In VitroStreptococcus pneumoniae Biofilm Formation and In Vivo Middle Ear Mucosal Biofilm in a Rat Model of Acute Otitis Induced by S. pneumoniae

Objectives

Streptococcus pneumoniae is one of the most common pathogens of otitis media (OM) that exists in biofilm, which enhances the resistance of bacteria against antibiotic killing and diagnosis, compared to the free-floating (planktonic) form. This study evaluated biofilm formation by S. pneumoniae on an abiotic surface and in the middle ear cavity in a rat model of OM.

Methods

In vitro biofilm formation was evaluated by inoculation of a 1:100 diluted S. pneumoniae cell suspension in a 96-well microplate. Adherent cells were quantified spectrophotometrically following staining with crystal violet by measurement of optical density at 570 nm. The ultrastructure of pneumococcal biofilm was assessed by scanning electron microscopy (SEM). For in vitro biofilm study, S. pneumoniae cell suspensions containing 1×10⁷ colony forming units were injected through transtympanic membrane into the middle ear cavity of Sprague Dawley rats. The ultrastructure of middle ear mucus was observed by SEM 1 and 2 weeks post-inoculation.

Results

The in vitro study revealed robust biofilm formation by S. pneumoniae after 12-18 hours of incubation in high glucose medium, independent of exogenously supplied competence stimulating peptide and medium replacement. Adherent cells formed three-dimensional structures approximately 20-30 µm thick. The in vivo study revealed that ciliated epithelium was relatively resistant to biofilm formation and that biofilm formation occurred mainly on non-ciliated epithelium of the middle ear cavity. One week after inoculation, biofilm formation was high in 50% of the treated rats and low in 25% of the rats. After 2 weeks, biofilm formation was high and low in 25% and 37.5% of rats, respectively.

Conclusion

The results imply that glucose level is important for the S. pneumoniae biofilm formation and S. pneumoniae biofilm formation may play important role in the pathophysiology of OM.     

Gentian violet and ferric ammonium citrate disrupt Pseudomonas aeruginosa biofilms

Objective/Hypothesis: Bacterial biofilms are resistant to antibiotics and may contribute to persistent infections including chronic otitis media and cholesteatoma. Discovery of substances to disrupt biofilms is necessary to treat these chronic infections. Gentian violet (GV) and ferric ammonium citrate (FAC) were tested against Pseudomonas aeruginosa biofilms to determine if either substance can reduce biofilm volume. Study Design: The biofilm volume and planktonic growth of PAO1 and otopathogenic P. aeruginosa (OPPA8) isolated from an infected cholesteatoma was measured in the presence of GV or FAC. Methods: OPPA8 and PAO1 expressing a green fluorescent protein plasmid (pMRP9‐1) was inoculated into a glass flow chamber. Biofilms were grown under low flow conditions for 48 hours and subsequently exposed to either GV or FAC for an additional 24 hours. Biofilm formation was visualized by confocal laser microscopy and biofilm volume was assayed by measuring fluorescence. Planktonic cultures were grown under standard conditions with GV or FAC. Statistical analysis was performed by Student t test and one‐way ANOVA. Results: GV reduced PAO1 and OPPA8 biofilm volume (P < .01). GV delayed the onset and rate of logarithmic growth in both strains. FAC reduced OPPA8 biofilm volume (P < .01), but did not effect of PAO1 biofilms. FAC had no effect on planktonic growth. Conclusions: The efficacy of GV in disrupting biofilms in vitro suggests that it may disrupt biofilms in vivo. The effect of FAC on Pseudomonas aeruginosa biofilms is strain dependent. Strain differences in response to increasing iron concentration and biofilm morphology stress the importance of studying clinically isolated strains in testing antibiofilm agents.     

Biofilm formation on tympanostomy tubes depends on methicillin-resistant Staphylococcus aureus genetic lineage

Bacterial biofilm formation has been implicated in the high incidence of persistent otorrhoea after tympanostomy tube insertion. The aim of the study was to investigate whether biofilm formation on tympanostomy tubes depends on the genetic profile of methicillin-resistant Staphylococcus aureus (MRSA) strains. Capacity of biofilm formation on fluoroplastic tympanostomy tubes (TTs) was tested on 30 MRSA strains. Identification and methicillin resistance were confirmed by PCR for nuc and mecA genes. Strains were genotypically characterised (SCCmec, agr and spa typing). Biofilm formation was tested in microtiter plate and on TTs. Tested MRSA strains were classified into SCCmec type I (36.7 %), III (23.3 %), IV (26.7 %) and V (13.3 %), agr type I (50 %), II (36.7 %) and III (13.3 %), and 5 clonal complexes (CCs). All tested MRSA strains showed ability to form biofilm on microtiter plate. Capacity of biofilm formation on TTs was as following: 13.3 % of strains belonged to the category of no biofilm producers, 50 % to the category of weak biofilm producers and 36.7 % to moderate biofilm producers. There was a statistically significant difference between CC, SCCmec and agr types and the category of biofilm production on TTs tubes (p < 0.001): CC5, SCCmecI type and agrII type with a moderate amount of biofilm, and CC8 and agrI type with a low amount of biofilm. Biofilm formation by MRSA on TTs is highly dependent on genetic characteristics of the strains. Therefore, MRSA genotyping may aid the determination of the possibility of biofilm-related post-tympanostomy tube otorrhea.     

Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media

Objectives

The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease.

Methods

Biofilm formations by NTHi clinical isolates from pediatric AOM patients were evaluated by a crystal violet microtiter plate and a 98 well pin-replicator assay with a confocal laser scanning microscopy (CLSM). Optical density values of clinical isolates were compared with a positive control and the ratio of clinical isolates to a positive control was defined as biofilm formation index (BFI).

Results

84.3% clinical isolates of NTHi were biofilm forming strains (BFI ≥ 0.4). The BFI represented the levels of biofilm formation and adherence on the surface. The identical strains isolated from both middle ear fluids (MEFs) and nasopharynx showed biofilm formation at the same level. The prevalence of biofilm forming isolates was significantly higher among the susceptible strains than resistant strains. The level of biofilm formation of NTHi isolated from AOM cases who was not improved by amoxicillin (AMPC) was significantly higher than that of NTHi isolated from AOM cases who was improved by AMPC.

Conclusion

We clearly showed the biofilm formation of clinical NTHi isolates from AOM children. In addition, the biofilm formed by NTHi would play an important role in persistent or intractable clinical course of AOM as a result of lowered treatment efficacy of antibiotics.     

In vivo biofilm formation on stainless steel bonded retainers during different oral health-care regimens

Retention wires permanently bonded to the anterior teeth are used after orthodontic treatment to prevent the teeth from relapsing to pre-treatment positions. A disadvantage of bonded retainers is biofi...     

Prevention of biofilm formation by polyquaternary polymer

ObjectiveBiofilm formation has been linked to device-associated infections in otolaryngology. This study was conducted to determine if a microbicidal polyquaternary polymer, poly diallyl-dimethylammonium chloride (pDADMAC) could prevent biofilm development by pathogens that commonly cause implant infections, Staphylococcus aureus and Pseudomonas aeruginosa.Methods and materialsThis study was prospective and controlled in vitro microbiological study. Polyurethane tubes (20 per treatment) with and without a polyquaternary polymer coating were briefly exposed to plasma or saline, then to S. aureus or P. aeruginosa. Polyurethane tubes were incubated in growth media. After 4 days, antibiotics were added to kill planktonic bacteria. S. aureus or P. aeruginosa bacterial counts and scanning electron microscopy (SEM) were performed.ResultsS. aureus biofilm counts were reduced by 8 logs on tubes with polyquaternary polymer coating compared to the control tubes, either with plasma (3.67E+01 ± 7.30E+01 vs 1.08E+09 ± 4.81E+08; P < 0.0001) or without plasma (3.70E+00 ± 1.10E+01 vs 6.50E+08 ± 2.79E+08; P < 0.0001). P. aeruginosa biofilm formation was also reduced on tubes with polyquaternary polymer, either with plasma (2.90E+07 ± 1.71E+07 vs 9.16E+08 ± 4.43E+08; P < 0.0001) or without plasma (2.50E+07 ± 9.54E+06 vs 3.35E+08 ± 2.18E+08; P < 0.001), but the reduction was only 1 log. On control tubes, plasma promoted S. aureus (1.08E+09 ± 4.81E+08 vs 6.05E+08 ± 2.79E+08; P < 0.0001) and P. aeruginosa (9.16E+08 ± 4.43E+08 vs 3.35E+08 ± 2.18E+08; P < 0.0001) bacterial counts but not on the tubes coated with polyquaternary polymer.ConclusionsIncorporation of the microbicidal polyquaternary polymer, pDADMAC, into polyurethane dramatically inhibits S. aureus biofilm formation. Further research is warranted to evaluate the efficacy and safety of this technology in otolaryngologic implants.     

The phenotypic and genetic biofilm formation characteristics of coagulase-negative staphylococci isolates in children with otitis media

Objective

Medical biofilms are involved in a number of chronic infections including otitis media with effusion and chronic rhinosinusitis, which are common pediatric infectious diseases. The purpose of the study was to analyze the phenotypic and genotypic indicators of biofilm formation of coagulase negative staphylococci isolates in children with otitis media with effusion, and in children with chronic rhinosinusitis as a comparison group by using three different detection methods.

Methods

Forty nine children aged from 2 to 6 years old, diagnosed with otitis media with effusion were enrolled to the study. The comparative group consisted of twenty three strains of coagulase-negative staphylococci from the strains collection isolated from nose swabs from children 3 to 7 years old suffering from rhinosinusitis for longer than 12 weeks. Cultured strains were tested for biofilm formation ability with three tests: Congo red agar, tissue culture plate methods and detection of ica operon.

Results

Out of 97 ear effusion specimens, obtained from 49 children suffering from OME, 38 were found positive in conventional culture resulting in isolation of 50 different bacterial species. Nested-PCR method confirmed bacterial presence in 95 (97.9%) cases. Among 50 different bacterial species isolated, 30 (30.9%) CNS and 20 (20.6%) other than CNS species.Detection of slime producing phenotype of CNS was performed with CRA plate test. Among OME isolates, 11 (36.7%) were CRA plate test positive. In case of isolates from CRS, 8 (34.8%) strains revealed black coloration on CRA.Using TCP method, strong adherence to microtiter plate was observed in two Staphylococcus epidermidis strains from OME and two S. epidermidis from CRS.By using the ica operon test, the genotypic ability to form biofilm was identified in 7 (23.3%) S. epidermidis strains cultured from ears effusion and in 3 (13%) strains from nose swabs.

Conclusions

CNS strains revealed genotypic and phenotypic features responsible for the ability to form the biofilm in vivo. The presence of ica genes and phenotypic ability to form a biofilm by CNS strains emphasizes the pathogenic character of these strains in some cases of otitis media with effusion.     

Laser disruption and killing of methicillin-resistant Staphylococcus aureus biofilms

Objective

The aim of the study was to study the efficacy of 2 different lasers in vitro, in disrupting biofilm and killing planktonic pathogenic bacteria.

Materials and methods

Biofilms of a stable bioluminescent of Staphylococcus aureus Xen 31 were grown in a 96-well microtiter plate for 3 days. The study included 7 arms: (a) control; (b) ciprofloxacin (3 mg/L, the established minimum inhibitory concentration [MIC]) alone; (c) shock wave (SW) laser alone; (d) near-infrared (NIR) laser alone; (e) SW laser and ciprofloxacin; (f) SW and NIR lasers; (g) SW, NIR lasers, and ciprofloxacin. The results were evaluated with an in vivo imaging system (IVIS) biophotonic system (for live bacteria) and optical density (OD) for total bacteria.

Results

Without antibiotics, there was a 43% reduction in OD (P < .05) caused by the combination of SW and NIR suggesting that biofilm had been disrupted. There was an 88% reduction (P < .05) in live biofilm. Ciprofloxacin alone resulted in a decrease of 28% of total live cells (biofilm remaining attached) and 58% of biofilm cells (both P > .05). Ciprofloxacin in combination with SW and SW + NIR lasers caused a decrease of more than 60% in total live biomass and more than 80% of biofilm cells, which was significantly greater than ciprofloxacin alone (P < .05).

Conclusions

We have demonstrated an effective nonpharmacologic treatment method for methicillin-resistant Staphylococcus aureus (MRSA) biofilm disruption and killing using 2 different lasers. The preferred treatment sequence is a SW laser disruption of biofilm followed by NIR laser illumination. Treatment optimization of biofilm is possible with the addition of ciprofloxacin in concentrations consistent with planktonic MIC.     

Role of saliva proteinase 3 in dental caries

Salivary analysis can be used to assess the severity of caries. Of the known salivary proteins, a paucity of information exists concerning the role of proteinase 3 (PR3), a serine protease of the chymotrypsin family, in dental caries. Whole, unstimulated saliva was collected from children with varying degrees of active caries and tested using a Human Protease Array Kit and an enzyme-linked immunosorbent assay. A significantly decreased concentration of salivary PR3 was noted with increasing severity of dental caries (P<0.01); a positive correlation (r=0.87; P<0.01; Pearson''s correlation analysis) was also observed between salivary pH and PR3 concentration. In an antibacterial test, a PR3 concentration of 250 ng·mL⁻¹ or higher significantly inhibited Streptococcus mutans UA159 growth after 12 h of incubation (P<0.05). These studies indicate that PR3 is a salivary factor associated with the severity of dental caries, as suggested by the negative relationship between salivary PR3 concentration and the severity of caries as well as the susceptibility of S. mutans to PR3.     

Determination of the biofilm formation capacity of bacterial pathogens associated with otorhinolaryngologic diseases in the Malaysian population

This study aims to assess the association between microbial composition, biofilm formation and chronic otorhinolaryngologic disorders in Malaysia. A total of 45 patients with chronic rhinosinusitis, chronic tonsillitis and chronic suppurative otitis media and 15 asymptomatic control patients were studied. Swab samples were obtained from these subjects. Samples were studied by conventional microbiological culturing, PCR-based microbial detection and Confocal Laser Scanning Microscopy (CLSM). Haemophilus influenzae, Staphylococcus aureus, Streptococcus pneumoniae, coagulase-negative staphylococci (CoNS) and other Streptococcus species were detected in subjects of both patient and control groups. Biofilm was observed in approximately half of the smear prepared from swab samples obtained from subjects of the patient group. Most of these were polymicrobial biofilms. S. aureus biofilm was most prevalent among nasal samples while H. influenzae biofilm was more common among ear and throat samples. Results from this study supported the hypothesis that chronic otorhinolaryngologic diseases may be biofilm related. Due to the presence of unculturable bacteria in biofilms present in specimens from ear, nose and throat, the use of molecular methods in combination with conventional microbiological culturing has demonstrated an improvement in the detection of bacteria from such specimens in this study.     

Effect of blood and mucus on tympanostomy tube biofilm formation

OBJECTIVES/HYPOTHESIS: Tympanostomy tube (TT) biofilm formation may lead to refractory otorrhea and occlusion. The aim of this study was to determine whether TT biofilm formation may be promoted by mucus or blood exposure. STUDY DESIGN: In vitro, controlled. METHODS: Fluoroplastic TTs were exposed to blood, mucoid effusion, or saline. Half were allowed to dry. TTs were cultured with Pseudomonas aeruginosa. After 4 days, gentamicin was added to kill planktonic bacteria. Biofilm formation was assessed by quantitative bacterial counts and scanning electron microscopy. RESULTS: Mucus pretreatment (dry and wet) did not increase biofilm formation. Both dry and wet blood exposure increased biofilm formation by bacterial counts (P < .0001). Biofilm formation was demonstrated by electron microscopy in all groups. CONCLUSIONS: P. aeruginosa biofilm formation on fluoroplastic TTs is enhanced by blood exposure. Care should be taken to minimize bleeding with TT placement to reduce the risk of biofilm formation.     

Association of adenoid hyperplasia and bacterial biofilm formation in children with adenoiditis in Taiwan

The adenoid is a bacterial reservoir that contributes to chronic otolaryngologic infections. Staphylococcus aureus (S. aureus) is a common pathogen in the adenoid. The increase of antibiotic resistance in S. aureus has become an important issue in public health. The aim of this study was to compare adenoid hyperplasia and biofilm formation in children with S. aureus adenoiditis in Taiwan. The patients were divided into methicillin-resistant and methicillin-sensitive S. aureus groups according to the S. aureus obtained from adenoid tissue after antibiotic susceptibility testing. Adenoid hyperplasia was assessed by lateral cephalometry, and the severity of sinusitis was evaluated by Water’s view. Microbiological investigation of available S. aureus isolates was performed by in vivo morphological observation and an in vitro bacterial biofilm assay. Sixty isolates of S. aureus were identified in 283 children (21.2%) after adenoidectomy, of which 21 (35%) were methicillin-resistant S. aureus (MRSA). The severity of adenoid hyperplasia and extensive biofilm formation were more prominent in patients infected with methicillin-resistant S. aureus than in those infected with methicillin-sensitive S. aureus (MSSA). The primary outcome of this study was to provide evidence that S. aureus constituted a significant portion of the adenoidal pathogens. The secondary outcome of this study was that MRSA adenoiditis may be associated with adenoid hyperplasia and biofilm formation.     

A new method for in vivo evaluation of biofilms on surface-modified silicone rubber voice prostheses

A new method is presented that permits a rapid and accurate in vivo evaluation of biofilm formation on surface-modified silicone rubber voice prostheses. The method is based on partial modification of a Groningen button voice prosthesis by exposing half of the prosthesis to an argon plasma. This results in one side of the prosthesis becoming hydrophilic while leaving the unmodified side hydrophobic as a control. Modified prostheses were placed in patients for an evaluation period of approximately 4 weeks. Despite making the silicone rubber surface hydrophilic, biofilm formation was stimulated when compared to unmodified, hydrophobic silicone rubber. Findings show that biofilm formation on voice prostheses is influenced by hydrophobicity of a silicone rubber surface. The method of partial surface modification used was seen to be suitable for demonstrating such influences regardless of nutrition and other variations in the patient’s lifestyle. Microbiological analysis of the biofilms on both sides of the prosthesis valve did not show any changes in microbial composition, withCandida albicans, streptococci and staphylococci being the most commonly isolated strains.     

In vitro activity of mupirocin on clinical isolates of Staphylococcus aureus and its potential implications in chronic rhinosinusitis

BACKGROUND: It has been postulated that bacterial biofilms are involved in the pathogenesis of chronic rhinosinusitis (CRS). Biofilms present on sinus mucosa are difficult to eradicate with conventional antibiotic therapy and are thought to provide a nidus for recurrent infection. Topical delivery of antibiotics via nasal irrigation may present a way of delivering high concentrations of antibiofilm agents with potentially low systemic absorption and side effects. This study investigates the effectiveness of mupirocin and two other antibiotics, ciprofloxacin and vancomycin, on established in vitro biofilms of Staphylococcus aureus isolated from patients with CRS. METHODS: S. aureus American Type Culture Collection 25923 and 12 clinical isolates were investigated for their ability to form biofilms in an in vitro setting using a 96 well microtiter crystal violet (CV) plate assay and confocal scanning laser microscopy (CSLM). Antimicrobial susceptibility tests to determine minimum inhibitory concentrations were performed on planktonic and biofilm forming strains. In addition, established biofilms were subjected to the antimicrobial agents at a twofold dilution series. A CV analysis of biofilm mass was performed after 1 and 24 hours of treatment, and minimum biofilm inhibition concentrations at 50% (MIB50) and 90% (MIB90) biofilm inhibition were recorded. RESULTS: With use of a 96-well microtiter plate CV assay, 8 of the 12 clinical isolates formed mature biofilms after 8 days of culture. These results correlated with findings from CSLM analysis of in vitro biofilms grown on Permanox chamber slides. Increased antimicrobial resistance was observed in the biofilm isolates when compared with planktonic counterparts. Mupirocin was capable of reducing biofilm mass by greater than 90% at concentrations of 125 mug/mL or less in all S. aureus isolates. Ciprofloxacin and vancomycin were largely ineffective in attaining MIB90 concentrations within safe dosage ranges. CONCLUSIONS: The topical application of mupirocin via nasal irrigation may be useful in eliminating S. aureus biofilms present on the sinus mucosa of patients with CRS and may offer an additional treatment to patients with recalcitrant sinusitis.     

Evaluation of three-dimensional biofilms on antibacterial bonding agents containing novel quaternary ammonium methacrylates

Antibacterial adhesives are promising to inhibit biofilms and secondary caries. The objectives of this study were to synthesize and incorporate quaternary ammonium methacrylates into adhesives, and investigate the alkyl chain length effects on three-dimensional biofilms adherent on adhesives for the first time. Six quaternary ammonium methacrylates with chain lengths of 3, 6, 9, 12, 16 and 18 were synthesized and incorporated into Scotchbond Multi-Purpose. Streptococcus mutans bacteria were cultured on resin to form biofilms. Confocal laser scanning microscopy was used to measure biofilm thickness, live/dead volumes and live-bacteria percentage vs. distance from resin surface. Biofilm thickness was the greatest for Scotchbond control; it decreased with increasing chain length, reaching a minimum at chain length 16. Live-biofilm volume had a similar trend. Dead-biofilm volume increased with increasing chain length. The adhesive with chain length 9 had 37% live bacteria near resin surface, but close to 100% live bacteria in the biofilm top section. For chain length 16, there were nearly 0% live bacteria throughout the three-dimensional biofilm. In conclusion, strong antibacterial activity was achieved by adding quaternary ammonium into adhesive, with biofilm thickness and live-biofilm volume decreasing as chain length was increased from 3 to 16. Antibacterial adhesives typically only inhibited bacteria close to its surface; however, adhesive with chain length 16 had mostly dead bacteria in the entire three-dimensional biofilm. Antibacterial adhesive with chain length 16 is promising to inhibit biofilms at the margins and combat secondary caries.     

Role of bacterial biofilms in idiopathic childhood epistaxis

The objective of the study is to conduct a prospective trial investigating the possible role of bacterial biofilms in the pathogenesis of severe idiopathic childhood epistaxis. This study included 84 cases of severe idiopathic epistaxis, aged below 16 years, who were prepared for cautery under general anesthesia. A nasal swab was taken for bacterial culture and a nasal mucosal specimen (≤3 mm²) was taken from the suspected site of bleeding just prior to cautery and sent for bacterial identification by pathogen specific fluorescence in situ hybridization (FISH) and also for detection of bacterial biofilms by scanning electron microscope (SEM). Nasal mucosal specimens from 20 children of the same age prepared for reduction of fracture nasal bones and have no nasal problems were taken as a control group. Bacterial culture was positive in 27.3 % of patients and the most common organism was Staphylococcus aureus (19 %). By SEM, biofilm formation was detected in only six patients (7.1 %). Evaluation of nasal specimens with FISH was positive for pathogenic bacteria in 37 % of cases; the most common organism was S. aureus (22.6 % of cases). In the control group, no biofilm was detected by SEM and no pathogenic bacteria were cultured or detected by FISH. The difference between the two groups was statistically significant. Bacterial biofilm does not seem to play a major role in the pathogenesis of idiopathic epistaxis in children (only positive in 7.1 % of cases by SEM) although a low-grade chronic inflammation is not infrequently present (37 % of cases detected by FISH). FISH is more sensitive than bacterial culture in detecting bacterial infections. S. aureus was the most common pathogen detected by both techniques.     

Influence of surface properties of Merocel® (polyvinyl acetal) and silicone nasal splints on biofilm formation

The objective of the study was to investigate biofilm formation on Merocel^® and silicone nasal splint after nasal septal surgery. 50 patients who were scheduled to undergo nasal septal surgery were included in this study. The patients were randomized into receiving an insert of Merocel^® or silicone splint after septoplasty. In group 1 (8 females, 17 males) and group 2 (10 females, 15 males), Merocel ^® packs or silicone splints were inserted into nasal cavities at the end of the procedures, respectively. All packs were removed 48 h after insertion, and samples were taken from the packs under sterilized conditions. Scanning electron microscopy was performed to observe biofilm formation on the surfaces of Merocel^® and silicone splints. Biofilm formation was observed in 25 (100 %) and 3 (12 %) of the Merocel^® and silicone splint samples, respectively. Our study revealed that biofilm formation on Merocel^® packs is significantly higher than silicone splints, mainly due to the different texture and surface properties of these materials. Considering the hazardous effects of biofilm formation on humans, our observations in this study may guide surgeons to choose the most appropriate packing material after nasal septal surgery.     

Resistance to Noise-Induced Hearing Loss in 129S6 and MOLF Mice: Identification of Independent,Overlapping, and Interacting Chromosomal Regions

Noise-induced hearing loss (NIHL) is a prevalent health risk. Inbred mouse strains 129S6/SvEvTac (129S6) and MOLF/EiJ (MOLF) show strong NIHL resistance (NR) relative to CBA/CaJ (CBACa). In this study, we developed quantitative trait locus (QTL) maps for NR. We generated F1 animals by intercrossing (129S6 × CBACa) and (MOLF × CBACa). In each intercross, NR was recessive. N2 animals were produced by backcrossing F1s to their respective parental strain. The 232 N2-129S6 and 225 N2-MOLF progenies were evaluated for NR using auditory brainstem response. In 129S6, five QTL were identified on chromosomes (Chr) 17, 18, 14, 11, and 4, referred to as loci nr1, nr2, nr3, nr4, and nr5, respectively. In MOLF, four QTL were found on Chr 4, 17, 6, and 12, referred to as nr7, nr8, nr9, and nr10, respectively. Given that NR QTL were discovered on Chr 4 and 17 in both the N2-129S6 and N2-MOLF cross, we generated two consomic strains by separately transferring 129S6-derived Chr 4 and 17 into an otherwise CBACa background and a double-consomic strain by crossing the two strains. Phenotypic analysis of the consomic strains indicated that whole 129S6 Chr 4 contributes strongly to mid-frequency NR, while whole 129S6 Chr 17 contributes markedly to high-frequency NR. Therefore, we anticipated that the double-consomic strain containing Chr 4 and 17 would demonstrate NR across the mid- and high-frequency range. However, whole 129S6 Chr 17 masks the expression of mid-frequency NR from whole 129S6 Chr 4. To further dissect NR on 129S6 Chr 4 and 17, CBACa.129S6 congenic strains were generated for each chromosome. Phenotypic analysis of the Chr 17 CBACa.129S6 congenic strains further defined the NR region on proximal Chr 17, uncovered another NR locus (nr6) on distal Chr 17, and revealed an epistatic interaction between proximal and distal 129S6 Chr 17.     

Nasopharyngeal Streptococcus pneumoniae carriage in Japanese children attending day-care centers

Objectives

We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Streptococcus pneumoniae carriage in healthy children in day-care centers (DCCs).

Methods

A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 181) and the following winter (n = 182). We obtained 157 S. pneumoniae isolates and analyzed them by antibiotic susceptibility testing, PCR assay for the penicillin-binding protein (PBP) genes and macrolide-resistance gene, and pulsed-field gel electrophoresis (PFGE).

Results

The overall carriage rate was 43.3% (157/363). The percentages of penicillin-intermediately resistant S. pneumoniae (PISP) strains, penicillin-resistant S. pneumoniae (PRSP) strains, erythromycin-intermediately resistant S. pneumoniae strains and erythromycin-resistant S. pneumoniae strains were 35.7% (56/157), 0.6% (1/157), 1.9% (3/157), and 69.4% (109/157), respectively. The percentages of S. pneumoniae strains with the pbp mutation(s) and mefA and/or ermB gene(s) were 92.4% (145/157) and 71.3% (112/157), respectively. Fifty strains with different PFGE patterns were obtained from among the 157 isolates. Thirteen strains were observed in both seasons, but only one of these strains was isolated from the same carrier. Twenty-one strains (42.0%) were isolated from two or more children, and 17 of these were each isolated from children attending the same DCC.

Conclusions

These results indicate the spread of S. pneumoniae, particularly those with antibiotic-resistance genes, and the vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan.     

In vitro antimicrobial activity of Luffa operculata

IntroductionLuffa operculata is probably one of the most popular herbal medicines used in the treatment of rhinitis and rhinosinusitis. However, its specific mechanism of action is still unknown.ObjectiveTo evaluate in vitro antibacterial activity of L. operculata against three ordinary agents of upper respiratory tract infection: Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes.MethodsDifferent concentrations of L. operculata alcoholic extract were applied to bacterial broth containing reference and community strains of the three described agents. After a 24-h incubation period, the bacterial culture turbidity was measured. The samples were then inoculated onto Mueller-Hinton and human blood agar plates. Bacterial growth was analyzed after 24- and 48-h incubation period. The test was considered negative when there was no environmental turbidity, confirmed by the absence of bacterial growth into the inoculated plates. Tests were considered positive when either turbidity changes were observed on the bacterial broth or when bacterial growth was detected on inoculated plates. Appropriate statistical analysis of the data was performed.ResultsL. operculata extracts showed antibacterial activity mainly to S. pyogenes followed by S. pneumoniae and S. aureus.ConclusionsL. operculata extract showed promising antibacterial activity in vitro against the studied agents.