Glucose controls manganese homeostasis through transcription factors regulating known and newly identified manganese transporter genes in Bacillus subtilis.

Mn2+ is an essential nutrient whose concentration is tightly controlled in bacteria. In Bacillus subtilis, the Mn2+-activated transcription factor MntR controls Mn2+ transporter genes. However, factors regulating intracellular Mn2+ concentration are incompletely understood. Here, we found that glucose addition induces an increase in intracellular Mn2+ concentration. We determined this upshift was mediated by glucose induction of the major Mn2+ importer gene mntH by the transcription factor AhrC, which is known to be involved in arginine metabolism and to be indirectly induced by glucose. In addition, we identified novel AhrC-regulated genes encoding the Mn2+ importer YcsG and the ABC-type exporter YknUV. We found the expression of these genes was also regulated by glucose and contributes to the glucose induction of Mn2+ concentrations. ycsG expression is regulated by MntR as well. Furthermore, we analyzed the interaction of AhrC and MntR with the promoter driving ycsG expression and examined the Mn2+-dependent induction of this promoter to identify the transcription factors responsible for the Mn2+ induction. RNA-Seq revealed that disruption of ahrC and mntR affected the expression of 502 and 478 genes, respectively (false discovery rate, <0.001, log2[fold change] ≥ |2|. The AhrC- and/or MntR-dependent expression of twenty promoters was confirmed by LacZ analysis, and AhrC or MntR binding to some of these promoters was observed via EMSA. The finding that glucose promotes an increase in intracellular Mn2+ levels without changes in extracellular Mn2+ concentrations is reasonable for the bacterium, as intracellular Mn2+ is required for enzymes and pathways mediating glucose metabolism.

Glucolipids and lipoteichoic acids affect the activity of SigI, an alternative sigma factor, and WalKR, an essential two-component system, in Bacillus subtilis.

In a Bacillus subtilis ugtP mutant lacking glucolipids, SigI was activated in the log phase, and the activation of SigI in the mutant was suppressed by the expression of native ugtP. By contrast, SigI was inhibited in a yfnI mutant lacking one of the lipoteichoic acid (LTA) synthase genes, and the inhibition was suppressed by the expression of yfnI. A series of mutation analyses of the sigI promoter revealed that the two WalR binding sites were involved in the increase of PsigI -lacZ activity in the ugtP mutant and decrease of the lacZ activity in the yfnI mutant. Transcription from the SigI recognition sequence was enhanced in the ugtP mutant, whereas yfnI disruption inhibited the transcription from the SigA recognition sequence in the sigI promoter. We found that not only SigI but also WalKR, the essential two-component system, was activated in the ugtP mutant and inhibited in the yfnI mutant. The walK mutants with activated WalR exhibited abnormal morphology, but this phenotype was suppressed by the addition of MgSO4 . We conclude that glucolipids and LTA are key compounds in the maintenance of normal cell surface structure in B. subtilis.

Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis.

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3, which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present.IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc-independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14 because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14, but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis.

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp0 strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp0 strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp0 suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp0 backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.

Distinct roles for U-type proteins in iron-sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon.

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.

Zinc-Ligand Swapping Mediated Complex Formation and Sulfur Transfer between SufS and SufU for Iron-Sulfur Cluster Biogenesis in Bacillus subtilis.

SufU is a zinc-containing protein involved in mobilization of sulfur from SufS for iron-sulfur cluster biogenesis of Bacillus subtilis. Structural basis for the sulfur transfer in SufS-SufU complex was revealed. A zinc-ligand exchange reaction upon SufS-SufU complexation provides a free thiol from Cys41 of SufU as a sulfur acceptor.

Linoleic acid content of human meibum is associated with telangiectasia and plugging of gland orifices in meibomian gland dysfunction.

To examine the relation between changes in the free fatty acid (FFA) composition of human meibum and both objective signs and subjective symptoms of meibomian gland dysfunction (MGD), we analyzed the FFA content of meibum collected from both MGD patients and control subjects. Thirty-eight patients with MGD (13 men and 25 women; mean age ± SD, 66.9 ± 15.0 years) were evaluated. Various objective signs and subjective symptoms of MGD were assessed. Meibum was analyzed by liquid chromatography-Fourier transform mass spectrometry, and the relation between the FFA composition of meibum and each objective sign and subjective symptom was examined by principal component analysis (PCA). No relation was apparent between the FFA composition of meibum and individual subjective symptoms or objective signs of MGD. However, a PCA score plot for meibum samples grouped on the basis of the severity of both telangiectasia and plugging of meibomian gland orifices revealed clear separation of mild and severe groups. This separation of the two groups was largely due to a significantly increased linoleic acid content in meibum of the severe group (3.56%, versus 0.70% of total FFAs in the mild group). The relative amount of linoleic acid in meibum was thus associated with the severity of telangiectasia and plugging of gland orifices in MGD, suggesting that this FFA might contribute to the pathogenesis of these signs.

SP10 infectivity is aborted after bacteriophage SP10 infection induces nonA transcription on the prophage SPß region of the Bacillus subtilis genome.

Bacteria have developed various strategies for phage resistance. Infection with phage induces the transcription of part of the phage resistance gene, but the regulatory mechanisms of such transcription remain largely unknown. The phage resistance gene nonA is located on the SPß prophage region of the Bacillus subtilis Marburg strain genome. The nonA transcript was detected at the late stage of SP10 infection but is undetectable in noninfected cells. The nonA transcript was detected after the induction of the sigma factor Orf199-Orf200 (σ(Orf199-200)), when sigma factors encoded in the SP10 genome were expressed from a xylose-inducible plasmid. Thus, the SP10 sigma factor is an activator of a set of SP10 genes and nonA. The nonA gene encodes a 72-amino-acid protein with a transmembrane motif and has no significant homology with any protein in any database. NonA overexpression halted cell growth and reduced the efficiency of B. subtilis colony formation and respiration activity. In addition, SP10 virion protein synthesis was inhibited in the nonA(+) strain, and SP10 virion particles were scarce in it. These results indicate that NonA is a novel protein that can abort SP10 infection, and its transcription was regulated by SP10 sigma factor.

Artificial activation of both σH and Spo0A in Bacillus subtilis enforced initiation of spore development at the vegetatively growing phase.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σH are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σH -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σA. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σA expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σA did not change drastically throughout growth. Mechanisms that sequester σA from the core RNA polymerase and help σH to become active exist, but this has not yet been elucidated.

A putative bactoprenol glycosyltransferase, CsbB, in Bacillus subtilis activates SigM in the absence of co-transcribed YfhO.

Bacteria are equipped with complex cell surface structures, such as cell walls. How they maintain cell surface integrity through cell wall metabolism during growth and adaptation to unfavorable environmental conditions is still elusive. In the Gram-positive soil bacterium Bacillus subtilis, one extracytoplasmic function (ECF) sigma factor, SigM, is believed to play a primary role in cell surface integrity. Here, we find that expression of CsbB, which is known to be involved in the extracellular stress response, causes constitutive activation of SigM when YfhO, a membrane protein with unknown function, is lost. CsbB has similarity with the well-characterized bactoprenol glucosyltransferase GtrB found in Gram-negative bacteria. Substitution of a single amino acid residue at the putative catalytic site of CsbB abolishes this constitutive activation, and expression of Escherichia coli GtrB in B. subtilis causes similar effects as expression of CsbB, suggesting that SigM is activated by the glycosyltransferase activity of CsbB. A comparison with the Gtr system in Gram-negative bacteria suggests that accumulation of glycosylated bactoprenol catalyzed by CsbB reduces the bactoprenol pool in the absence of YfhO. Reduction of bactoprenol synthesis causes similar effects as expression of CsbB. We propose that it is the shortage of available bactoprenol within a cell that induces SigM activity.

Induction of extracytoplasmic function sigma factors in Bacillus subtilis cells with defects in lipoteichoic acid synthesis.

Lipoteichoic acid (LTA) is an important cell envelope component of Gram-positive bacteria. Bacillus subtilis has four homologous genes for LTA synthesis: ltaS (yflE), yfnI, yqgS and yvgJ. The products LtaS (YflE), YfnI and YqgS are bona fide LTA synthetases, whereas YvgJ functions only as an LTA primase. To clarify whether defects in LTA on the cell envelope trigger extracytoplasmic function (ECF) sigma factors, mRNA levels of the autoregulated ECF sigma factors in cells with singly and multiply deleted alleles of the ltaS homologues were examined by real-time RT-PCR. This revealed that sigM and sigX were induced in cells with a null allele of ΔltaS and ΔyfnI, respectively, and that no ECF sigma factor was induced in cells with a single null allele of ΔyqgS or ΔyvgJ. In cells with double null alleles (ΔltaS and ΔyfnI), sigW and ylaC were induced in addition to sigM and sigX. Cells with triple null alleles (ΔltaS ΔyfnI and ΔyqgS) showed a pattern of induction similar to that of the double null. In cells with quadruple null alleles, sigV and sigY were newly induced. Cells with ΔltaS had approximately 1/4 the diglucosyldiacylglycerol and over 10 times the CDP-diacylglycerol of wild-type cells. Compensatory elevation of the mRNA level of other homologues was observed (in ΔltaS cells the level of yfnI was elevated; in ΔyfnI cells that of yqgS and yvgJ was elevated; both were even higher in ΔltaS ΔyfnI cells). In ΔltaS cells, the mRNA level of yfnI was corroborated to be regulated by σ(M), which is activated in the null mutant cells. In ΔyfnI cells, the mRNA levels of yqgS and yvgJ reverted to less than those of wild-type when a defective sigX allele was introduced. Since sigX was activated in cells with ΔyfnI, this suggests that the induction of yqgS and yvgJ is dependent on σ(X). The LTAs produced by the four ltaS homologues seem to play distinct physiological roles to maintain the full function of LTA on the B. subtilis cell envelope.

Analysis of cell death in Bacillus subtilis caused by sesquiterpenes from Chrysopogon zizanioides (L.) Roberty.

Recently, the antibacterial effects of essential oils have been investigated in addition to their therapeutic purposes. Owing to their hydrophobic nature, they are thought to perturb the integrity of the bacterial cell membrane, leading to cell death. Against such antibiotic challenges, bacteria develop mechanisms for cell envelope stress responses (CESR). In Bacillus subtilis, a gram-positive sporulating soil bacterium, the extracytoplasmic function (ECF) sigma factor-mediated response system plays a pivotal role in CESR. Among them, σM is strongly involved in response to cell envelope stress, including a shortage of available bactoprenol. Vetiver essential oil, a product of Chrysopogon zizanioides (L.) Roberty root, is also known to possess bactericidal activity. σM was exclusively and strongly induced when the cells were exposed to Vetiver extract, and depletion of multi-ECF sigma factors (ΔsigM, ΔsigW, ΔsigX, and ΔsigV) enhanced sensitivity to it. From this quadruple mutant strain, the suppressor strains, which restored resistance to the bactericidal activity of Vetiver extract, emerged, although attempts to obtain resistant strains from the wild type did not succeed. Whole-genome resequencing of the suppressor strains and genetic analysis revealed inactivation of xseB or pnpA, which code for exodeoxyribonuclease or polynucleotide phosphorylase, respectively. This allowed the quadruple mutant strain to escape from cell death caused by Vetiver extract. Composition analysis suggested that the sesquiterpene, khusimol, might contribute to the bactericidal activity of the Vetiver extract.

The genome of Bacillus subtilis phage SP10: a comparative analysis with phage SPO1.

A nucleotide sequence of the whole genome of Bacillus subtilis phage SP10 was determined. It was composed of 143,986 bp with 236 putative open reading frames (ORFs). Sixty-five of 236 predicted ORFs showed high similarity to that of SPO1, and the genome organizations of the two phages were similar to each other. SP10 belongs to the Myoviridae family, for which the well-studied phage SPO1 is the representative phage. Hence, we compared SP10 to SPO1. The SP10 genome DNA showed different sensitivity to restriction enzymes than SPO1, due to differences in base modification. According to transcriptional analysis, the gene expression of regulatory network of SP10 was similar to SPO1. It was observed that RNA polymerase containing sigma-A was inactive in directing the host genes but active in directing the phage genes. It appeared that the association of sigma-A with the core enzyme complex of RNA polymerase was strengthened during development.

Heterologous expression of the Oceanobacillus iheyensis SigW and its anti-protein RsiW in Bacillus subtilis.

Pairs of the ECF sigma factor and its anti-sigma factor, SigW and RsiW, of Bacillus-related species that inhabit extreme environments were heterologously expressed in B. subtilis. All the RsiWs, membrane proteins, failed to fill their function of repressing cognate SigW activity, despite their close structural similarities. Particularly, uncontrolled expression of Oceanobacillus iheyensis OISigW due to abortive OIRsiW was harmful to B. subtilis. Analysis of revertants of this growth defect and site-directed mutagenesis indicated that the insertion of six and a minimum of three hydrophobic amino acid residues occurring in the transmembrane region allowed OIRsiW to function as anti-OISigW. Subcellular localization of OIRsiW was detected by immunoblot analysis, suggesting that both the wild-type and the mutant form of OIRsiW were localized to the membrane. An appropriate length of a transmembrane region required for proper integration into the membrane after translocation might vary among these Bacillus-related species.

Characterization of the nuclear- and plastid-encoded secA-homologous genes in the unicellular red alga Cyanidioschyzon merolae.

SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.

Wheeze Recognition Algorithm for Remote Medical Care Device in Children: Validation Study.

BACKGROUND: Since 2020, peoples' lifestyles have been largely changed due to the COVID-19 pandemic worldwide. In the medical field, although many patients prefer remote medical care, this prevents the physician from examining the patient directly; thus, it is important for patients to accurately convey their condition to the physician. Accordingly, remote medical care should be implemented and adaptable home medical devices are required. However, only a few highly accurate home medical devices are available for automatic wheeze detection as an exacerbation sign. OBJECTIVE: We developed a new handy home medical device with an automatic wheeze recognition algorithm, which is available for clinical use in noisy environments such as a pediatric consultation room or at home. Moreover, the examination time is only 30 seconds, since young children cannot endure a long examination time without crying or moving. The aim of this study was to validate the developed automatic wheeze recognition algorithm as a clinical medical device in children at different institutions. METHODS: A total of 374 children aged 4-107 months in pediatric consultation rooms of 10 institutions were enrolled in this study. All participants aged ≥6 years were diagnosed with bronchial asthma and patients ≤5 years had reported at least three episodes of wheezes. Wheezes were detected by auscultation with a stethoscope and recorded for 30 seconds using the wheeze recognition algorithm device (HWZ-1000T) developed based on wheeze characteristics following the Computerized Respiratory Sound Analysis guideline, where the dominant frequency and duration of a wheeze were >100 Hz and >100 ms, respectively. Files containing recorded lung sounds were assessed by each specialist physician and divided into two groups: 177 designated as "wheeze" files and 197 as "no-wheeze" files. Wheeze recognitions were compared between specialist physicians who recorded lung sounds and those recorded using the wheeze recognition algorithm. We calculated the sensitivity, specificity, positive predictive value, and negative predictive value for all recorded sound files, and evaluated the influence of age and sex on the wheeze detection sensitivity. RESULTS: Detection of wheezes was not influenced by age and sex. In all files, wheezes were differentiated from noise using the wheeze recognition algorithm. The sensitivity, specificity, positive predictive value, and negative predictive value of the wheeze recognition algorithm were 96.6%, 98.5%, 98.3%, and 97.0%, respectively. Wheezes were automatically detected, and heartbeat sounds, voices, and crying were automatically identified as no-wheeze sounds by the wheeze recognition algorithm. CONCLUSIONS: The wheeze recognition algorithm was verified to identify wheezing with high accuracy; therefore, it might be useful in the practical implementation of asthma management at home. Only a few home medical devices are available for automatic wheeze detection. The wheeze recognition algorithm was verified to identify wheezing with high accuracy and will be useful for wheezing management at home and in remote medical care.

Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function σ factors.

Bacillus subtilis encodes seven extracytoplasmic function (ECF) σ factors. Three (σ(M), σ(W), and σ(X)) mediate responses to cell envelope-active antibiotics. The functions of σ(V), σ(Y), σ(Z), and σ(YlaC) remain largely unknown, and strong inducers of these σ factors and their regulons have yet to be defined. Here, we define transcriptomic and phenotypic differences under nonstress conditions between a strain carrying deletions in all seven ECF σ factor genes (the Δ7ECF mutant), a ΔMWX triple mutant, and the parental 168 strain. Our results identify >80 genes as at least partially dependent on ECF σ factors, and as expected, most of these are dependent on σ(M), σ(W), or σ(X), which are active at a significant basal level during growth. Several genes, including the eps operon encoding enzymes for exopolysaccharide (EPS) production, were decreased in expression in the Δ7ECF mutant but affected less in the ΔMWX mutant. Consistent with this observation, the Δ7ECF mutant (but not the ΔMWX mutant) showed reduced biofilm formation. Extending previous observations, we also note that the ΔMWX mutant is sensitive to a variety of antibiotics and the Δ7ECF mutant is either as sensitive as, or slightly more sensitive than, the ΔMWX strain to these stressors. These findings emphasize the overlapping nature of the seven ECF σ factor regulons in B. subtilis, confirm that three of these (σ(M), σ(W), and σ(X)) play the dominant role in conferring intrinsic resistance to antibiotics, and provide initial insights into the roles of the remaining ECF σ factors.

Partial functional redundancy of MreB isoforms, MreB, Mbl and MreBH, in cell morphogenesis of Bacillus subtilis.

MreB proteins are bacterial actin homologues thought to have a role in cell shape determination by positioning the cell wall synthetic machinery. Many bacteria, particularly Gram-positives, have more than one MreB isoform. Bacillus subtilis has three, MreB, Mbl and MreBH, which colocalize in a single helical structure. We now show that the helical pattern of peptidoglycan (PG) synthesis in the cylindrical part of the rod-shaped cell is governed by the redundant action of the three MreB isoforms. Single mutants for any one of mreB isoforms can still incorporate PG in a helical pattern and generate a rod shape. However, after depletion of MreB in an mbl mutant (or depletion of all three isoforms) lateral wall PG synthesis was impaired and the cells became spherical and lytic. Overexpression of any one of the MreB isoforms overcame the lethality as well as the defects in lateral PG synthesis and cell shape. Furthermore, MreB and Mbl can associate with the peptidoglycan biosynthetic machinery independently. However, no single MreB isoform was able to support normal growth under various stress conditions, suggesting that the multiple isoforms are used to allow cells to maintain proper growth and morphogenesis under changing and sometimes adverse conditions.

A wheeze recognition algorithm for practical implementation in children.

BACKGROUND: The detection of wheezes as an exacerbation sign is important in certain respiratory diseases. However, few highly accurate clinical methods are available for automatic detection of wheezes in children. This study aimed to develop a wheeze detection algorithm for practical implementation in children. METHODS: A wheeze recognition algorithm was developed based on wheezes features following the Computerized Respiratory Sound Analysis guidelines. Wheezes can be detected by auscultation with a stethoscope and using an automatic computerized lung sound analysis. Lung sounds were recorded for 30 s in 214 children aged 2 months to 12 years and 11 months in a pediatric consultation room. Files containing recorded lung sounds were assessed by two specialist physicians and divided into two groups: 65 were designated as "wheeze" files, and 149 were designated as "no-wheeze" files. All lung sound judgments were agreed between two specialist physicians. We compared wheeze recognition between the specialist physicians and using the wheeze recognition algorithm and calculated the sensitivity, specificity, positive predictive value, and negative predictive value for all recorded sound files to evaluate the influence of age on the wheeze detection sensitivity. RESULTS: The detection of wheezes was not influenced by age. In all files, wheezes were differentiated from noise using the wheeze recognition algorithm. The sensitivity, specificity, positive predictive value, and negative predictive value of the wheeze recognition algorithm were 100%, 95.7%, 90.3%, and 100%, respectively. CONCLUSIONS: The wheeze recognition algorithm could identify wheezes in sound files and therefore may be useful in the practical implementation of respiratory illness management at home using properly developed devices.

Wheeze sound characteristics are associated with nighttime sleep disturbances in younger children.

BACKGROUND: Wheezing is a typical symptom of respiratory conditions. Few objective methods are available for predicting sleep disturbance in young children with wheezing. OBJECTIVE: We investigated whether wheezing characteristics, detected by lung-sound analysis, were associated with risk of sleep disturbance. METHODS: We recorded the lung sounds of 66 young children (4-59 months) every morning, for the entire duration of a wheezing episode. On lung-sound analysis, wheezing was displayed as horizontal bars of intensity with corresponding sharp peaks of power. The sharp peak of power was defined as a wheeze band. Wheezing characteristics (e.g., number, frequency, duration, and frequency of maximum intensity of wheeze bands) were analyzed using lung-sound analysis. Patients were divided into 3 groups based on sleep disturbance on the first night after wheezing was recorded: mild group (no sleep disturbance and disappearance of wheezing within 2 days), moderate group (no sleep disturbance but disappearance of wheezing after 3 or more days), and severe group (sleep disturbance and disappearance of wheezing after 3 or more days). Wheezing characteristics on the first morning were compared among the 3 groups based on sleep disturbance on the first night. RESULTS: The highest frequency, the frequency of maximum intensity, and the number of wheeze bands per 30 seconds were significantly higher in the severe group than in the mild group (p < 0.005, p < 0.005, p < 0.001, respectively). The number of wheeze bands per 30 seconds was a predictor of nighttime sleep disturbance, with a cutoff value of 11.1. The sensitivity, specificity, and positive- and negative-predictive values were 100%, 65%, 32%, and 100% (p < 0.001), respectively, with an area under the curve of 0.86 ± 0.05. CONCLUSIONS: The number of wheeze bands per 30 seconds on lung-sound analysis was a useful indicator of risk of prolonged exacerbation.