Evidence-based occupational hearing screening II: validation of a screening methodology using measures of functional hearing ability.

OBJECTIVE: Validate use of the Extended Speech Intelligibility Index (ESII) for prediction of speech intelligibility in non-stationary real-world noise environments. Define a means of using these predictions for objective occupational hearing screening for hearing-critical public safety and law enforcement jobs. DESIGN: Analyses of predicted and measured speech intelligibility in recordings of real-world noise environments were performed in two studies using speech recognition thresholds (SRTs) and intelligibility measures. ESII analyses of the recordings were used to predict intelligibility. Noise recordings were made in prison environments and at US Army facilities for training ground and airborne forces. Speech materials included full bandwidth sentences and bandpass filtered sentences that simulated radio transmissions. STUDY SAMPLE: A total of 22 adults with normal hearing (NH) and 15 with mild-moderate hearing impairment (HI) participated in the two studies. RESULTS: Average intelligibility predictions for individual NH and HI subjects were accurate in both studies (r2 ≥ 0.94). Pooled predictions were slightly less accurate (0.78 ≤ r2 ≤ 0.92). CONCLUSIONS: An individual's SRT and audiogram can accurately predict the likelihood of effective speech communication in noise environments with known ESII characteristics, where essential hearing-critical tasks are performed. These predictions provide an objective means of occupational hearing screening.

Whole-Genome Sequences of Eight Campylobacter jejuni Isolates from Wild Birds.

We present here the draft genome sequences of 8 Campylobacter jejuni strains isolated from wild birds. The strains were initially isolated from swabs taken from resident wild birds in the Tokachi area of Japan. The genome sizes range from 1.65 to 1.77 Mbp.

Virulence characterization of Campylobacter jejuni isolated from resident wild birds in Tokachi area, Japan.

The prevalence of Campylobacter jejuni in wild birds is a potential hazard for human and animal health. The aim of this study was to establish the prevalence of C. jejuni in wild birds in Tokachi area, Hokkaido, Japan and investigate their virulence in vitro. In total, 173 cloacal swabs from individual wild birds were collected for the detection of Campylobacter spp. Thirty four samples (19.7%) were positive for Campylobacter of which 94.1% (32/34 samples) were C. jejuni. Additionally, one C. coli and one C. fetus were isolated. Seven C. jejuni isolates (one from crows and the other from pigeons) had important virulence genes including all three CDT genes (cdtA, cdtB and cdtC) and flaA, flaB, ciaB and cadF, and the other isolates were lacking cdtA gene. Further studies on in vitro virulence-associated phenotypes, such as motility assay on soft agar and invasion assay in Caco-2 cells, were performed. The wild bird C. jejuni isolates adhered and invaded human cells. Although the numbers of viable intracellular bacteria of wild bird isolates were lower than a type strain NCTC11168, they persisted at 48-hr and underwent replication in host cells.

The effect of interleaved filters on normal hearing listeners' perception of binaural cues.

OBJECTIVES: Hearing-impaired individuals often have difficulty in noisy environments. Interleaved filters, where signals from neighboring frequency regions are sent to opposite ears, may benefit those individuals but may also reduce the benefits of spatial cues. This study investigated the effect of interleaved filters on the use of spatial cues. DESIGN: Normal-hearing subjects' sound localization abilities were tested with and without interleaved filters. RESULTS: Participants' localization performance was worse with interleaved filters but better than chance. Interleaving in high-frequency regions primarily affected interaural level difference cues, and interleaving in low-frequency regions primarily affected interaural time difference cues. CONCLUSIONS: Interleaved filters reduced but did not eliminate the benefits of spatial cues. The effect was dependent on the frequency region they were used in, indicating that it may be possible to use interleaved filters in a subset of frequency regions to selectively preserve different binaural cues.

Determining the relevance of different aspects of formant contours to intelligibility.

Previous studies have shown that "clear" speech, where the speaker intentionally tries to enunciate, has better intelligibility than "conversational" speech, which is produced in regular conversation. However, conversational and clear speech vary along a number of acoustic dimensions and it is unclear what aspects of clear speech lead to better intelligibility. Previously, Kain et al. [J. Acoust. Soc. Am. 124 (4), 2308-2319 (2008)] showed that a combination of short-term spectra and duration was responsible for the improved intelligibility of one speaker. This study investigates subsets of specific features of short-term spectra including temporal aspects. Similar to Kain's study, hybrid stimuli were synthesized with a combination of features from clear speech and complementary features from conversational speech to determine which acoustic features cause the improved intelligibility of clear speech. Our results indicate that, although steady-state formant values of tense vowels contributed to the intelligibility of clear speech, neither the steady-state portion nor the formant transition was sufficient to yield comparable intelligibility to that of clear speech. In contrast, when the entire formant contour of conversational speech including the phoneme duration was replaced by that of clear speech, intelligibility was comparable to that of clear speech. It indicated that the combination of formant contour and duration information was relevant to the improved intelligibility of clear speech. The study provides a better understanding of the relevance of different aspects of formant contours to the improved intelligibility of clear speech.

Deletion in the C-terminal domain of ClpX delayed entry of Salmonella enterica into a viable but non-culturable state.

Under stressful conditions, bacteria enter a viable but non-culturable (VBNC) state in which they are alive but fail to grow on conventional media. The molecular basis underlying this state is unknown. To identify the key gene responsible for the VBNC state in Salmonella spp., we examined a S. Typhimurium LT2 VBNC mutant, which shows a characteristic delay in entering the VBNC state. The mutant showed a higher level of expression of general stress sigma factor RpoS than wild-type LT2. The mutant carried a 99-bp in-frame deletion in the clpX gene (clpXΔ323-355). ClpX is known to form a ClpXP protease complex with ClpP, which plays a role in the degradation of RpoS. To investigate the effect of clpXΔ323-355 on VBNC induction, ΔclpX and clpXΔ323-355 strains were generated from LT2 cells. Compared to LT2, the ΔclpX and clpXΔ323-355 strains showed greater amounts of RpoS and required a longer incubation time for induction into the VBNC state. These results suggest that residues 323-355 of ClpX play a major role in the hexameric formation or function of ClpX and in the rate of induction of the VBNC state.

Microbial assessment and prevalence of foodborne pathogens in natural cheeses in Japan.

The production and consumption of domestic natural cheese in Japan is increasing year by year. More than ninety percent of domestic natural cheese is produced in Hokkaido region of Japan, while information on its quality and safety related to foodborne pathogens is limited. To assess the microbiological safety of domestic natural cheese, a total of 126 natural cheese samples produced in Hokkaido were collected from December, 2012, to July, 2013. In addition to standard plate count (SPC) and coliform counts, the prevalence study of three pathogens (Listeria monocytogenes, pathogenic Escherichia coli, and Salmonella spp.) was performed on each sample. Real-time PCR and matrix-assisted laser desorption-ionization time-of-flight mass spectrometer methods were employed for identification of presumptive pathogens. Coliform was detected in 25 samples (19.8%) with a minimum of 25 cfu/g and a maximum of more than 3.0 × 10(6) cfu/g. Salmonella spp. and L. monocytogenes were not isolated from any of the samples. Only one sample (0.80%) showed positive PCR amplification for ipaH gene suggesting possible contamination of enteroinvasive E. coli or Shigella in this product. Overall results indicate that natural cheeses produced in Hokkaido region were satisfactory microbiological quality according to existing international standards.

Protective effect of Bacillus anthracis surface protein EA1 against anthrax in mice.

Bacillus anthracis spores germinate to vegetative forms in host cells, and produced fatal toxins. A toxin-targeting prophylaxis blocks the effect of toxin, but may allow to grow vegetative cells which create subsequent toxemia. In this study, we examined protective effect of extractable antigen 1 (EA1), a major S-layer component of B. anthracis, against anthrax. Mice were intranasally immunized with recombinant EA1, followed by a lethal challenge of B. anthracis spores. Mucosal immunization with EA1 resulted in a significant level of anti-EA1 antibodies in feces, saliva and serum. It also delayed the onset of anthrax and remarkably decreased the mortality rate. In addition, the combination of EA1 and protective antigen (PA) protected all immunized mice from a lethal challenge with B. anthracis spores. The numbers of bacteria in tissues of EA1-immunized mice were significantly decreased compared to those in the control and PA alone-immunized mice. Immunity to EA1 might contribute to protection at the early phase of infection, i.e., before massive multiplication and toxin production by vegetative cells. These results suggest that EA1 is a novel candidate for anthrax vaccine and provides a more effective protection when used in combination with PA.

General stress sigma factor RpoS influences time required to enter the viable but non-culturable state in Salmonella enterica.

In stressful conditions, bacteria enter into the viable but non-culturable (VBNC) state; in this state, they are alive but fail to grow on conventional media on which they normally grow and develop into colonies. The molecular basis underlying this state is unknown. We investigated the role of the alternative sigma factor RpoS (σ(38)) in the VBNC induction using Salmonella Dublin, Salmonella Oranienburg and Salmonella Typhimurium LT2. VBNC was induced by osmotic stress in LT2 and Oranienburg. Dublin also entered the VBNC state, but more slowly than LT2 and Oranienburg did. The LT2 rpoS gene was initiated from an alternative initiation codon, TTG; therefore, LT2 had smaller amounts of RpoS than Dublin and Oranienburg. Oranienburg had a single amino acid substitution (D118N) in RpoS (RpoS(SO)). Disruption of rpoS caused rapid VBNC induction. VBNC induction was significantly delayed by Dublin-type RpoS (RpoS(SD)), but only slightly by RpoS(SO). These results indicate that RpoS delays VBNC induction and that the rapid induction of VBNC in LT2 and Oranienburg may be due to lower levels of RpoS and to the D118N amino acid substitution, respectively. Reduced RpoS intracellular level was observed during VBNC induction. During the VBNC induction, Salmonella might regulate RpoS which is important for maintenance of culturablity under stresses.

Physiological characterization of Campylobacter jejuni under cold stresses conditions: its potential for public threat.

Campylobacter jejuni is the major cause of human gastroenteritis worldwide. Under stress conditions, C. jejuni can enter a viable but non-culturable (VBNC) state. We found that the C. jejuni was able to enter a VBNC state by prolonged incubation at 4°C. The standard isolation methods using pre-enrichment steps in Bolton broth or Preston broth could not detect the VBNC cells in spiked chicken meat. The transcription levels of virulence-associated genes (flaA, flaB, cadF, ciaB, cdtA, cdtB and cdtC) were expressed in VBNC cells but in low levels. The VBNC cells retained the ability to invade Caco-2 human intestinal epithelial cells in vitro. In most cases, VBNC cells failed to resuscitate in Caco-2 cells, but in some experiments, they formed colonies after co-incubation with host cells. Collectively, C. jejuni enters into a VBNC state at 4°C and the VBNC C. jejuni remains virulent which may possibly lead to disease in humans. C. jejuni in VBNC state is a potential concern for food safety.

Conversion of mono-polar to peritrichous flagellation in Vibrio alginolyticus.

Precise regulation of the number and positioning of flagella are critical in order for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. It has been shown that, in V. alginolyticus cells, the putative GTPase FlhF determines the polar location and production of flagella, while the putative ATPase FlhG interacts with FlhF, preventing it from localizing at the pole, and thus negatively regulating the flagellar number. In fact, no ΔflhF cells have flagella, while a very small fraction of ΔflhFG cells possess peritrichous flagella. In this study, the mutants that suppress inhibition of the swarming ability of ΔflhFG cells were isolated. The mutation induced an increase in the flagellar number and, furthermore, most Vibrio cells appeared to have peritrichous flagella. The sequence of the flagella related genes was successfully determined, however, the location of the suppressor mutation could not been found. When the flhF gene was introduced into the suppressor mutant, multiple polar flagella were generated in addition to peritrichous flagella. On the other hand, introduction of the flhG gene resulted in the loss of most flagella. These results suggest that the role of FlhF is bypassed through a suppressor mutation which is not related to the flagellar genes.

Isolation and characterization of Listeria monocytogenes from commercial asazuke (Japanese light pickles).

Asazuke is a ready-to-eat Japanese light pickle, mainly made of vegetables which are known to be one of the sources of Listeria monocytogenes contamination. Although asazuke is a popular side-dish in Japan, the hazard of bacterial contamination has not been evaluated yet. In this study, we investigated the prevalence of L. monocytogenes, Salmonella spp., verotoxigenic E. coli (VTEC) and coliforms in 108 asazuke samples that randomly collected from supermarkets in Obihiro (Hokkaido prefecture, Japan) during the period of June to November 2007. Twelve (11.11%) L. monocytogenes were isolated with predominant serotype 4b (seven isolates) followed by 1/2a (two isolates), 1/2b, 3b and 4c (one isolate each) while Salmonella spp., VTEC and coliforms were not detected. All L. monocytogenes isolates demonstrated hemolytic activity by CAMP test and possessed all the virulence-associated genes (prfA, actA, mpl, inlA, inlC, plcA, plcB, hly, iap, clpC and opuCA) as resulted in PCR, thus revealed their potential pathogenicity. Moreover, 7 out of 12 isolates were from asazuke samples produced by the same factory and their pulsed-field gel electrophoresis (PFGE) profiles suggested that 6 of them were indistinguishable and one was different. L. monocytogenes contamination in the asazuke factory environment was further investigated and 23 out of 60 environmental swabs (38.33%) contained the bacterium. Comparison of PFGE profiles showed relatedness between food and environmental isolates indicating that contamination probably occurred in the asazuke factory during manufacturing. Interestingly, after HACCP training course conducted to the factory workers, 20 samples collected during the period of November to December 2008 were negative to L. monocytogenes revealing that the hygienic status has improved.

Mutational analysis of the GTP-binding motif of FlhF which regulates the number and placement of the polar flagellum in Vibrio alginolyticus.

Precise regulation of the number and placement of flagella is critical for the mono-flagellated bacterium Vibrio alginolyticus to swim efficiently. We previously proposed a model in which the putative GTPase FlhF determines the polar location and generation of the flagellum, the putative ATPase FlhG interacts with FlhF to prevent FlhF from localizing to the pole, and thus FlhG negatively regulates the flagellar number in V. alginolyticus cells. To investigate the role of the GTP-binding motif of FlhF, we generated a series of alanine-replacement mutations at the positions that are highly conserved among homologous proteins. The results indicate that there is a correlation between the polar localization and the ability to produce flagella in the mutants. We investigated whether the mutations in the GTP-binding motif affected the ability to interact with FlhG. In contrast to our prediction, no significant difference was detected in the interaction with FlhG between the wild-type and mutant FlhFs. We showed that the GTP-binding motif of FlhF is important for polar localization of the flagellum but not for the interaction with FlhG.

Hybridizing conversational and clear speech to determine the degree of contribution of acoustic features to intelligibility.

Speakers naturally adopt a special "clear" (CLR) speaking style in order to be better understood by listeners who are moderately impaired in their ability to understand speech due to a hearing impairment, the presence of background noise, or both. In contrast, speech intended for nonimpaired listeners in quiet environments is referred to as "conversational" (CNV). Studies have shown that the intelligibility of CLR speech is usually higher than that of CNV speech in adverse circumstances. It is not known which individual acoustic features or combinations of features cause the higher intelligibility of CLR speech. The objective of this study is to determine the contribution of some acoustic features to intelligibility for a single speaker. The proposed method creates "hybrid" (HYB) speech stimuli that selectively combine acoustic features of one sentence spoken in the CNV and CLR styles. The intelligibility of these stimuli is then measured in perceptual tests, using 96 phonetically balanced sentences. Results for one speaker show significant sentence-level intelligibility improvements over CNV speech when replacing certain combinations of short-term spectra, phoneme identities, and phoneme durations of CNV speech with those from CLR speech, but no improvements for combinations involving fundamental frequency, energy, or nonspeech events (pauses).

Collaboration of FlhF and FlhG to regulate polar-flagella number and localization in Vibrio alginolyticus.

Precise regulation of the number and placement of flagella is critical for the mono-polar-flagellated bacterium Vibrio alginolyticus to swim efficiently. We have shown previously that the number of polar flagella is positively regulated by FlhF and negatively regulated by FlhG. We now show that DeltaflhF cells are non-flagellated as are most DeltaflhFG cells; however, some of the DeltaflhFG cells have several flagella at lateral positions. We found that FlhF-GFP was localized at the flagellated pole, and its polar localization was seen more intensely in DeltaflhFG cells. On the other hand, most of the FlhG-GFP was diffused throughout the cytoplasm, although some was localized at the pole. To investigate the FlhF-FlhG interaction, immunoprecipitation was performed by using an anti-FlhF antibody, and FlhG co-precipitated with FlhF. From these results we propose a model in which FlhF localization at the pole determines polar location and production of a flagellum, FlhG interacts with FlhF to prevent FlhF from localizing at the pole, and thus FlhG negatively regulates flagellar number in V. alginolyticus cells.

Improving syllable identification by a preprocessing method reducing overlap-masking in reverberant environments.

Overlap-masking degrades speech intelligibility in reverberation [R. H. Bolt and A. D. MacDonald, J. Acoust. Soc. Am. 21(6), 577-580 (1949)]. To reduce the effect of this degradation, steady-state suppression has been proposed as a preprocessing technique [Arai et al., Proc. Autumn Meet. Acoust. Soc. Jpn., 2001; Acoust. Sci. Tech. 23(8), 229-232 (2002)]. This technique automatically suppresses steady-state portions of speech that have more energy but are less crucial for speech perception. The present paper explores the effect of steady-state suppression on syllable identification preceded by /a/ under various reverberant conditions. In each of two perception experiments, stimuli were presented to 22 subjects with normal hearing. The stimuli consisted of mono-syllables in a carrier phrase with and without steady-state suppression and were presented under different reverberant conditions using artificial impulse responses. The results indicate that steady-state suppression statistically improves consonant identification for reverberation times of 0.7 to 1.2 s. Analysis of confusion matrices shows that identification of voiced consonants, stop and nasal consonants, and bilabial, alveolar, and velar consonants were especially improved by steady-state suppression. The steady-state suppression is demonstrated to be an effective preprocessing method for improving syllable identification by reducing the effect of overlap-masking under specific reverberant conditions.

Regulation of polar flagellar number by the flhF and flhG genes in Vibrio alginolyticus.

The number and location of bacterial flagella vary with the species. The Vibrio alginolyticus cell has a single polar flagellum, which is driven by sodium ions. We selected mutants on the basis of reduced swarming ability on soft agar plates. Among them, we found two mutants with multiple polar flagella, and named them KK148 and NMB155. In Pseudomonas species, it is known that FlhF and FleN, which are FtsY and MinD homologs, respectively, are involved in regulation of flagellar placement and number, respectively. We cloned homologous genes of V. alginolyticus, flhF and flhG. KK148 cells had a nonsense mutation in flhG; cells expressing transgenic flhG recovered the swarming ability and had a reduced number of polar flagella. NMB155 cells did not have a mutation in either flhF or flhG. In wild-type cells, expression of flhF increased the number of polar flagella; in contrast, expression of flhG reduced both the number of polar flagella and the swarming ability. These results suggest that FlhG negatively regulates the number of polar flagella in V. alginolyticus. KK148 cells expressing both flhF and flhG exhibited fewer polar flagella and better swarming ability than KK148 cells expressing flhG alone, suggesting that FlhG acts with FlhF.

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor.

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.

Cell surface differentiation of Mycoplasma mobile visualized by surface protein localization.

Mycoplasma mobile has a flask-shaped cell morphology and glides toward its tapered end at a rate of 3-7 cell lengths per s (2.0-4.5 microm s(-1)) by an unknown mechanism. Gliding requires that the surface of the cell is in contact with a solid substrate, such as glass or plastic. In order to characterize the nature of the outer surface of M. mobile, monoclonal antibodies were raised against intact cells and screened for their ability to recognize surface proteins. Four antibodies were identified and their protein targets were determined. One antibody recognized the Gli349 protein, which is known to be involved in glass binding and gliding. This antibody was also able to displace attached M. mobile cells from glass, suggesting that Gli349 is the major adhesion protein in M. mobile. The other three antibodies recognized members of the Mvsp family of proteins, which are presumably the major surface antigens of M. mobile. Immunofluorescence studies were performed to localize these proteins on the surface of M. mobile cells. Gli349 localized to the proximal region of the tapered part of the cell (the 'neck'), while the various Mvsp family members showed several distinct patterns of subcellular localization. MvspN and MvspO localized to the distal end of the tapered part of the cell (the 'head'), MvspK localized to the main part of the cell (the 'body'), and MvspI localized to both the head and body but not the neck. This analysis shows that M. mobile surprisingly expresses multiple versions of its major surface antigen at once but differentiates its surface by differential localization of the various paralogues.