2 isolates. Three nontypeable strains were also tested for their ability to adhere to endothelial cells and were found to attach in higher amounts compared with the serotype 2 isolates. Electron microscopy analysis revealed the absence of a capsule in the seven nontypeable isolates, which correlated with a much higher cell surface hydrophobicity than that of serotype 2 isolates. All nontypeable isolates of S. suis also showed the capacity to form a biofilm while serotype 2 isolates were unable to do so. In conclusion, the nontypeable isolates of S. suis examined in this study possess surface properties different from those of serotype 2 isolates.

Recently, 2 serologically and biochemically distinct subtypes-11Aalpha and 11Abeta-were discovered among serotype 11A isolates of Streptococcus pneumoniae. Sequence comparison of the capsular polysaccharide synthesis (cps) loci of the 2 subtypes identified disruption of the wcjE gene, a putative O-acetyltransferase, as the genetic hallmark of the 11Abeta phenotype. Directed disruption of wcjE in vitro in an 11Aalpha strain switched the strain to the 11Abeta phenotype, confirming the role played by the gene in the divergence between the subtypes. Furthermore, sequences from 7 11Abeta clinical strains each contained unrelated disruptive mutations in the wcjE gene, displaying an unprecedented degree of genetic heterogeneity in a pneumococcal serotype. We propose to name the 11Aalpha subtype as serotype 11A and the 11Abeta subtype as 11E, a new serotype. Our findings also suggest that the diversity of pneumococcal capsules is much greater than was previously recognized.

Group A streptococci (GAS) may engage different sets of virulence strategies, depending on the site of infection and host context. We previously isolated 2 phenotypic variants of a globally disseminated M1T1 GAS clone: a virulent wild-type (WT) strain, characterized by a SpeB(+)/SpeA(-)/Sda1(low) phenotype, and a hypervirulent animal-passaged (AP) strain, better adapted to survive in vivo, with a SpeB(-)/SpeA(+)/Sda1(high) phenotype. This AP strain arises in vivo due to the selection of bacteria with mutations in covS, the sensor part of a key 2-component regulatory system, CovR/S. To determine whether covS mutations explain the hypervirulence of the AP strain, we deleted covS from WT bacteria (DeltaCovS) and were able to simulate the hypervirulence and gene expression phenotype of naturally selected AP bacteria. Correction of the covS mutation in AP bacteria reverted them back to the WT phenotype. Our data confirm that covS plays a direct role in regulating GAS virulence.

A total of 171 Streptococcus pneumoniae isolates causing invasive disease were isolated from Chinese children. The serotype distribution and antimicrobial resistance were tested. The results suggested that the 7-valent pneumococcal conjugate vaccine has a preventive effect among children and that there should be long-term surveillance for serotype 19A.

Aim The aim was to isolate and characterize Lactococcus strains with new properties compared to those of usual Lactococcus dairy starters derived from cow's milk. Methods and results Algerian dromedary's milk was screened for proteolytic isolates able to grow rapidly on agar milk medium. PCR experiments revealed that 74 proteolytic isolates belonged to the genus Lactococcus and harboured the prtP gene encoding the lactococcal cell-surface proteinase. Among these, 85% were able to ferment citrate (Cit(+) phenotype) and were classified as Lactococcus lactis ssp. lactis biovar. diacetylactis. This classification was confirmed after sequencing of the 16S rDNA gene of five Cit(+) isolates. In contrast to dairy lactococci described in the literature, several Cit(+) isolates exhibited a tolerance to 50 degrees C (Ther(+)) and alkaline pH. Two genetic approaches allowed to show the presence of four independent plasmids (so-called pTher, pPrt, pLac, pCit) associated with the four respective phenotypes: Ther(+), cell-surface proteinase activity PrtP (PrtP(+)), lactose catabolism (Lac(+)) and citrate utilization (Cit(+)). Two types of pCit plasmid were amplified by inverse PCR: class 1 was characterized by a 9-kb plasmid harbouring the expected lactococcal citQRP operon and class 2 by a 23-kb plasmid harbouring the Leuconostoc cit cluster (citI-CitMCDEFGRP). Conclusions This work enlarges knowledge of the biovariety diacetylactis by far mainly limited to the citrate-fermenting ability and suggests that the cit plasmid system of some lactococcal strains could have been acquired from another lactic acid bacteria (Leuconostoc spp.). Significance and impact of the study This study reveals new potential dairy lactococci starters of the biovariety diacetylactis able to grow rapidly in milk at a higher temperature in addition to their casein, lactose and citrate-utilizing abilities.

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm-mediated collagen type I adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (20% of the 60 isolates), fibrinogen (17%) and laminin (13%), while only one or two of the isolates adhered to collagen type V, transferrin or lactoferrin and none to the other host components tested. Adherence to fibronectin and laminin was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to fibronectin and laminin, in addition to collagen type I, may have contributed to the emergence and adaptation of E. faecium, in particular CC17, as a nosocomial pathogen.

Aims: Genotypic and technological characterization of wild lactococci isolated from artisanal Manchego cheese during the ripening process for selection of suitable starter cultures. Methods and Results: A total of 114 isolates of lactococci were typed using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). Sixteen distinct RAPD-PCR patterns, at a similarity level of 73%, were obtained. On the basis of species-specific PCR reaction, the isolates were assigned to the species Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris with L. lactis subsp. lactis being predominant at both dairies. Twenty-six isolates were technologically characterized to select those with the best properties. Most of them showed good technological properties although some could produce tyramine. Conclusions: The presence of coincident genotypes at both dairies has been demonstrated, which would suggest that they are well adapted to the Manchego cheese environment. Interesting differences were found in the technological characterization and the potential role of autochthonous lactococci strains as starter culture has been displayed. Significance and Impact of the Study: The great economic importance of Manchego cheese encouraged a deeper knowledge of its microbiota, to select strains with the best properties to use as starter cultures in industrial Manchego cheeses, preserving the autochthonous characteristics.

Aims: To study the ability of daily applications of Streptococcus rattus strain JH145 to affect the numbers of an implanted Streptococcus mutans strain in a rat model. Methods and Results: A spontaneous L(+)-lactate dehydrogenase (LDH)-deficient mutant of Streptococcus rattus, JH146, was isolated by screening on selective medium and compared with a previously isolated spontaneous LDH deficient strain, JH145. Both strains were shown to have single base pair deletion mutations in the structural gene (ldh) for LDH, and reversion frequencies were approximately the same. Animals treated once daily with ≥106 CFU (colony forming units) of JH145 showed a statistically significant decrease in the proportion of implanted S. mutans to total cultivable bacteria in oral swab samples. The rate of decrease in S. mutans levels was dose-dependent. No adverse effects were observed by in-life observation of treated animals, and histopathological, haematological and blood chemistry analyses were unremarkable. Conclusions: The results presented indicate that daily application of JH145, a naturally occurring LDH-deficient variant of S. rattus, can compete with S. mutans for its habitat on the tooth surface. Significance and Impact of the Study: S. rattus JH145 has potential as a probiotic for use in the prevention of dental caries.

Diverse immune responses to an orally administered heat-killed cell preparation of Enterococcus faecalis strain EC-12 (EC-12) among jejunal-Peyer's patches (PPs), ileal-PPs, mesenteric lymph nodes (MLNs), and spleen were compared by real-time PCR in mice. Intriguingly, distinct responses to EC-12 were observed in the various tissues. This study indicates a site-specific response to orally administered bacteria, particularly in jejunal- and ileal-PPs.

Aims: Dental caries is caused by the disturbance in oral homeostasis, marked by a notable increase in the population of Streptococcus mutans. Lectins are a group of plant proteins that are capable of recognizing the glycoconjugates present on the bacterial surface. The aim of this study was to evaluate the effect of seven plant lectins on the growth and initial adhesion of S. mutans. Methods and Results: Lectins of different carbohydrate specificities were isolated from plant sources by conventional methods of protein purification. The effect on growth of S. mutans was evaluated following CLSI guidelines. None of the lectins used in this study inhibited the bacterial growth and multiplication. The adherence and biofilm formation of bacteria to saliva-coated polystyrene plates was tested in the presence of plant lectins. All the plant lectins tested, inhibited both the adherence and biofilm in a concentration dependent manner. Confocal microscopy and scanning electron microscopy were employed to assess the biofilm formation in the presence of plant lectin (glucose ⁄mannose-specific) at sub-minimal inhibitory concentrations. These evaluations revealed that lectins inhibited the clumping and attachment of S. mutans. Conclusions: Lectins tested here inhibited initial biofilm formation by S. mutans. Glucose ⁄Mannose-specific lectin altered the adhesion arrangement of the bacteria on the saliva-coated surfaces. Significance and Impact of the Study: The plant lectins used in this study may offer a novel strategy to reduce development of dental caries by inhibiting the initial adhesion and subsequent biofilm formation of S. mutans.

Nisin production in continuous cultures of bioengineered Lactococcus lactis strains that incorporate additional immunity and regulation genes was studied. Highest nisin activities were observed at 0.2 h-1 dilution rate and 12.5 g l-1 fructose concentration for all strains. Recombinant strains were able to produce greater amounts of nisin at dilution rates below 0.3 h-1 compared to the control strain. However, this significant difference disappeared at dilution rates of 0.4 and 0.5 h-1. For the strains LL27, LAC338, LAC339, and LAC340, optimum conditions for nisin production were determined to be at 0.29, 0.26, 0.27, and 0.27 h-1 dilution rates and 11.95, 12.01, 11.63, and 12.50 g l-1 fructose concentrations, respectively. The highest nisin productivity, 496 IU ml -1 h-1, was achieved with LAC339. The results of this study suggest that low dilution rates stabilize the high specific nisin productivity of the bioengineered strains in continuous fermentation. Moreover, response surface methodology analysis showed that regulation genes yielded high nisin productivity at wide ranges of dilution rates and fructose concentrations. © 2009 Society for Industrial Microbiology.

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis, respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges-Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.

The aim of this paper was to describe an increased occurrence of vancomycin-resistant enterococci (VRE) in Swedish broilers since 2000 and to investigate the genetic relatedness of isolates. Caecal content from slaughtered broilers was cultured for VRE on medium supplemented with vancomycin (16 mg/L). Species identification, antibiotic susceptibility determination, vancomycin resistance genotyping, multilocus sequence typing (MLST) and characterization of Tn1546 were performed. The proportion of VRE-positive samples increased gradually from

Streptococcus bovis HC5 produces a broad spectrum lantibiotic (bovicin HC5) that inhibits pure cultures of hyper ammonia-producing bacteria (HAB). Experiments were preformed to see if: (1) S. bovis HC5 cells could inhibit the deamination of amino acids by mixed ruminal bacteria taken directly from a cow, (2) semi-purified bovicin was as effective as S. bovis HC5 cells, and 3) semi-purified and the feed additive monensin were affecting the same types of ammonia-producing ruminal bacteria. Because purified and semi-purified bovicin HC5 was as effective as S. bovis HC5 cells, it appeared that bovicin HC5 was penetrating the cell membranes of HAB before it could be degraded by peptidases and proteinases. Mixed ruminal bacteria that were successively transferred and enriched nine times with trypticase did not become significantly more resistant to either bovicin HC5 (50 AU mL(-1)) or monensin (5 microM), and amplified rDNA restriction analysis indicated that bovicin HC5 and monensin appeared to be selecting against the same types of bacteria.

Aims: This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1. Methods and Results: Eight prtF1-positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced (P

Aims: To investigate clonality among clinical Enterococcus faecium isolates and normal intestinal microflora isolates as well as cross-transmission between patients in relation to the presence of the esp gene and antibiotic resistance. Methods and Results: Blood-culture isolates (n = 101) deriving from tertiary, secondary and primary hospitals were analysed. Antibiotic susceptibility was investigated. Polymerase chain reaction and pulsed-field gel electrophoresis were used for detection of esp and genotyping, respectively. Nearly half (43%) of the patients included were involved in a cross-transmission event with Ent. faecium. These strains disseminated both within and between all hospitals. The antibiotic resistance and presence of esp were highest in isolates from the tertiary hospital. Isolates harbouring esp showed less genetic diversity compared with esp negative ones. Conclusions: Cross-transmission with Ent. faecium between patients was readily detected, indicating that hospital-adapted clones circulate within and between hospitals. Acquired characteristics, such as antibiotic resistance and esp, seem to accumulate in the isolates disseminating in the tertiary hospital. Significance and Impact of the Study: It is important to characterize Ent. faecium isolates causing infections and to determine the extent of dissemination in order to prevent further spread of these pathogens.

Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ-introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study: Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.

We investigated the preventive effect of Streptococcus thermophilus YIT 2001, a lactic acid bacterum having high antioxidative activity, on acute colitis induced by 2.5% dextran sulfate sodium in mice, and compared the effect with that of S. thermophilus YIT 2084 which has lower antioxidative activity. Feeding S. thermophilus YIT 2001 decreased the disease activity index and level of lipid peroxide (the thiobarbituric acid reactive substance content) in the colonic mucosa. The hematocrit and hemoglobin concentrations in the blood of S. thermophilus YIT 2001-fed mice were higher than those of the control mice. S. thermophilus YIT 2084 had no significant effect on these parameters. The results suggest that the antioxidative activity of S. thermophilus YIT 2001 was involved in the improving effect on colitis.

Background: Studies have shown the efficacy of intra-partum antibiotics in preventing early-onset group B streptococcal sepsis. This approach results in a high intra-partum antibiotic use. Worryingly, the same antibiotics used in prophylaxis are also first-line treatment for neonatal sepsis, and antibiotic exposure in the peri-natal period has been shown to be a risk factor for late-onset serious bacterial infections and allergic disease. Antibiotic exposure in the peri-natal period is becoming a major public health issue; alternative strategies are needed. Garlic has been traditionally used to treat vaginal infections. Allicin is the main antibacterial agent isolated from garlic. Objectives: The aim of the study was to investigate the in vitro activity of a novel allicin extract in aqueous and gel formulation against 76 clinical isolates of Lancefield group B streptococci (GBS). Methods: MICs and MBCs of allicin were determined for 76 GBS isolates by agar dilution and microtitre plate methods. Killing kinetics were determined for a selected 16 of the 76 strains. Agar diffusion tests were compared for allicin liquid and gel (500 mg/L). Results and conclusions: MICs and MBCs of allicin liquid were 35 to 95 mg/L and 75 to 315 mg/L, respectively. Time/dose kill curves produced a 2–3 log reduction in cfu/mL within 3 h and no detectable growth at 8 and 24 h. A novel 500 mg/L allicin gel produced an average zone size of 23+-6 mm compared with 21+-6 mm for allicin in water. Aqueous allicin is bactericidal against GBS isolates and maintains activity in a novel gel formulation.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.

Objectives: The present study focused on isolation, characterization and evaluation of purified compounds from Morus alba against Streptococcus mutans biofilm formation. Methods: The effect of crude extract from M. alba leaves was evaluated against oral pathogens, chiefly S. mutans. MICs were determined by the microdilution method. The compound was purified by employing silica gel chromatography and critically analysed with GC‐MS, NMR and IR spectroscopy. The S. mutans traits of adherence and biofilm formation were assessed at sub-MIC concentrations of the crude extract and purified compound. Both water-soluble and alkali-soluble polysaccharide were estimated to determine the effect of the purified compound on the extracellular polysaccharide secretion of S. mutans. Its effect on biofilm architecture was also investigated with the help of confocal microscopy. Results: The purified compound of M. alba showed an 8-fold greater reduction of MIC against S. mutans than the crude extract (MICs, 15.6 and 125 mg/L, respectively). The extract strongly inhibited biofilm formation of S. mutans at its active accumulation and plateau phases. The purified compound led to a 22% greater reduction in alkali-soluble polysaccharide than in water-soluble polysaccharide. The purified compound was found to be 1-deoxynojirimycin (DNJ). Confocal microscopy revealed that DNJ distorts the biofilm architecture of S. mutans. Conclusions: The whole study reflects a prospective role of DNJ as a therapeutic agent by controlling the overgrowth and biofilm formation of S. mutans.

In a previous study, preparations of polyclonal antibodies (PAP) against Fusobacterium necrophorum (PAP-Fn) or Streptococcus bovis (PAP-Sb) were successful in decreasing ruminal counts of target bacteria and increasing ruminal pH in steers fed high-grain diets. The objective of this study was to evaluate the effects of feeding PAP-Fn or PAP-Sb on performance, carcass characteristics, and ruminal fermentation variables of feedlot steers. In Exp. 1, during 2 consecutive years, 226 or 192 Angus and Angus crossbred steers were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. When measured on a BW basis, steers fed only PAP-Sb had a greater G:F (P 0.10) to steers fed no PAP or only PAP-Sb. Steers receiving PAP-Fn (alone or in combination with PAP-Sb) had a decreased (P 0.10) were observed in any other carcass characteristics. In Exp. 2, sixteen ruminally cannulated Angus crossbred steers (BW = 665 +/- 86 kg) were fed a high-grain diet containing either PAP-Sb or PAP-Fn, or both. Feeding only PAP-Fn or PAP-Sb for 19 d decreased (P < 0.05) ruminal counts of S. bovis when compared with steers fed both or no PAP. The ruminal counts of F. necrophorum in steers fed PAP-Fn alone or in combination with PAP-Sb were decreased by 98% (P < 0.05) after 19 d, when compared with the counts in control steers. Mean daily ruminal pH was greater (P < 0.05) in steers fed both PAP when compared with feeding either or no PAP. Ruminal pH in the first 4 h after feeding was greater (P < 0.05) for steers receiving PAP-Fn alone or in combination with PAP-Sb. Steers receiving either PAP alone or in combination had less (P < 0.05) ruminal NH(3)-N concentrations in the first 4 h after feeding when compared with those of control steers. Polyclonal antibody preparations against S. bovis were effective in enhancing G:F of steers fed high-grain diets, but dressing percentage was decreased. Mechanisms of enhancement of G:F remain unknown but may be related to changes in ruminal counts of target bacteria and associated effects on ruminal fermentation products.

Treatment of bacterial meningitis caused by Streptococcus pneumoniae is increasingly difficult, because of emerging resistance to antibiotics. Recombinant Cpl-1, a phage lysin specific for S. pneumoniae, was evaluated for antimicrobial therapy in experimental pneumococcal meningitis using infant Wistar rats. A single intracisternal injection (20 mg/kg) of Cpl-1 resulted in a rapid (within 30 min) decrease in pneumococci in cerebrospinal fluid (CSF) by 3 orders of magnitude lasting for 2 h. Intraperitoneal administration of Cpl-1 (200 mg/kg) led to an antibacterial effect in CSF of 2 orders of magnitude for 3 h. Cpl-1 may hold promise as an alternative treatment option in pneumococcal meningitis.

Streptococcus bovis HC5 cultures released a broad spectrum lantibiotic, bovicin HC5, into the cell-fee culture supernatant after they reached stationary phase, but most of the antibacterial activity remained cell-associated. Cell-associated bovicin HC5 was more resistant to degradation by Pronase E than the cell-free activity. Acidic NaCl (pH 2.0, 100 mM) did not release all of the cell-associated activity, and cells that were sequentially treated with acidic NaCl and Pronase E still had antibacterial activity. Cell-associated activity retained after acidic NaCl treatment was still able to catalyze potassium efflux from S. bovis JB1, a sensitive strain. These results indicate that cell-associated bovicin HC5 is more active and stable than cell-free bovicin HC5.

During the last 20 years, a dramatic increase in antibiotic resistance among pneumococci has been observed and linked to increasing antibiotic consumption [1-4]. Several studies have suggested that reduction in antibiotic consumption leads to a reduction in the antibiotic resistance of Streptococcus pneumoniae in the community [2, 5-7]. However, other studies could not demonstrate such an effect [8-10]. The relationship between antibiotic consumption and resistance is complex. Some antimicrobial agents select resistant pneumococcal strains more effectively than others [8, 11-15]. Moreover, the rate of the decrease in resistance expected after a decrease in antibiotic use depends on both the fitness cost of resistance for the bacteria and the extent of the reduction in consumption [16, 17]. If fitness cost is minimal or if the reduction in antibiotic use is small, antibiotic resistance may continue increasing, albeit perhaps more slowly, or it may decline gradually. However, if resistance exacts a strong fitness cost (with respect to transmissibility) on the bacteria, then a quick and significant decrease in resistance would be expected after a major decrease in antibiotic consumption [18]. A recent study in rats suggested that penicillin resistance in S. pneumoniae, particularly high-level resistance, carries a substantial fitness cost [19]. Another recent study showed a fitness cost of fluoroquinolone-resistant mutants of S. pneumoniae [20]. A complicating factor is the presence of dual and multiple resistance, because reduction mainly in the use of a single antibiotic may not result in the expected reduction in resistance, because of coselection of resistance caused by the continued use of other antibiotics [18]. Surveillance of antibiotic prescriptions in children

BACKGROUND: To prepare for national introduction of a pneumococcal conjugate vaccine of restricted valency, we studied nasopharyngeal carriage of Streptococcus pneumoniae in Gambian infants. METHODS: We studied 236 infants in 21 villages. We collected nasopharyngeal swab samples at birth, twice per month for 6 months, and every second month until 1 year of age. We studied time to acquisition and duration of pneumococcal carriage according to serotype. RESULTS: All infants carried S. pneumoniae at some point. Sixty-five serotypes were found, and the 5 most common serotypes (6B, 19F, 6A, 14, and 23F) accounted for 51% of isolates. The mean age at first acquisition of carriage was 33 days (95% confidence interval, 29-36 days). There were no significant differences in acquisition rates between the 6 most common serotypes (P = .067) or between vaccine serotypes, vaccine-related serotypes, or nonvaccine serotypes (P = .317). However, the duration of carriage differed significantly between the 6 most common serotypes (P = .004). The rate of reacquisition of carriage and the duration of carriage did not differ significantly between the 6 most common serotypes (P = .229 and P = .669 respectively). However, nonvaccine types were acquired faster (P = .004) and were carried for a shorter duration (P < .001) than were vaccine serotypes. A previous episode of serotype 14 carriage was associated with delayed reacquisition of this serotype (P = .005) and longer duration of carriage (P = .017). CONCLUSIONS: The data provided in this study regarding time to acquisition and duration of pneumococcal carriage in Gambian infants provide an important baseline for evaluating the impact of the introduction of a pneumococcal conjugate vaccine in The Gambia and elsewhere in Africa.

IFN-gamma is a cytokine produced primarily by both T lymphocytes and natural killer cells and it is considered to be an attractive therapeutic molecule. In the present study, a DNA sequence encoding the mature murine IFN-gamma (muIFN-gamma) protein was cloned and expressed in the food-grade lactic acid bacterium Lactococcus lactis. The activity of recombinant muIFN-gamma produced by genetically engineered L. lactis was confirmed in an antiviral assay using MoV cells infected with Vesicular Stomatitis Virus. The data provide the first demonstration that a Gram-positive bacterium, L. lactis, is able to produce functional muIFN-gamma. This recombinant strain could lead to the development of a new, well-tolerated vector to deliver active muIFN-gamma at the mucosal level.

A group A Streptococcus (GAS) isolate, serotype M12, recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing phi149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of phi149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage phi149::Km(r). Phage phi149::Km(r) from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones.

Aims: To improve the production of sweet-tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin-controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni-NTA plates and Western blot. Use of the M-17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml−1 nisin at OD 3·0 led to an approx. 17-fold increase in brazzein per cell production compared to non-optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.

Antibodies to pneumococcus are thought to represent the primary mechanism of naturally acquired resistance to colonization. Here, however, we show that, in patients with chronic obstructive pulmonary disease (COPD), resistance to pneumococcal colonization is not associated with higher concentrations of serum anti-capsular or -noncapsular antibodies. We compared preacquisition serum antibody concentrations to capsular antigens 6B, 7F, 14, 19F, and 23F from patients with COPD who did and did not acquire pneumococcal respiratory tree colonization over the course of 2 years. Colonized patients did not have lower anti-capsular antibody concentrations than control subjects who did not acquire pneumococcus. We found no difference in functional antibody concentrations between colonized patients and control subjects. Furthermore, colonized patients had significantly higher preacquisition concentration of antibody directed against the whole cell and pneumococcal surface protein A than control subjects. We thus conclude that, in adult patients with COPD, resistance to pneumococcal colonization is unlikely to be determined by higher serum antibody concentrations to pneumococcal antigens.

Aims: To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco-2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219. Methods and Results: Bacteriocin HV219 activity against Gram-positive and Gram-negative bacteria was confirmed by leakage of DNA and β-galactosidase, and atomic force microscopy. Adsorption of bacteriocin HV219 to bacteria is influenced by pH, temperature, surfactants and salts. Initially, only 3% of HV219 cells adhered to Caco-2 cells. However, after 2 h, adherence increased to 7%. Strain HV219 and Listeria monocytogenes ScottA did not compete for colonization. Strain HV219 is sensitive to most antibiotics tested, but resistant to amikacin, ceftazidime, nalidixic acid, metronidazole, neomycin, oxacillin, streptomycin, sulphafurazole, sulphamethoxazole, sulphonamides, tetracycline and tobramycin. Ibuprofen, ciprofloxacin, diklofenak and nonoxylol-9 inhibited the growth of strain HV219. Conclusion: Strain HV219 is resistant to hostile conditions in the intestinal tract, including therapeutic levels of specific antibiotics and binds to Caco-2 cells, but not in competition with L. monocytogenes. Significance and Impact of the Study: Strain HV219 will only be effective as probiotic if taken with specific antibiotics and not with anti-inflammatory drugs and spermicides.

The plasmid stability of three wild type Lactococcus lactis strains and their mutants was investigated at different incubation time and temperatures in two different media [M17 broth and reconstituted skim milk (RSM)]. The results showed that both incubation times and temperature are effective on plasmid loss. The plasmid profiles of wild type strains exhibited 8 to 9 distinct plasmid species with molecular weights from 2.1 to 24.0 kb. Lactose fermentation ability was found to be encoded by 22.2 (strain U70), 23.6 (strain U29) and 24.0 (strain U52) kb plasmids in the wild type strains, respectively. The stabilities of the plasmids were explained by applying a second-order polynomial modeling system. Reasonable fittings were obtained for the model and the adjusted regression coefficients (R 2 adj) were between 0.76 and 0.99 for the overall data. Overall, it was found that incubation time had the most profound effect on plasmid stability, with plasmid loss occurring after 72 h, while temperatures in the range of 15–40°C also induced plasmid instability.

Aims: To investigate the effects of two prebiotics and trehalose on the production of bacteriocins. Methods and Results: Four carbohydrates [dextrose, fructo-oligosaccharides (FOS), raffinose, and trehalose] were used as the sole carbon source in a simple broth. Five bacteriocin-producing strains of bacteria, including those producing nisin, enteriocin, and other bacteriocins, were used, and their inhibitory activities when grown on each carbohydrate were determined. The inhibitory activity assay was performed using the agar well diffusion method, and Lactobacillus sakei JCM 1157T was used as the indicator strain. Effective enhancement of bacteriocin production was observed with FOS and trehalose incubation. Conclusions: The results suggest that FOS and trehalose can effectively enhance the production of the five kinds of bacteriocins evaluated in this study. Significance and Impact of the Study: This study offers useful information for not only a new application of FOS and trehalose, but also the potential improvement of food preservation.

Recently, we showed that the in vitro lag phase correlates with the pneumococcal serotype. This study investigated the role of capsule genes in bacterial growth using strain D39. Deletion of the entire capsule operon induced a significantly prolonged lag phase in Todd Hewitt broth (P=0.0002). However, partial deletions showed a different influence on the lag phase. Supplementation of media with 5% fetal bovine serum restored normal growth, at least partially, in mutants with a prolonged lag phase. Therefore, pneumococcal capsule gene products influence bacterial growth in vitro in strain D39.

Objectives: In a previous study, we observed marked synergy between daptomycin and rifampicin against 73% of rifampicin-resistant, vancomycin-resistant Enterococcus faecium (VRE), with approximately 100-fold reductions in rifampicin MICs observed at one-eighth to one-fourth daptomycin MIC. The purpose of this study was to determine whether the synergy between daptomycin and rifampicin could be explained by enhanced entry of rifampicin into the cell or was related to amino acid substitutions in the rifampicin-binding site in the b subunit (rpob) of the RNA polymerase. Methods: We developed a bioassay for rifampicin to measure cell-bound rifampicin levels as well as metabolic inactivation of rifampicin. In addition, we sequenced the rifampicin-binding site in the rpob of VRE strains with and without synergy between daptomycin and rifampicin. Results: Cell-bound rifampicin levels were the same in rifampicin-susceptible VRE as in rifampicinresistant VRE showing daptomycin synergy and were not affected by the presence of daptomycin. In contrast, rifampicin-resistant VRE without daptomycin synergy had undetectable cell-bound rifampicin. Sequencing the rpob rifampicin-binding site revealed that the synergistic strains had the same sequence as rifampicin-susceptible wild-type E. faecium. The daptomycin synergy-resistant strains all had mutations in known rifampicin-binding sites. Conclusions: Daptomycin is able to reverse rifampicin resistance in some strains of VRE, but the mechanism could not be explained by an effect of daptomycin on entry of rifampicin into or transport out of the cell, by inactivation of rifampicin or by mutation involving the rifampicin-binding site.