The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae.

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to ß-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 102 to 106 colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with ß-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as ß-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae.

BACKGROUND: In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. METHODS: A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. RESULTS: By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. CONCLUSIONS: These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

Quinolone Resistance Is Transferred Horizontally via Uptake Signal Sequence Recognition in Haemophilus influenzae.

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA. Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC, respectively, contributed to the horizontal transfer of resistance in H. influenzae. Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae.

BACKGROUND: Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. OBJECTIVES: To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. METHODS: Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. RESULTS: By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. CONCLUSIONS: These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.

Salmonella Fimbrial Protein FimH Is Involved in Expression of Proinflammatory Cytokines in a Toll-Like Receptor 4-Dependent Manner.

Type 1 fimbriae are proteinaceous filamentous structures present on bacterial surfaces and are mainly composed of the major fimbrial protein subunit FimA and the adhesive protein FimH, which is located at the tip of the fimbrial shaft. Here, we investigated the involvement of type 1 fimbriae in the expression of proinflammatory cytokines in macrophages infected with Salmonella enterica serovar Typhimurium. The level of interleukin-1ß (IL-1ß) mRNA was lower in macrophages infected with fimA or fimH mutant strains than in those infected with wild-type Salmonella Treatment of macrophages with purified recombinant FimH protein, but not FimA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor κB signaling pathways, leading to the expression of not only IL-1ß but also other proinflammatory cytokines, such as IL-6 and tumor necrosis factor alpha. However, FimH carrying an N-terminal region deletion or heat-treated FimH did not show such effects. The expression of FimH-induced IL-1ß was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242 but not by treatment with polymyxin B, a lipopolysaccharide antagonist. Furthermore, FimH treatment stimulated HEK293 cells expressing TLR4 and MD-2/CD14 but did not stimulate HEK293 cells expressing only TLR4. Collectively, FimH is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 in the presence of MD-2 and CD14 and plays a significant role in the expression of proinflammatory cytokines in Salmonella-infected macrophages.

Moxifloxacin resistance and genotyping of Mycobacterium avium and Mycobacterium intracellulare isolates in Japan.

BACKGROUND: Although fluoroquinolones are considered as alternative therapies of pulmonary Mycobacterium avium complex (MAC) disease, the association between fluoroquinolone resistance and MAC genotypes in clinical isolates from individuals not previously treated for MAC infection is not fully clear. METHODS: Totals of 154 M. avium isolates and 35 Mycobacterium intracellulare isolates were obtained from treatment-naïve patients with pulmonary MAC disease at the diagnosis of MAC infection at 8 hospitals in Japan. Their susceptibilities of moxifloxacin were determined by broth microdilution methods. Moxifloxacin-resistant isolates were examined for mutations of gyrA and gyrB. Variable numbers of tandem repeats (VNTR) assay was performed using 15 M. avium VNTR loci and 16 M. intracellulare VNTR loci. RESULTS: Moxifloxacin susceptibility was categorized as resistant and intermediate for 6.5% and 16.9%, respectively, of M. avium isolates and 8.6% and 17.1% of M. intracellulare isolates. Although the isolates of both species had amino acid substitutions of Thr 96 and Thr 522 at the sites corresponding to Ser 95 in the M. tuberculosis GyrA and Gly 520 in the M. tuberculosis GyrB, respectively, these substitutions were observed irrespective of susceptibility and did not confer resistance. The VNTR assays showed revealed three clusters among M. avium isolates and two clusters among M. intracellulare isolates. No significant differences in moxifloxacin resistance were observed among these clusters. CONCLUSIONS: Although resistance or intermediate resistance to moxifloxacin was observed in approximately one-fourth of M. avium and M. intracellulare isolates, this resistance was not associated with mutations in gyrA and gyrB or with VNTR genotypes.

Antibiotic Susceptibility and Genotyping of Mycobacterium avium Strains That Cause Pulmonary and Disseminated Infection.

Mycobacterium avium subsp. hominissuis mainly causes disseminated infection in immunocompromised hosts, such as individuals with human immunodeficiency virus (HIV) infection, and pulmonary infection in immunocompetent hosts. However, many aspects of the different types of M. avium subsp. hominissuis infection remain unclear. We examined the antibiotic susceptibilities and genotypes of M. avium subsp. hominissuis isolates from different hosts by performing drug susceptibility testing using eight antibiotics (clarithromycin, rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, and levofloxacin) and variable-number tandem-repeat (VNTR) typing analysis for 46 isolates from the sputa of HIV-negative patients with pulmonary M. avium subsp. hominissuis disease without previous antibiotic treatment and 30 isolates from the blood of HIV-positive patients with disseminated M. avium subsp. hominissuis disease. Interestingly, isolates from pulmonary M. avium subsp. hominissuis disease patients were more resistant to seven of the eight drugs, with the exception being rifampin, than isolates from HIV-positive patients. Moreover, VNTR typing analysis showed that the strains examined in this study were roughly classified into three clusters, and the genetic distance from reference strain 104 for isolates from pulmonary M. avium subsp. hominissuis disease patients was statistically significantly different from that for isolates from HIV-positive patients (P = 0.0018), suggesting that M. avium subsp. hominissuis strains that cause pulmonary and disseminated disease have genetically distinct features. Significant differences in susceptibility to seven of the eight drugs, with the exception being ethambutol, were noted among the three clusters. Collectively, these results suggest that an association between the type of M. avium subsp. hominissuis infection, drug susceptibility, and the VNTR genotype and the properties of M. avium subsp. hominissuis strains associated with the development of pulmonary disease are involved in higher levels of antibiotic resistance.

ASSOCIATION BETWEEN A pMAH135 PLASMID AND THE PROGRESSION OF PULMONARY DISEASE CAUSED BY MYCOBACTERIUM AVIUM.

BACKGROUND: Pulmonary disease caused by nontuberculous mycobacteria has a variable clinical course. Although this is possibly the result of not only host factors, but also bacterial factors, many questions remain to be answered regarding these manifestations. METHODS: To assess the relationship between the progression of pulmonary Mycobacterium avium disease and bacterial factors we performed variable number tandem repeats (VNTR) typing analysis of M. avium tandem repeats (MATR) in M. avium isolates from 46 patients with different clinical courses, and furthermore, examined the association between disease progression and a pMAH135 plasmid derived from M. avium. RESULTS: In patients whose treatment was initiated because of worsenedchest radiograph findings and/or clinical symptoms within 18 months after being diagnosed with pulmonary M. avium disease, the detection rate of 6 genes located in pMAH135 was 35.3-47.1% for 17 isolates. However, in untreated patients with a stable condition, these rates were 10.3-13.8% in 29 isolates. MATR-VNTR typing analysis showed that isolates from patients with worsened disease and those with stable disease are clustered differently. In cluster III, the number of isolates from patients with worsened disease was higher than that from patients with stable disease (p = 0.019), and furthermore, the number of isolates carrying pMAH135 genes was higher than that not carrying pMAH135 genes (p ≤ 0.001). CONCLUSION: These results indicate an association between the progression of pulmonary M. avium disease and pMAH135. The presence of pMAH135 genes might be a useful prognostic indicator for pulmonary M. avium disease and may serve as one criterion for treatment initiation.

X-ray structure analysis and characterization of AFUEI, an elastase inhibitor from Aspergillus fumigatus.

Elastase from Aspergillus sp. is an important factor for aspergillosis. AFUEI is an inhibitor of the elastase derived from Aspergillus fumigatus. AFUEI is a member of the I78 inhibitor family and has a high inhibitory activity against elastases of Aspergillus fumigatus and Aspergillus flavus, human neutrophil elastase and bovine chymotrypsin, but does not inhibit bovine trypsin. Here we report the crystal structure of AFUEI in two crystal forms. AFUEI is a wedge-shaped protein composed of an extended loop and a scaffold protein core. The structure of AFUEI shows remarkable similarity to serine protease inhibitors of the potato inhibitor I family, although they are classified into different inhibitor families. A structural comparison with the potato I family inhibitors suggests that the extended loop of AFUEI corresponds to the binding loop of the potato inhibitor I family, and AFUEI inhibits its cognate proteases through the same mechanism as the potato I family inhibitors.

Development of a rapid detection method for the macrolide resistance gene in Mycobacterium avium using the amplification refractory mutation system-loop-mediated isothermal amplification method.

Macrolide antibiotics such as clarithromycin (CLR) and azithromycin are the key drugs used in multidrug therapy for Mycobacterium avium complex (MAC) diseases. For these antibacterial drugs, drug susceptibility has been correlated with clinical response in MAC diseases. We have previously demonstrated the correlation between drug susceptibility and mutations in the 23S rRNA gene, which confers resistance to macrolides. Herein, we developed a rapid detection method using the amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) technique to identify mutations in the 23S rRNA gene of M. avium. We examined the applicability of the ARMS-LAMP method to genomic DNA extracted from six genotypes of M. avium clinical isolates. The M. avium isolates were classified into 21 CLR-resistant and 9 CLR-susceptible strains based on the results of drug susceptibility tests; the 23S rRNA genes of these strains were sequenced and analyzed using the ARMS-LAMP method. Sequence analysis revealed that the 9 CLR-sensitive strains were wild-type strains, whereas the 21 CLR-resistant strains comprised 20 mutant-type strains and one wild-type strain. Using ARMS-LAMP, no amplification from genomic DNAs of the 10 wild-type strains was observed using the mutant-type mismatch primer sets (MTPSs); however, amplification from the 20 mutant-type strain DNAs was observed using the MTPSs. The rapid detection method developed by us integrates ARMS-LAMP with a real-time turbidimeter, which can help determine drug resistance in a few hours. In conclusion, ARMS-LAMP might be a new clinically beneficial technology for rapid detection of mutations.IMPORTANCEMultidrug therapy for pulmonary Mycobacterium avium complex disease is centered on the macrolide antibiotics clarithromycin and azithromycin, and resistance to macrolides is an important prognosticator for clinical aggravation. Therefore, it is important to develop a quick and easy method for detecting resistance to macrolides. Drug resistance is known to be correlated with mutations in macrolide resistance genes. We developed a rapid detection method using amplification refractory mutation system (ARMS)-loop-mediated isothermal amplification (LAMP) to identify a mutation in the 23S rRNA gene, which is a macrolide resistance gene. Furthermore, we examined the applicability of this method using M. avium clinical isolates. The rapid method developed by us for detection of the macrolide resistance gene by integrating ARMS-LAMP and a real-time turbidimeter can help in detection of drug resistance within a few hours. Since this method does not require expensive equipment or special techniques and shows high analytical speed, it would be very useful in clinical practice.

[Extensive genetic analysis of clinical tuberculosis isolates and analysis of host factors to evaluate rapid development of multidrug resistance during initial treatment].

INTRODUCTION: In this study, we aimed at determining the cause of resistance to tuberculosis treatment by performing genetic analyses of bacteria obtained from a patient who developed multidrug-resistant tuberculosis (MDR-TB) during the initial course of treatment for tuberculosis. METHODS: Specimens obtained before and after the development of MDR-TB were subjected to spoligotyping, drug-resistance gene analysis, and variable-number tandem repeat (VNTR) typing. The patient's clinical background was also reviewed. RESULTS: After the development of resistance, the bacterial genome had changed with regard to only 1 mutation: S531L in the rpoB gene. Spoligotyping revealed that the genotype was that of the Beijing strain. VNTR typing confirmed all 35 loci. Review of the patient's clinical background showed that diabetes mellitus was present as a complication. DISCUSSION: There was no evidence of reinfection or polyclonal infection. The strain belonged to a sublineage of the Beijing genotype that is a common precipitating cause of MDR-TB due to this genotype. The patient had diabetes mellitus and was thus vulnerable to the development of resistance. Factors associated with both the host and bacteria, therefore, contributed to the development of resistance in this case, which seemed to result in the rapid development of MDR-TB.

A Rapid Screening Assay for Clarithromycin-Resistant Mycobacterium avium Complex Using Melting Curve Analysis with Nonfluorescent Labeled Probes.

Mycobacterium avium complex (MAC) thrives in various environments and mainly causes lung disease in humans. Because macrolide antibiotics such as clarithromycin or azithromycin are key drugs for MAC lung disease, the emergence of macrolide-resistant strains prevents the treatment of MAC. More than 95% of macrolide-resistant MAC strains are reported to have a point mutation in 23S rRNA domain V. This study successfully developed a melting curve assay using nonfluorescent labeled probes to detect the MAC mutation at positions 2058 to 2059 of the 23S rRNA gene (AA genotype, clarithromycin susceptible; TA, GA, AG, CA, AC, and AT genotypes, clarithromycin resistant). In the AA-specific probe assay, the melting peak of the DNA fragment of the AA genotype was higher than that of DNA fragments of other genotypes. Melting temperature (Tm) values of the AA genotype and the other genotypes were about 80°C and 77°C, respectively. DNA fragments of each genotype were identified correctly in six other genotype-specific probes (TA, GA, AG, CA, AC, and AT) assays. Using genomic DNA from six genotype strains of M. avium and four genotype strains of M. intracellulare, we confirmed that all genomic DNAs could be correctly identified as individual genotypes according to the highest Tm values among the same probe assays. These results indicate that this melting curve-based assay is able to determine MAC genotypes at positions 2058 to 2059 of the 23S rRNA gene. This simple method could contribute to the rapid detection of clarithromycin-resistant MAC strains and help to provide accurate drug therapy for MAC lung disease. IMPORTANCE Since macrolide antibiotics such as clarithromycin or azithromycin are key drugs in multidrug therapy for Mycobacterium avium complex (MAC) lung diseases, the rapid detection of macrolide-resistant MAC strains has important implications for the treatment of MAC. Previous studies have reported a correlation between drug susceptibility testing and the mutation of macrolide resistance genes. In this study, we developed a novel melting curve-based assay using nonfluorescent labeled probes to identify both clarithromycin-resistant M. avium and M. intracellulare with mutations in the 23S rRNA gene, which is the clarithromycin or azithromycin resistance gene. This assay contributed to not only the detection of MAC mutations but also the determination of all genotypes at positions 2058 to 2059 of the 23S rRNA gene. Furthermore, because nonfluorescent labeled probes are used, this assay is more easily and more immediately available than other methods.

[Multicenter study on clinical features and genetic characteristics of Mycobacterium avium strains from patients in Japan with lung disease caused by M. avium].

BACKGROUND: The pulmonary disease caused by Mycobacterium avium shows diverse clinical manifestations. Little is known about the potential association between the genetic characteristics of M. avium strains and disease progression. SUBJECTS AND METHODS: We enrolled 89 patients with disease caused by M. avium, from 12 hospitals in Japan and collected the corresponding M. avium isolates and clinical data. We divided the 89 patients into 2 groups: one group comprising 43 patients with progressive disease despite chemotherapy ("progressive"), and the other group comprising 46 patients with untreated disease ("untreated"). We compared clinical and bacteriological characteristics between these groups. The bacteriological characteristics that we examined were drug susceptibility, variable-number tandem-repeat (VNTR) typing, and presence of the insertion sequence ISMav6. Seventeen patients in the "untreated" group were started on chemotherapy because their condition had clinically deteriorated during follow-up. RESULTS: The result of VNTR typing showed that there was no specific clustering according to geographical region or clinical group in the "untreated" or "progressive" groups. Six out of eight cases those of polyclonal infection, and 11 of 12 isolates that were highly resistant to clarithromycin were isolated from patients with progressive disease. The frequency of isolates with ISMav6 inserted into upstream region of the cfp29 gene, which is involved in the induction of interferon-gamma production, was significantly higher in patients with deteriorating disease than in stable patients in the "untreated" group (p = 0.002). CONCLUSION: Polyclonal infection and clarithromycin resistance may be involved in disease progression. ISMav6 inserted into the cfp29 gene is also suggested to be a factor related to the deterioration of pulmonary Mycobacterium avium complex disease.

[Identification of novel variable number tandem repeat (VNTR) loci in Mycobacterium avium and development of an effective means of VNTR typing].

INTRODUCTION: To make more effective use of variable number tandem repeat (VNTR) typing, we identified novel VNTR loci in Mycobacterium avium and used them for modified M. avium tandem repeat-VNTR (MATR-VNTR) typing. METHOD: Analysis of a DNA sample extracted from a clinical isolate (strain HN135) with the FLX system genome sequencer (Roche Diagnostic System) led to discovery of several novel VNTR loci. The allelic diversity of the novel VNTR loci was evaluated for 71 clinical isolates and compared with the diversity of the MATR-VNTR loci. To improve efficacy of MATR-VNTR typing, we tested typing using 2 sets of loci selected from the newly identified loci and the MATR loci, i.e., one set containing 7 and another 16 loci. Hunter Gaston's discriminatory index (HGDI) was calculated for these sets. RESULTS: Six VNTR loci were newly identified, of which 5 showed a high diversity. The HGDI was 0.980 for the improved new typing using a set of 7 loci, and 0.995 for another set of 16 loci, while it was 0.992 for the conventional MATR-VNTR typing. DISCUSSION: VNTR typing with the set of the 7 loci enabled a rapid analysis, and another set of 16 loci enabled a precise analysis, as compared with conventional MATR-VNTR typing. A method that uses only VNTR loci with relatively high allelic diversity is considered to be a useful tool for VNTR typing of MAC isolates.

[A study of genetic characteristics of Mycobacterium avium strains from patients with pulmonary M. avium disease in Japan and Korea].

INTRODUCTION: To determine the characteristics of Mycobacterium avium in Japan, we compared the genetic properties of M. avium isolated in different countries. METHODS: A Mycobacterium avium tandem-repeat variable-number tandem-repeat (MATR-VNTR) analysis was performed using South Korean strains (n = 119) and Japanese strains (n = 76). In addition, we compared the frequencies of a new insertion sequence, ISMav6. RESULTS: A phylogenetic analysis identified different clusters between the two countries' strains. The prevalence of ISMav6 was significantly different between them, i.e., 75.0% in Japanese strains and 59.8% in the Korean ones (P < 0.035). The frequency of strains with IS Mav6 in the Shine-Dalgarno (SD) sequence of the cfp29 gene that is involved in the interferon-gamma induction was also different, with stronger significance (Japan: 38.2%, Korea: 12.4%, P < 0.001). DISCUSSION: It is possible that M. avium strains prevalent in Japan and in Korea are genetically distinct. The analyses of the presence of ISMav6, as well as the VNTR patterns of M.avium strains from many different countries would be a promising methodology in elucidating the causes of the recent increase in cases of pulmonary MAC diseases.

Salmonella fimbrial protein StcD induces cyclooxygenase-2 expression via Toll-like receptor 4.

INTRODUCTION: The genome of Salmonella enterica serovar Typhimurium contains 13 operons with homology to fimbrial genes. METHODS: To investigate the involvement of these fimbrial gene clusters in the expression of cyclooxygenase-2 (COX-2), which is an inducible enzyme involved in the synthesis of prostanoids, in J774 macrophages infected with S. enterica serovar Typhimurium, we constructed strains carrying a mutation in genes encoding the putative subunit proteins in 12 fimbrial operons. RESULTS: The level of COX-2 expression was lower in macrophages infected with fimA or stcA mutant Salmonella than in those infected with wild-type Salmonella. Therefore, we focused on putative subunit protein StcA and adhesive like protein StcD encoded in the stc operon. Treatment of macrophages with purified recombinant StcD protein, but not StcA, resulted in the activation of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, leading to the expression of not only COX-2 but also of pro-inflammatory cytokines such as interleukin (IL)-1ß, IL-6, and tumor necrosis factor alpha. The expression of StcD-induced COX-2 was inhibited by treatment with the Toll-like receptor 4 (TLR4) inhibitor TAK-242, but not by treatment with the lipopolysaccharide (LPS) antagonist polymyxin B. Furthermore, StcD treatment stimulated HEK293 cells expressing TLR4 in the presence of CD14 and MD-2. CONCLUSION: StcD is a pathogen-associated molecular pattern of S. enterica serovar Typhimurium that is recognized by TLR4 and plays a significant role in the induction of COX-2 expression in macrophages.

Evaluation of a rapid detection method of clarithromycin resistance genes in Mycobacterium avium complex isolates.

OBJECTIVES: Clarithromycin is the key drug in the various treatment regimens of Mycobacterium avium complex (MAC) diseases, and is the only drug for which drug susceptibility has been shown to correlate with clinical response in these diseases. A point mutation at either positions 2058 or 2059 of the 23S rRNA gene has been reported to occur in high-level clarithromycin-resistant isolates. In this study, we examined the correlation between the results from a drug susceptibility test and the mutation of the 23S rRNA gene involved in drug resistance in MAC. Furthermore, we adapted a rapid detection method using amplification refractory mutation system (ARMS)-PCR to identify mutations in the 23S rRNA gene in MAC isolates. METHODS: Using a microdilution method based on the NCCLS/CLSI recommendation, the MIC of clarithromycin was determined for 245 clinical MAC isolates. Of these, 219 clarithromycin-susceptible and 26 clarithromycin-resistant strains were analysed by sequencing of the 23S rRNA gene and ARMS-PCR. RESULTS: The drug susceptibility test revealed a bimodal distribution of MICs for both the susceptible and resistant strains. Sequence analysis of the 23S rRNA gene revealed that all of the clarithromycin-susceptible strains were wild-type whereas 24 of the clarithromycin-resistant strains were mutant type. The sensitivity of the sequence and ARMS-PCR analyses was 92.3% and 84.6%, respectively, and the specificity of both was 100%. CONCLUSIONS: We found a correlation between MICs of clarithromycin and 23S rRNA gene mutations. ARMS-PCR for 23S rRNA mutations of MAC isolates is useful for rapid detection of clarithromycin resistance.

Molecular typing of Mycobacterium intracellulare using multilocus variable-number of tandem-repeat analysis: identification of loci and analysis of clinical isolates.

In addition to its known status as a disseminated disease in HIV-positive patients, Mycobacterium avium complex (MAC) is increasingly recognized as a causative pathogen of respiratory disease in HIV-negative patients. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, of which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 50 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter Gaston's discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay are highly discriminating and stable over time.

[Clinical application of line probe assay (LiPA) for rifampicin (RFP)-resistant gene examination in sputum from tuberculosis patients].

INTRODUCTION: Preventing the spread of drug-resistant tuberculosis is a clinically important challenge. In this effort, rifampicin (RFP)-resistant gene examination by line probe assay (LiPA) was evaluated for its clinical application for rapid detection of tuberculosis. METHODS: The RFP-resistant gene was examined in a total of 110 samples of sputum obtained from patients that were definitively diagnosed with pulmonary tuberculosis by auto-LiPA. The difference in detection sensitivity between the results of the smear and culture examinations was evaluated. Culture-positive samples were compared with the results of the drug susceptibility test. RESULTS: Smear-positive samples were LiPA positive in 69 of 73 samples (sensitivity: 94.5%), and smear-negative samples were LiPA positive in 25 of 37 samples (67.6%). More than half of the samples were LiPA positive, even those that were culture-negative or contaminated. Comparison of the 76 culture-positive samples with the results of the drug susceptibility test found that all samples were wild type among the RFP-sensitive strains. Among the 8 RFP-resistant strains, 6 were mutation type. All samples shown to be mutation type were obtained from patients with multi-drug resistant tuberculosis. DISCUSSION: Using LiPA, the amount of smear can be used as a factor for detection of RFP-resistant genes. Detection was possible even with culture-negative and contaminated samples, allowing more rapid diagnosis of patients with multi-drug resistant tuberculosis.

Comparison of a variable-number tandem-repeat (VNTR) method for typing Mycobacterium avium with mycobacterial interspersed repetitive-unit-VNTR and IS1245 restriction fragment length polymorphism typing.

Mycobacterium avium complex (MAC) infections are increasing annually in various countries, including Japan, but the route of transmission and pathophysiology of the infection remain unclear. Currently, a variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (V. C. Thibault, M. Grayon, M. L. Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, J. Clin. Microbiol. 45:2404-2410, 2007). Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections.