Interferon-γ stimulates CD14, TLR2 and TLR4 mRNA expression in gingival fibroblasts increasing responsiveness to bacterial challenge.

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.

Comparison of antibiotic susceptibility in viridans group streptococci in low and high antibiotic-prescribing General Practices.

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) ß-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 ß-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.

Reliability of self-reporting of antibiotic consumption in the community - Index of Reliability.

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.

Molecular characterisation of the quinolone resistance-determining regions (QRDR) including gyrA, gyrB, parC and parE genes in Streptococcus pneumoniae.

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.

Exposure to clinical X-ray radiation does not alter antibiotic susceptibility or genotype profile in gram-negative and gram-positive clinical pathogens.

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.

Construction and validation of two parent-report scales for the evaluation of early intervention programs.

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.

Molecular characterisation of the quinolone resistance-determining region (QRDR) of the parC gene locus in viridans-group streptococci.

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.

Prevalence of clustered regulatory interspaced short palindromic repeat (CRISPR)-like sequences in mitis-group streptococci.

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.

Measuring schools' efforts to partner with parents of children served under IDEA: scaling and standard setting for accountability reporting.

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.

Comparasion of five gene loci (rnpB, 16S rRNA, 16S-23S rRNA, sodA and dnaJ) to aid the molecular identification of viridans-group streptococci and pneumococci.

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.

Optimisation of storage conditions for maintaining culturability of penicillin-susceptible and penicillin-resistant isolates of Streptococcus pneumoniae in transport medium.

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.