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The aim of this study was to examine the survival dynamics of several epidemic health care-associated (HA) and community-associated (CA) methicillin-resistant Staphylococcus aureus (MRSA) in planktonic state in widely employed denture-cleaning solutions. The bacteriocidal activity of five widely employed denture-cleaning formulations were examined against five phage-types of HA-MRSA (EMRSA 15, EMRSA 16, Irish 1, Irish 2, unique type), as well as a CA-MRSA strain, in this study. Viable MRSA cells (circa 10(5) cfu/mL) were coincubated with optimum recommended working concentrations of denture-cleaning solutions for up to 17 hours (overnight). Recovery experiments were unable to isolate any of the inoculated MRSA organisms 10 minutes post inoculation. The significance and impact of this short study indicates that HA-MRSA and CA-MRSA are not able to remain culturable for 10 minutes in planktonic form, in commonly used denture-cleaning formulations widely available on the UK High Street, suggesting that these formulations may be useful in lowering the numbers of MRSA. Further work is however required to examine the more complex survival dynamics of MRSA in naturally derived denture biofilm, associated with dental plaque and the use of such cleaning formulations. [ABSTRACT FROM AUTHOR]

Porphyromonas gingivalis produces different LPS isoforms with significant structural variations of their lipid A and O-antigen moieties that can affect its pro-inflammatory and bone-resorbing potential. We show here, for the first time, that P. gingivalis LPS isolated from W83 strain is highly sialylated and possesses significantly reduced inflammatory potential compared with less sialylated ATCC 33277 strain LPS. Nevertheless, the reduction in the endotoxin activity is not mediated by the presence of sialic acid LPS moieties as the sialic acid-free LPS produced by the mutant W83 strain exhibits a similar inflammatory potential to the wild type strain. Furthermore, our findings suggest that the interaction between the sialic acid LPS moieties and the inhibitory CD33 receptor is prevented by endogenously expressed sialic acid on the surface of THP-1 cells that cannot be out-competed by sialic acid containing P. gingivalis LPS. The present study also highlights the importance of endogenous sialic acid as a 'self-associated molecular pattern' and CD33 receptors in modulation of innate immune response as human gingival fibroblasts, which do not express CD33 receptors, and desialylated THP-1 cells have both been found to have much higher spontaneous IL-8 production than naïve THP-1 cells.

Objective: To investigate the potential effects of IFN-03A5 on the responsiveness of human gingival fibroblasts to bacterial challenge., Design: mRNA and protein expression of CD14, TLR2 and TLR4 in human gingival fibroblasts was detected by quantitative polymerase chain reaction (Q-PCR) and flow cytometry. The effect of preincubation with IFN-03A5 on subsequent bacterial LPS-induced expression of IL-6 and IL-8 by gingival fibroblasts was determined by ELISA. Bacterial LPS-induced IκBα degradation in human gingival fibroblasts was investigated by western blot., Results: Human gingival fibroblasts express CD14, TLR2 and TLR4 mRNAs. IFN-03A5, but not IL-103B2, induced mRNA expression of all three receptors and the expression of membrane bound CD14 protein. Pre-incubation of fibroblasts with IFN-03A5 and subsequent stimulation with Escherichia coli LPS or Porphyromonas gingivalis LPS led to increased production of IL-6 and IL-8. LPS-induced pro-inflammatory cytokine production was abrogated by a blocking antibody to CD14. Both E. coli LPS and P. gingivalis LPS induced IκBα degradation in human gingival fibroblasts., Conclusion: Our data indicate that IFN-03A5 primes human gingival fibroblasts, through the upregulation of CD14 expression, which results in increased responsiveness to bacterial LPS challenge, as determined by pro-inflammatory cytokine production., (Copyright © 2015. Published by Elsevier Ltd.)

What Is Known and Objective: Antibiotic resistance has become a global public health issue. Most antibiotics are prescribed in the community, although there is less stewardship of such agents in the community compared to secondary and tertiary care. Few studies have attempted to examine the prescribing practices in General Practice and its impact on antibiotic resistance and, therefore, a study was performed in order to compare antibiotic susceptibilities of commensal viridans group streptococci (VGS) obtained from patient cohorts in General Practices (GP), who were high and low prescribers of oral antibiotics., Method: Sixty-five patients (<1 month-81 years; 77% female: 23% male) were enrolled onto the study, and viridans group streptococci (n = 5/patient) were collected from each patient's nasal passages and oropharynx region and tested for antibiotic susceptibility against (i) tetracyclines (doxycycline); (ii) macrolides (erythromycin); (iii) β-lactams (penicillin G); and (iv) fluoroquinolones (ofloxacin & levofloxacin)., Results and Discussion: There were no significant differences in MICs between high and low GP prescribers with doxycycline (P = 0·094), erythromycin (P = 0·122), ofloxacin (P = 0·193) and levofloxacin (P = 0·058). However, there was a significant difference between high and low GP practices with regard to penicillin G (P = 0·031). This finding is important as the β-lactams are the most commonly prescribed oral antibiotic in the community., What Is New and Conclusion: This study demonstrates that high prescribing practices may lead to an altered (higher) level of resistance to these agents in the commensal VGS population, which may be important as reservoirs of antibiotic resistance determinants in subsequent horizontal gene transfer events, particularly with newly colonizing pathogens, including pneumococci. Primary care physicians should be aware that increased prescribing of antibiotics may led to increased level of penicillin resistance., (© 2015 John Wiley & Sons Ltd.)

What Is Known and Objective: To date, there is no evidence to indicate the reliability of how patients self-report their own antibiotic usage in the community. Such data are fundamental in supporting antimicrobial stewardship practices, and so there is a need to determine its accuracy and reliability., Comment: Patients in the community (n = 476) were required to recollect their antibiotic usage in the past three months. Simultaneously, similar information was obtained by careful extraction from their respective medical notes, which was qualitatively compared with the patient's recollection. Overall, concordance was high (88·1%), but age (<20 and >80 years) and sex (female) were significant factors of reliability., What Is New and Conclusion: This study suggests that basic self-reporting of antibiotic usage amongst patients is relatively reliable, with increasing accuracy with years until 80 years. Where such information is critical, the current study can help decide who to interview and whose notes to interrogate, in the quest to obtain reliable and accurate information., (© 2014 John Wiley & Sons Ltd.)

Aim: Recent UK National Institute for Health and Clinical Excellence guidelines state that there is no longer a need for oral antibiotic prophylaxis in patients undergoing dental procedures who are at risk of infective endocarditis (IE), and advocate the importance of maintaining good oral health. As viridans group streptococci (VGS) are common etiological agents of IE and inhabitants of the mouth, the purpose of this study was to examine the efficacy of common high-street mouthwashes against four classes of VGS organisms (salivarius, mitis, anginosus, and mutans groupings)., Methods: The survival of VGS, Streptococcus gordonii (National Collection of Type Cultures [NCTC] 7865), Streptococcus intermedius (NCTC 11324), Streptococcus mutans (NCTC 10449), Streptococcus oralis (NCTC 11427), Streptococcus pneumoniae (NCTC 7465, NCTC 7978, & American Type Culture Collection 49619) and Streptococcus salivarius (NCTC 8618) was assessed in vitro following treatment of approximately 10(7) c.f.u. in planktonic state with four mouthwashes., Results: No organisms were culturable following 1-min exposure, and were not recovered following non-selective enrichment following incubation in Brain Heart Infusion broth supplemented with 0.8% (w/v) yeast extract., Conclusions: These data indicate that such mouthwashes are able to completely kill VGS organisms tested in planktonic solution, where their use would promote good oral hygiene in patients at risk of IE., (© 2014 Wiley Publishing Asia Pty Ltd.)

Aim: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease., Materials and Methods: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry., Results: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites., Conclusions: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A study was undertaken to examine the population structure of viridans group streptococci (VGS) isolated the upper respiratory tract of adult and paediatric patients within the community. VGS are common commensal bacterial inhabitants of the upper respiratory tract and valuable sentinel reporters of underlying antibiotic resistance (AR). Laboratory examination of the colonising VGS species may provide a valuable ecological description of the species isolated from the upper respiratory tract and their antibiotic susceptibility, including an estimation of the AR reservoir in this population. Freshly obtained nasal and oropharyngeal swabs from 84 patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 363 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 10 species of VGS and included S. anginosus, S. australis, S. constellatus, S. infantis, S. mitis, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis and S. vestibularis. The most frequent VGS organisms isolated was S. salivarius (282/363; 78.0%), followed by S. sanguinis (23/363; 6.3%), S. parasanguinis (21/363; 5.8%), S. mitis (18/363; 5.0%), S. anginosus (5/363; 1.4%), S. vestibularis (5/363; 1.4%), S. australis (3/363; 0.8%), S. oralis (3/363; 0.8%), S. infantis (1/363; 0.3%) and S. constellatus (1/363; 0.3%). All patients examined carried at least one VGS organism, where there were 17 combination patterns of carriage of the 10 species of VGS species isolated, where 54.2%, 37.3%, 7.2% and 1.2% of patients harboured one, two, three and four different VGS species, respectively. Antibiotic susceptibility was determined by standard disk diffusion assay testing against four classes of antibiotics, including the b-lactams [cefotaxime, cefuroxime], the tetracyclines [doxycycline], the fluoroquinolones [levofloxacin] and the macrolides [erythromycin]. Overall, there was no resistance to levofloxacin and cefuroxime, with limited resistance to cefotaxime (3.3%) and doxycycline (9.8%). Antibiotic resistance was highest in erythromycin, where 40.9% of isolates were resistant. S. vestibularis was the most antibiotic resistance of all VGS species examined (S. vestibularis v S. salivarius p=0.011), followed by S. anginosis. S. salivarius was the most antibiotic susceptible VGS species examined. Overall, given their infrequency in causing infection, relatively few studies to date have attempted to examine their ecology in their preferred body niche, namely the upper respiratory tract. However, knowing their prevalence is becoming increasingly important in relation to their ability to exclude significant respiratory pathogens, including Streptococcus pneumoniae. In conclusion, these data indicate that VGS colonisation of the upper respiratory tract in individuals within the community is dominated mainly with relatively antibiotic susceptible S. salivarius.

Aim: Previous work has indicated that environmental stresses on bacteria might lead to an upregulation of stress response. LED curing lights (315-400 nm) and other UV lights used in tooth whitening cosmetic procedures might act as stresses. We examined the effect of UV-C light, as a high-energy surrogate to the lower-energy UV-A light used in such instruments, to examine its effect on the antibiotic susceptibility of viridans group streptococci., Methods: Twelve species of viridans group streptococci were examined in this study: Streptococcus anginosus, Streptococcus australis, Streptococcus cristatus, Streptococcus gordonii, Streptococcus infantis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus pneumoniae, Streptococcus salivarius, and Streptococcus sanguinis. These organisms were exposed to varying degrees of sublethal UV-C radiation, and their minimum inhibitory concentration susceptibility was determined by broth dilution assay against three classes of commonly-used antibiotics: β-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin)., Results: There was no significant difference between antibiotic susceptibility before UV-C exposure and following maximum sublethal stress, prior to cell death due to fatal UV-C exposure., Conclusions: Exposure to UV-C light will not result in altered antibiotic susceptibility patterns on viridans group streptococci. Given that UV-C is more toxic and mutagenic than UV-A light, it is unlikely than UV-A light would yield any difference in response to such exposure., (© 2011 Blackwell Publishing Asia Pty Ltd.)

Skin tanning, either by exposure to natural sunlight or through use of UV sunbeds, has become a popular practice in the US, where it is estimated that approximately 1 million times per day someone in the US uses UV radiation for skin tanning, equating to 30 million Americans (circa 10% of the US population) who use a tanning bed. As well as exposing the host to periods of UV radiation, such practices also expose commensal skin bacteria, including Staphylococcus aureus, to such UV radiation. Previous work has indicated that environmental stresses on bacteria may lead to an upregulation of stress responses, in an attempt for the organism to combat the applied stress and remain viable. UV light may act as an environmental stress on bacteria, and so it was the aim of this study to examine the effect of UVc light on the antibiotic susceptibility of commensal skin bacteria, to determine if UV radiation would increase the antibiotic resistance of such skin flora and thus lead to a potential skin flora with increased antibiotic resistance. Previously, it has been shown that UVc light has a greater mutational effect on bacteria compared to lower-energy UV forms, including UVa and UVb light. Therefore, we decided to employ UVc light in our study to amplify the potential for mutational events occurring in skin staphylococci organisms (n=8) including methicillin-sensitive Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (n=4), and coagulase-negative staphylococci (Staphylococcus haemolyticus) (n=2) were exposed to varying degrees of sublethal radiation via UVc light, and their minimum inhibitory concentration (MIC) susceptibility was determined by broth dilution assay against three classes of commonly used antibiotics, namely β-lactams (penicillin), macrolides (erythromycin), and fluoroquinolones (ciprofloxacin). There was no significant difference between antibiotic susceptibility before UVc exposure and until maximum sublethal stress, prior to cell death due to fatal UVc exposure with the cells. These results indicate that UV environmental stress/exposure does not upregulate antibiotic resistance, and therefore these data indicate that UVc radiation does not lead to a more antibiotic-resistant population in the staphylococci organisms post-exposure.

Antibiotic resistance in clinical pathogens in humans may be traced back to resistance mechanisms in environmental bacteria and any factors, which are likely to alter (upregulate) resistance in environmental organisms, is of potential and eventual consequence to human pathogens. Furthermore, sublethal doses of gamma radiation to environmental organisms may cause sublethal stress and a selective pressure, which may lead to mutational events that alter the bacterium's susceptibility profile. A gamma (γ) radiation simulation experiment was performed to emulate the exposure of four environmental bacteria, including Listeria innocua, Bacillus subtilis, E. coli and Pseudomonas aeruginosa, to levels of radiation in and around Fukushima, Japan, equating to 1, 10 and 100 years equivalence exposure. Alteration to susceptibility to 14 antibiotics was measured as the primary endpoint. There was no significant alteration in the susceptibility of the Gram-positive organisms, whereas both Gram-negative organisms became slightly more susceptible to the antibiotics tested over time. These data indicate that such radiation exposure will not increase the antibiotic resistance profile of these organisms and hence not add to the global public health burden of increased antibiotic resistance in human bacterial pathogens., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia (CAP). Currently, empirical treatment with quinolones is being used due to the emergence of beta-lactam and macrolide resistance in S. pneumonaie. Although the prevalence of quinolone-resistant S. pneumoniae remains low, increasing numbers of resistant isolates are being seen. Genetic mechanisms leading to fluoroquinolone resistance in pneumococci are complex. This study aims to use molecular methods to characterise all isolates through sequence analysis of their QRDR regions. Thirty-two S. pneumoniae isolates were obtained from nasal swabs from adult and paediatric patients attending local general practices in Northern Ireland. Phenotypic minimum inhibitory concentration (MIC) was determined for Clinical and Laboratory Standards Institute (CLSI) broth microdilution against ciprofloxacin, levofloxacin and norfloxacin. Simultaneously, the QRDR regions of gyrA, gyrB, parC and parE were analysed by sequence typing for all pneumococci obtained. Only one isolate (3.1%) showed reduced susceptibility to ciprofloxacin and levofloxacin. Two amino acid positions were discordant in the S. pneumoniae R6 strain and eight (25%) and 23 (71.9%) isolates contained the mutations Ile460Val in gyrA and Lys137Asn in parC (deposited in GenBank, accession numbers GQ999587-GQ999589), respectively. No mutations were found in either the gyrB or parE loci. In conclusion, the study demonstrated increased fluoroquinolone resistance which could not be accounted for simply through QRDR mutations, and, reciprocally, that mutations in the QRDR region do not necessarily result in overt phenotypic resistance.

The State Performance Plan (SPP) developed under the 2004 reauthorization of the Individuals with Disabilities Education Act (IDEA 2004, Public Law 108-446) requires states to collect data and report on the impact of early intervention services on three key outcomes for participating families. The NCSEAM Impact on Family Scale (NIFS) and the NCSEAM Family Centered Services Scale (NFCSS) were developed to provide states with a means to address this new reporting requirement and to collect additional data that would inform program improvement efforts. Items suggested by stakeholder groups were piloted with a nationally representative sample of parents of children with developmental delays or disabilities ages birth to three participating in early intervention services in eight states. The 28-item NIFS had measurement reliabilities ranging from .93-.96 in a sample of 1,750; measurement reliabilities for the 135-item NFCSS ranged from .94 to .97 in a sample of 1,755 respondents. A 29-item version of the NFCSS had measurement reliabilities ranging from .87 to .92. Using data from the pilot study, stakeholders established a recommended performance standard, set at a meaningful point in the NIFS item hierarchy, for each of the three established outcome areas.

Inadvertent exposure of bacterial pathogens to X-ray radiation may be an environmental stress, where the bacterium may respond by increasing mutational events, thereby potentially resulting in increased antibiotic resistance and alteration to genotypic profile. In order to examine this, four clinical pathogens, including the Gram-negative organisms Escherichia coli O157:H7 NCTC12900 and Pseudomonas aeruginosa NCTC10662, as well as the Gram-positive organisms Staphylococcus aureus NCTC6571 and Enterococcus faecium were exposed to X-rays (35,495 cGy/cm2) over a seven-day period. Antibiotic susceptibility was assessed before, during and after exposure by examining susceptibility, as quantified by E-test with six antibiotics, as well as to a further 11 antibiotics by measurement of susceptibility zone sizes (mm). Additionally, the DNA profile of each organism was compared before, during and after exposure employing the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC PCR). Results indicated that exposure of these organisms to this amount of X-ray radiation did not alter their antibiotic susceptibility, nor their genomic DNA profile. Overall, these data indicate that exposure of bacteria to X-ray radiation does not alter the test organisms' antibiotic susceptibility profiles, nor alter genomic DNA profiles of bacteria, which therefore does not compromise molecular epidemiological tracking of bacteria within healthcare environments in which patients have been exposed to X-ray radiation.

The aim of this study was to investigate the reliability of disc diffusion testing with penicillin, erythromycin and ciprofloxacin within the viridans group streptococci (VGS). In total, the antibiotic susceptibilities of 167 VGS isolates were compared by standard disc diffusion and broth microdilution methods, and these phenotypic data were compared to the carriage of the respective gene resistance determinants [ermB and mefA/E (macrolides); QRDR, gyrA, gyrB, parC and parE (quinolones)]. Overall, there were 35 discrepancies [resistant by MIC and susceptible by zone diameter (21.0%)] between MIC and disc diameter when penicillin susceptibility was interpreted by Clinical and Laboratory Standards Institute criteria. Scattergrams showed a bimodal distribution between non-susceptible and susceptible strains when erythromycin susceptibility was tested by both methods. Thirty-four (20.4%) isolates were categorized as resistant by MIC breakpoints, while disc diameter defined these as having intermediate resistance. With ciprofloxacin, three isolates (1.8%) showed minor discrepancies between MIC breakpoints and disc diameter. Isolates non-susceptible to all three antimicrobial agents tested were reliably distinguished from susceptible isolates by disc diffusion testing, except for the detection of low-level resistance to penicillin, where broth microdilution or an alternative quantitative MIC method should be used. Otherwise, we conclude that disc diffusion testing is a reliable method to detect strains of VGS non-susceptible to penicillin, erythromycin and ciprofloxacin, as demonstrated with their concordance to their gene resistance characteristics., (© 2011 SGM)

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-α and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-α after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-α but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-κB activation, whereas TNF-α expression is blocked at the gene transcription level. Interferon β plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon β. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon β and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Objectives: Ciprofloxacin is the most frequently used member of the fluoroquinolones during initial eradication therapy of Pseudomonas aeruginosa, as well as during acute pulmonary exacerbations. However, its long-term effect on the susceptibility of the commensal flora within the cystic fibrosis (CF) airways has not yet been examined. The aim of this study was therefore to examine the consequence of oral ciprofloxacin usage on the resistance of the commensal viridans group streptococci (VGS), in terms of MICs and mutational analysis of the quinolone resistance-determining regions (QRDRs)., Methods: The MICs of ciprofloxacin, efflux activities and amino acid substitutions in the QRDRs for 190 isolates of VGS, originating from the sputa of adult CF patients who had been exposed constantly to ciprofloxacin, were examined. VGS organisms included Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, Streptococcus oralis, Streptococcus parasanguinis, Streptococcus infantis, Streptococcus gordonii, Streptococcus anginosus, Streptococcus cristatus, Streptococcus australis and Streptococcus mutans. Ciprofloxacin susceptibility was determined by broth microdilution and QRDRs within the gyrA, gyrB, parC and parE gene loci were explored using sequence analysis., Results: Twenty-seven (14.2%) streptococcal isolates were resistant to ciprofloxacin (MICs ≥8 mg/L) and 21 (11.1%) had reduced susceptibility (MICs 4 mg/L). As a comparator, clinically non-significant and non-invasive VGS organisms were examined in 12 consecutive non-CF patients in the community, where no resistance to ciprofloxacin was observed. Five novel QRDR PCR assays were developed to elucidate mutations within the CF VGS population, where there were six positions, which corresponded to previously reported quinolone resistance responsible mutations, and eight novel potential QRDR resistance mutations. Double mutations in gyrA and parC/parE led to MICs of 16 to >64 mg/L, while single mutations in parC or parE resulted in MICs of 8-32 mg/L and 8 mg/L, respectively. The mean homologies of each species to Streptococcus pneumoniae R6 were: gyrA, 70.3%-95%; gyrB, 69.6%-96.2%; parC, 76.1%-94.8%; and parE, 70.7%-94.7%. The close relatives of S. pneumoniae, S. mitis and S. oralis, showed high similarity for all four genes (more than 86%)., Conclusions: Treatment of P. aeruginosa with oral ciprofloxacin in patients with CF may concurrently reduce antibiotic susceptibility in the commensal VGS flora, where these organisms may potentially act as a reservoir of fluoroquinolone resistance gene determinants for newly acquired and antibiotic-susceptible pathogens, particularly the Streptococcus milleri group.

A study was undertaken to examine the population structure of viridans group streptococci (VGS) in the sputum of adult patients with cystic fibrosis (CF). Freshly expectorated sputa (n=58) from 45 adult CF patients were examined by selective conventional culture on Mitis-Salivarius agar and yielded 190 isolates of VGS. Sequence analyses of the rpnB and 16-23S rRNA ITS genes identified these isolates to belong to 12 species of VGS and included S. anginosus, S. australis, S. cristatus, S. gordonii, S. infantis, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. pneumoniae, S. salivarius and S. sanguinis. The most frequently VGS organism isolated was S. salivarius (47/190; 24.7%), followed by S. mitis (36/190; 19%), S. sanguinis (25/190; 13.2%), S. oralis (20/190; 11.0%), S. pneumoniae (19/190; 10.0%), S. parasanguinis (16/190; 8.4%), S. infantis (11/190; 5.8%), S. gordonii (7/190; 3.7%), S. anginosus (4/190; 2.1%), S. cristatus (2/190; 1.1%), S. australis (1/190; 0.5%), S. mutans (1/190; 0.5%) and S. agalactiae (1/190; 0.5%). All, but four, patients harboured at least one VGS species, which ranged from one to five streptococcal species, with a mean of 2.85 species per patient. There was no clonality at the subspecies level employing ERIC RAPD PCR. Antibiotic susceptibility was determined by Minimum Inhibitory Concentration (MIC) testing against penicillin, erythromycin and ciprofloxacin. Overall, resistance to penicillin with all VGS was 73/190 (38.4%) and 167/190 (87.9%) for erythromycin. With regard to ciprofloxacin, 27/190 (14.2%) were fully resistant, whilst a further 21/190 (11.1%) showed intermediate resistance, which equated to approximately three quarters (74.7%) of isolates being fully sensitive to this agent. In addition, as a comparator control population, we examined antibiotic susceptibility, as above, in a non-CF population comprising 12 individuals (50 VGS isolates), who were not receiving chronic antibiotics. In comparison, 8% and 38% of VGS isolates from non-CF individuals were resistant by disk susceptibility testing to penicillin and erythromycin, respectively. None of the non-CF VGS organisms were resistant to ciprofloxacin, but 42% showed intermediate resistance., (Copyright © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

Indicator 8 of the State Performance Plan (SPP), developed under the 2004 reauthorization of the Individuals with Disabilities Education Act (IDEA 2004, Public Law 108-446) requires states to collect data and report findings related to schools' facilitation of parent involvement. The Schools' Efforts to Partner with Parents Scale (SEPPS) was developed to provide states with a means to address this new reporting requirement. Items suggested by stakeholder groups were piloted with a nationally representative sample of 2,634 parents of students with disabilities ages 5-21 in six states. Rasch scaling was used to calibrate a meaningful and invariant item hierarchy. The 78 calibrated items had measurement reliabilities ranging from .94-.97. Using data from the pilot study, stakeholders established a recommended performance standard set at a meaningful point in the item hierarchy. Implications of the findings are discussed in relation to the need for rigorous metrics within state accountability systems.

Viridans-group streptococci (VGS) consist of several taxa which historically have been highly diverse. However, at times it may become necessary to have a reliable scheme for the identification of these organisms to the species level. The aim of this study is to compare the ability of five gene loci, namely rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ, to speciate such organisms through a sequence typing-based approach. Reference organisms consisting of six VGS species were compared based on sequence typing, followed by comparison of 31 wild-type respiratory isolates, and showed that employment of sequence typing using the rnpB gene locus was the most specific and reliable. Therefore, the use of rnpB sequencing for the identification of VGS to species level is a reliable and feasible option, based on a single gene target.

Clustered regulatory interspaced short palindromic repeats (CRISPRs) have been discovered in many bacteria and archaea. Many CRISPR-like sequences have been identified in an increasing number of studies on the function of CRISPRs. One CRISPR-like sequence of approximately 240 base pairs has been found to be highly conserved within 11 genome sequences of Streptococcus pneumoniae. A specific CRISPR-like polymerase chain reaction (PCR) assay was designed with the novel primers CRISPR 5F (forward primer) 5'-CTA ATY TCA TAA CCA TAR GAA TC-3' and CRISPR 3R (reverse primer) 5'-GAT AAR ATC CTY TAA WCT TCT AG-3' to detect the presence of this CRISPR-like sequence in pneumococci, as well as in viridans-group streptococci (VGS). This study investigates the prevalence of this CRISPR-like sequence in S. pneumoniae and 12 viridans-group streptococcal species and shows its existence to be shared by the majority of S. pneumoniae and, to a lesser extent, S. mitis. This CRISPR-like sequence was also found in S. australis and it is highly conserved among these strains, suggesting possible biological functional differences from true CRISPR because this CRISPR-like sequence has relatively few repeat numbers, and adjacent homology of CRISPR-associated (cas) genes was absent. The sharing of this CRISPR-like sequence between pneumococci, the mitis group and other VGS, as well as its high sequence homology, may suggest close evolutionary emergence of this sequence between these species.

Forty-eight isolates of viridans-group streptococci (VGS) from adults and children in the community are examined for their resistance to ciprofloxacin phenotypically by determination of the minimum inhibitory concentration (MIC). In addition, the parC gene locus is amplified and sequenced in all isolates and mutations noted. Overall, 44 VGS organisms were found to be susceptible to ciprofloxacin by the broth microdilution method, and the remaining four strains had intermediate susceptibility. Reduced MICs were observed with intermediate strains when reserpine was added to the broth, inhibiting any efflux activity. Overall, the effect of adding reserpine to the broth medium was to add one doubling dilution to the MIC in the case of Streptococcus mitis, S. oralis and S. salivarius, as well as to increase the MIC by two doubling dilutions in two of the three S. parasanguinis isolates. Amino acid sequence analysis of the quinolone resistance-determining region (QRDR) of the parC gene locus showed good correlation to the phenotypic resistance to ciprofloxacin, where no confirmed mutation conferring quinolone resistance was found. Eleven amino acid positions showed discordance with S. pneumoniae R6 and eight (S52, F55, S58, N91, E135, K137, F141 and S167) were common in the VGS species examined. In addition, minor substitutions were found at three positions (D51, T54 and V86). In conclusion, this study demonstrates the low occurrence of ciprofloxacin resistance in a population of VGS isolated from the community. In addition, several silent mutations were noted in VGS organisms without any increase in MIC against ciprofloxacin.

Periodontitis, a chronic inflammatory disease of the tissues supporting the teeth, is characterized by an exaggerated host immune and inflammatory response to periopathogenic bacteria. Toll-like receptor activation, cytokine network induction, and accumulation of neutrophils at the site of inflammation are important in the host defense against infection. At the same time, induction of immune tolerance and the clearance of neutrophils from the site of infection are essential in the control of the immune response, resolution of inflammation, and prevention of tissue destruction. Using a human monocytic cell line, we demonstrate that Porphyromonas gingivalis lipopolysaccharide (LPS), which is a major etiological factor in periodontal disease, induces only partial immune tolerance, with continued high production of interleukin-8 (IL-8) but diminished secretion of tumor necrosis factor alpha (TNF-α) after repeated challenge. This cytokine response has functional consequences for other immune cells involved in the response to infection. Primary human neutrophils incubated with P. gingivalis LPS-treated naïve monocyte supernatant displayed a high migration index and increased apoptosis. In contrast, neutrophils treated with P. gingivalis LPS-tolerized monocyte supernatant showed a high migration index but significantly decreased apoptosis. Overall, these findings suggest that induction of an imbalanced immune tolerance in monocytes by P. gingivalis LPS, which favors continued secretion of IL-8 but decreased TNF-α production, may be associated with enhanced migration of neutrophils to the site of infection but also with decreased apoptosis and may play a role in the chronic inflammatory state seen in periodontal disease.

A novel microarray was constructed with DNA PCR product probes targeting species specific functional genes of nine clinically significant respiratory pathogens, including the Gram-positive organisms (Streptococcus pneumoniae, Streptococcus pyogenes), the Gram-negative organisms (Chlamydia pneumoniae, Coxiella burnetii Haemophilus spp., Legionella pneumophila, Moraxella catarrhalis, and Pseudomonas aeruginosa), as well as the atypical bacterium, Mycoplasma pneumoniae. In a "proof-of-concept" evaluation of the developed microarray, the microarray was compared with real-time PCR from 14 sputum specimens from COPD patients. All of the samples positive for bacterial species in real-time PCR were also positive for the same bacterial species using the microarray. This study shows that a microarray using PCR probes is a potentially useful method to monitor the populations of bacteria in respiratory specimens and can be tailored to specific clinical needs such as respiratory infections of particular patient populations, including patients with cystic fibrosis and bronchiectasis., (Copyright 2010 Elsevier B.V. All rights reserved.)

Methods employed by the World Health Organization (WHO) are used during this study to determine the optimum storage conditions for maintaining the culturability of Streptococcus pneumoniae in skimmed milk, tryptone, glucose and glycerin (STGG) transport medium. A comparison of S. pneumoniae strains sensitive and resistant to penicillin showed no significant difference in their survival ability in STGG medium. Furthermore, it is confirmed that storage at -70 degrees C remains most effective for maintaining viability by culture of S. pneumoniae. Storage at -20 degrees C would only be acceptable in the short-term, while storage at +4 degrees C is not recommended. Of note, this study has shown STGG medium at room temperature to be an efficient growth medium for pneumococci in the short-term. This work will help to establish robust sampling protocols when performing community studies to ensure culturability of comparison between community and laboratory pneumococci survival.

Although production of cytokines by TLR is essential for viral and bacterial clearance, overproduction can be detrimental, thus controlling these responses is essential. CD33-related sialic acid binding Ig-like lectin receptors (Siglecs) have been implicated in the control of leukocyte responses. In this study, we report that murine Siglec-E is induced by TLRs in a MyD88-specific manner, is tyrosine phosphorylated following LPS stimulation, and negatively regulates TLR responses. Specifically, we demonstrate the Siglec-E expression inhibits TLR-induced NF-kappaB and more importantly, the induction of the antiviral cytokines IFN-beta and RANTES. Siglec-E mediates its inhibitory effects on TIR domain containing adaptor inducing IFN-beta (TRIF)-dependent cytokine production via recruitment of the tyrosine [corrected] phosphatase SHP2 and subsequent inhibition of TBK1 activity as evidenced by enhanced TBK1 phosphorylation in cells following knockdown of Siglec-E expression. Taken together, our results demonstrate a novel role for Siglec-E in controlling the antiviral response to TLRs and thus helping to maintain a healthy cytokine balance following infection.

Background: Smoking is correlated with a large number of oral conditions such as tooth staining and bad breath, periodontal diseases, impaired healing of wounds, precancer and oral cancer. These effects are often visible and in the early stages they are reversible after cessation of smoking. Dentists, as part of the health profession, are frequently in contact with the general population and there is evidence that they are as effective in providing smoking cessation counselling as any other healthcare group., Aims and Methods: Patients' knowledge of the effects of smoking and their attitudes towards the role of dentists in smoking cessation activities were analysed via a self-completing questionnaire and compared depending on their smoking status (smokers and non-smokers)., Results: The results show that patients hold very positive attitudes towards dentists' role in smoking cessation. The results also show that although patients have a good knowledge of the effects of smoking on general health, smokers are significantly less aware of the relationship between smoking and gum disease and on wound healing., Conclusions: Dentists should inform their patients about the oral effects of smoking and strongly advise them not to smoke, especially in patients diagnosed with periodontal disease and requiring surgical procedures.

Objectives: Although long-term use of azithromycin has shown a significant clinical improvement for patients with cystic fibrosis (CF), its long-term effect on the susceptibility of commensal flora within CF airways has not yet been examined. We therefore suggest that long-term use of azithromycin increases macrolide resistance in commensal streptococci., Methods: Erythromycin susceptibility in naturally colonizing viridans group streptococci (VGS) was characterized, as well as macrolide resistance gene determinants through sequence analysis, in pneumococci (n = 15) and VGS [n = 84; i.e. Streptococcus salivarius (n = 30), Streptococcus mitis (n = 17), Streptococcus sanguinis (n = 11), Streptococcus oralis (n = 10), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 3), Streptococcus infantis (n = 3), Streptococcus cristatus (n = 2), Streptococcus anginosus (n = 1) and Streptococcus australis (n = 1)] isolated from sputum from 24 adult CF patients, who were on oral azithromycin therapy for at least the previous 7 months., Results: Almost three-quarters of isolates (74; 74.7%) were resistant to erythromycin, whilst a further 15 (15.2%) had reduced susceptibility, leaving only 10 (10.1%) isolates susceptible to erythromycin. The majority (89.8%) were not susceptible to erythromycin, as demonstrated by possession of the erm(B) gene in 25/99 (25.3%), the mef(A) gene in 1/99 (1.0%), the mef(E) gene in 75/99 (75.8%) and both erm(B) and mef(E) genes simultaneously in 11/99 (11.1%). These results indicate that genotypic resistance for macrolides is common in VGS in adult CF patients, with efflux being over three times more frequent., Conclusions: Long-term treatment with azithromycin in CF patients may reduce antibiotic susceptibility in commensal VGS, where these organisms may potentially act as a reservoir of macrolide resistance determinants for newly acquired and antibiotic-susceptible pathogens.

The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on inflammatory cytokine signaling has not been explored. In this study, we find that CCL11 induces suppressor of cytokine signaling (SOCS)1 and SOCS3 expression in murine macrophages, human monocytes, and dendritic cells (DCs). We also discover that CCL11 inhibits GM-CSF-mediated STAT5 activation and IL-4-induced STAT6 activation in a range of hematopoietic cells. This blockade of cytokine signaling by CCL11 results in reduced differentiation and endocytic ability of DCs, implicating CCL11-induced SOCS as mediators of chemotactic inflammatory control. These findings demonstrate cross-talk between chemokine and cytokine responses, suggesting that myeloid cells tracking to the inflammatory site do not differentiate in the presence of this chemokine, revealing another role for SOCS in inflammatory regulation.

Background: Chaperones are ubiquitous conserved proteins critical in stabilization of new proteins, repair/removal of defective proteins and immunodominant antigens in innate and adaptive immunity. Periodontal disease is a chronic inflammatory infection associated with infection by Porphyromonas gingivalis that culminates in the destruction of the supporting structures of the teeth. We previously reported studies of serum antibodies reactive with the human chaperone Hsp90 in gingivitis, a reversible form of gingival disease confined to the oral soft tissues. In those studies, antibodies were at their highest levels in subjects with the best oral health. We hypothesized that antibodies to the HSP90 homologue of P. gingivalis (HtpG) might be associated with protection/resistance against destructive periodontitis., Methodology/principal Findings: ELISA assays using cloned HtpG and peptide antigens confirmed gingivitis subjects colonized with P. gingivalis had higher serum levels of anti-HtpG and, concomitantly, lower levels of attachment loss. Additionally, serum antibody levels to P. gingivalis HtpG protein were higher in healthy subjects compared to patients with either chronic or aggressive periodontitis. We found a negative association between tooth attachment loss and anti-P. gingivalis HtpG (p = 0.043) but not anti-Fusobacterium nucleatum (an oral opportunistic commensal) HtpG levels. Furthermore, response to periodontal therapy was more successful in subjects having higher levels of anti-P. gingivalis HtpG before treatment (p = 0.018). There was no similar relationship to anti-F. nucleatum HtpG levels. Similar results were obtained when these experiments were repeated with a synthetic peptide of a region of P. gingivalis HtpG., Conclusions/significance: OUR RESULTS SUGGEST: 1) anti-P. gingivalis HtpG antibodies are protective and therefore predict health periodontitis-susceptable patients; 2) may augment the host defence to periodontitis and 3) a unique peptide of P. gingivalis HtpG offers significant potential as an effective diagnostic target and vaccine candidate. These results are compatible with a novel immune control mechanism unrelated to direct binding of bacteria.

Objectives: To review the evidence that the dental unit waterlines are a source of occupational and healthcare acquired infection in the dental surgery., Data: Transmission of infection from contaminated dental unit waterlines (DUWL) is by aerosol droplet inhalation or rarely imbibing or wound contamination in susceptible individuals. Most of the organisms isolated from DUWL are of low pathogenicity. However, data from a small number of studies described infection or colonisation in susceptible hosts with Legionella spp., Pseudomonas spp. and environmental mycobacteria isolated from DUWL. The reported prevalence of legionellae in DUWL varies widely from 0 to 68%. The risk from prolonged occupational exposure to legionellae has been evaluated. Earlier studies measuring surrogate evidence of exposure to legionellae in dental personnel found a significant increase in legionella antibody levels but in recent multicentre studies undertaken in primary dental care legionellae were isolated at very low rate and the corresponding serological titres were not above background levels. Whereas, a case of fatal Legionellosis in a dental surgeon concluded that the DUWL was the likely source of the infection. The dominant species isolated from dental unit waterlines (DUWL) are Gram-negative bacteria, which are a potent source of cell wall endotoxin. A consequence of indoor endotoxin exposure is the triggering or exacerbation of asthma. Data from a single large practice-based cross-sectional study reported a temporal association between occupational exposure to contaminated DUWL with aerobic counts of >200cfu/mL at 37 degrees C and development of asthma in the sub-group of dentists in whom asthma arose following the commencement of dental training., Sources: Medline 1966 to February 2007 was used to identify studies for this paper., Study Selection: Design criteria included randomised control trials, cohort, and observational studies in English., Conclusions: Although the number of published cases of infection or respiratory symptoms resulting from exposure to water from contaminated DUWL is limited, there is a medico-legal requirement to comply with potable water standards and to conform to public perceptions on water safety.

Chronic obstructive pulmonary disease (COPD) embraces a number of pathological processes including chronic bronchitis, chronic bronchiolitis and emphysema. The chronic and progressive course of COPD is often aggravated by short periods of increasing symptoms. Respiratory tract infections (RTIs) are the most common causes of COPD exacerbations. Detection and enumeration of respiratory bacteria are important techniques in diagnosing RTIs and in the validation of new treatment methods. We describe here the development and evaluation of real-time PCR assays for the simultaneous direct detection and quantification of a range of respiratory bacteria in individuals with COPD during stable periods and during acute exacerbations of the disease. Sputum samples from 30 subjects in a COPD study were analysed, and results compared with the current gold standard of culture. Real-time PCR assays proved highly sensitive, with no cross-reactivity with other species. The prevalence of bacteria detected by real-time PCR compared with that by culture was substantially higher for Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus spp. and Moraxella catarrhalis. Multiple pathogens were also found with real-time PCR but were not detected by culture. This study demonstrates the potential of such methods in the detection and enumeration of respiratory bacteria.

Background: The aim of this study was to investigate the influence of current hormonal contraceptive medication on periodontal health in young females., Methods: Fifty women aged 20 to 35 years (mean +/- SD: 29.7 +/- 4.7 years) had a comprehensive periodontal examination. Current and previous contraceptive pill use was assessed by a questionnaire. Periodontal assessment included plaque index, gingival index, probing depth, and attachment level at six sites per tooth. The periodontal health of current pill users was compared to that of women not taking the pill., Results: Forty-two percent of subjects were taking the contraceptive pill at the time of periodontal examination. Current pill users had deeper mean probing depths compared to non-users (3.3 mm versus 2.7 mm; P = 0.006) and more severe attachment loss (2.6 mm versus 1.7 mm; P = 0.015). Pill users had more sites with bleeding on probing (44.0% versus 31.1%; P = 0.017)., Conclusion: Current users of oral contraceptives had poorer periodontal health.

A genus-specific polymerase chain reaction (PCR)-based assay is developed for the detection and identification of clinically relevant Fusobacterium species, including F. nucleatum and F. necrophorum. Two 16S ribosomal DNA (rDNA) primers, FUSO1 (forward primer: 5'-GAG AGA GCT TTG CGT CC-3' [17-mer]) and FUSO 2 (reverse primer: 5'-TGG GCG CTG AGG TTC GAC -3' [18-mer]) are designed to target conserved regions of the 16S rDNA gene for Fusobacterium spp. Subsequent proof-of-principle studies employing this assay detected Fusobacterium spp. in the faeces of eight (10%) out of 80 patients with suspected gastrointestinal infection. This assay may be used for the genus-specific detection of Fusobacterium spp. from clinical specimens and for subsequent species identification.

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

The in vitro susceptibility of 618 Candida isolates to fluconazole, itraconazole, voriconazole, ketoconazole, miconazole, amphotericin B, and nystatin was determined. The isolates were obtained from 559 patients who had attended the UK dental hospital departments in Cardiff, Belfast, Glasgow or London. Antifungal susceptibility was assessed using a broth microdilution method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. The majority of the test strains were C. albicans (n = 521) with few of these being resistant to fluconazole (0.3%). A low incidence of fluconazole resistance (0-6.8%) was similarly evident with all non albicans species (Candida glabrata, 5 of 59 resistant; Candida krusei, 0 of 7 resistant; Candida tropicalis, 0 of 13 resistant; Candida parapsilosis, 0 of 12 resistant; other Candida species, 0 of 6 resistant). Voriconazole, ketoconazole, and miconazole also revealed high activity against both C. albicans and non albicans isolates, and 23.7% of C. glabrata isolates were found to be resistant to itraconazole. There was little difference in the antifungal susceptibilities of Candida isolated from patients who had a history of previous antifungal therapy compared with those who had not received antifungal treatment. In summary, this surveillance study of antifungal susceptibility of oral candidal isolates in the UK, through the collaboration of four dental hospitals, demonstrates that oral Candida species have a high level of susceptibilities to a range of antifungal agents.

Background/aims: The etiologic relationship between periodontitis and Porphyromonas gingivalis is attributed to the ability of the organism to express a variety of virulence factors, many of which are cell surface components including lipopolysaccharide and arginine-specific cysteine proteases (Arg-gingipains, RgpA, and RgpB). P. gingivalis responds to the stress of rapid elevation in temperature by activating a set of genes to produce heat shock proteins that mediate the effects of sudden changes in environmental temperatures by repairing or eliminating cellular proteins denatured by that stress., Methods: We used restriction fragment differential display (RFDD) to identify and measure the genes expressed by surrogates of environmental stresses, heat and oxidative stress. The results were then confirmed using quantitative reverse-transcription polymerase chain reaction., Results: We selected 16 genes differentially induced from over 800 total expression fragments on the RFDD gels for further characterization. With primers designed from those fragments we found that a + 5 degrees C heat shock caused a statistically significant increase in expression compared 12 of 18 untreated genes tested. The exposure of P. gingivalis to atmospheric oxygen resulted in statistically significant increases in five of the target genes. These genes are likely involved in transport and synthesis of components of the lipopolysaccharide biosynthetic pathway important in anchoring the Arg-gingipains required for virulence-related activities., Conclusion: These results emphasize the need for studies to measure the coordinated responses of bacteria like P. gingivalis which use a multitude of interrelated metabolic activities to survive the environmental hazards of the infection process.

Induction of resistance of oral anaerobes to the effects of human beta-defensin 1 (hbetaD-1) to hbetaD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures of Porphyromonas gingivalis were (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42 degrees C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 microl) were distributed among the wells of sterile 384-well plates containing hbetaD-1 to -4 (100 microg/ml). Plates were incubated at 37 degrees C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins for P. gingivalis were 3 to 12 microg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 microg of defensin per ml in P. gingivalis. Resistance induced by sublethal levels of hbetaD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hbetaD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogen P. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.