Chronic neutrophilic leukemia preceded by myelodysplastic syndromes.

Chronic neutrophilic leukemia (CNL) is primarily diagnosed by excluding myelodysplastic syndromes (MDS). We report the case of a patient who developed secondary CNL 3 years after hypoplastic MDS. We used droplet digital polymerase chain reaction mutation detection assay to analyze genomic alterations during the progression from MDS to CNL. At the time of MDS diagnosis, U2AF1 Q157P and SETBP1 D868N were dominant and additional mutation of ASXL1 1934_insG was observed. CSF3R T618I and SETBP1 D868N were increasing at the time of CNL diagnosis. We revealed the accumulation of multiple gene mutations during CNL development from MDS. This suggests that CNL was clonally developed from the founding clone of MDS and CSF3R mutation contributes to the development of CNL in the present case. These findings provide insights into the pathology of CNL.

Classification of gases around Pseudomonas aeruginosa and Acinetobacter baumannii by infrared spectroscopy.

Gas samples were collected from the air surrounding single and mixed laboratory cultures, and preliminary data on human breath samples were also obtained. The infrared spectra of a variety of gasses were measured at high resolution (0.5 cm-1) to obtain information about the infrared absorption bands to be used as indicators. These key bands enable bacterial classification, and the discrimination rates can be improved by observing multiple infrared absorptions. The air around Pseudomonas aeruginosa was distinguished from the other gas samples by the infrared absorptions at wavenumbers of 778.4 cm-1 and 2213.2 cm-1. For Acinetobacter baumannii, infrared absorptions at 1215.0 cm-1 and 2982.3 cm-1 were used; furthermore, adding those at 4768.7 cm-1 and 5353.8 cm-1 was shown to improve identification.

Lignosulfonate Rapidly Inactivates Human Immunodeficiency and Herpes Simplex Viruses.

Background: Very few studies of the antiviral potential of lignosulfonates have been published. With the aim of oral application, among various groups of natural products, the relative antiviral potency of lignosulfonate and its ability to rapidly inactivate viruses were investigated. Methods: As target cells, MT-4 cells in suspension and attached Vero cells were used for infections with human immunodeficiency virus (HIV) and human herpes simplex type-1 virus (HSV). Mock- or virus-infected cells were incubated for 3-5 days with various concentrations of test samples, and the viable cell number was determined with the MTT method. For the shorter exposure experiments, higher titers of HIV or HSV were exposed to test samples for 10 or 3 min, diluted to a normal multiplicity of infection (MOI), and applied to the cells. Antiviral activity was quantified by using the chemotherapy index. Results: In the long-exposure system, lignosulfonates showed comparable anti-HIV activity with those of AZT, ddC, and sulfated polysaccharides, and it exceeded those of hundreds of tannins and flavonoids. When the exposure time was shortened, the chemotherapeutic index of the lignosulfonates for HIV was increased 27-fold. At a physiological pH, lignosulfonate showed higher anti-HIV activity than commercial alkali-lignin, dealkali-lignin, and humic acid, possibly due to the higher solubility and purity. Conclusions: With their rapid virus-inactivation capabilities, lignosulfonates may be useful for the prevention or treatment of virally induced oral diseases.

Re-Evaluation of Chemotherapeutic Potential of Pyoktanin Blue.

Background: Pyoktanin blue (PB) is used for staining tissues and cells, and it is applied in photodynamic therapy due to its potent bactericidal activity. However, clinical application of PB as an antiviral and antitumor agent has been limited due to its potent toxicity. For clinical application, the antitumor and antiviral activity as well as the neurotoxicity of PB were re-evaluated with a chemotherapeutic index. Methods: Tumor-specificity (TS) was determined by the ratio of CC50 against normal oral cells/oral squamous cell carcinoma (OSCC); neurotoxicity by that of normal oral/neuronal cells; antiviral activity by that of mock-infected/virus-infected cells; and potency-selectivity expression (PSE) by dividing TS by CC50 (OSCC). Results: Antitumor activity of PB (assessed by TS and PSE) was comparable with that of DXR and much higher than that of 5-FU and melphalan. PB induced caspase-3 activation and subG1 cell accumulation in an OSCC cell line (Ca9-22). PB and anticancer drugs showed comparable cytotoxicity against both neuronal cells and OSCC cell lines. PB showed no detectable anti-HIV/HSV activity, in contrast to reverse transferase inhibitors, sulfated glucans, and alkaline extract of leaves of S.P. Conclusions: PB showed first-class anticancer activity and neurotoxicity, suggesting the importance of establishing the safe treatment schedule.

Quantification of the Ability of Natural Products to Prevent Herpes Virus Infection.

Background: Herpes simplex virus (HSV) is usually dormant and becomes apparent when body conditions decline. We investigated the anti-HSV activity of various natural and synthetic compounds for future clinical application. Methods: Mock- and HSV-infected Vero cells were treated for three days with various concentrations of samples. For short exposure, 100-fold concentrated virus were preincubated for 3 min with samples, diluted to normal multiplicity of infection (MOI), before the addition to the cells. Anti-HSV activity was evaluated by the chemotherapy index. Results: Alkaline extracts of the leaves of Sasa sp. (SE) and pine cone (PCE) showed higher anti-HSV activity than 20 Japanese traditional herb medicines (Kampo formulas), four popular polyphenols, and 119 chromone-related compounds. Exposure of HSV to SE or PCE for 3 min almost completely eliminated the infectivity of HSV, whereas much longer exposure time was required for Kakkonto, the most active Kampo formulae. Anti-HSV activity of PCE and Kakkonto could be detected only when they were dissolved by alkaline solution (pH 8.0), but not by neutral buffer (pH 7.4). Anti-HSV activity of SE and povidone iodine was stable if they were diluted with neutral buffer. Conclusions: The present study suggests the applicability of SE and PCE for treatment of oral HSV and possibly other viruses.

PCR amplification of the erm(41) gene can be used to predict the sensitivity of Mycobacterium abscessus complex strains to clarithromycin.

A worldwide increase in the Mycobacterium abscessus (M. abscessus) complex has been observed. Therefore, the aim of the present study was to investigate the diversity of the rrl and erm(41) genes, both of which are associated with macrolide sensitivity in the M. abscessus complex. The current study also examined the efficacy of mass spectrometry as an alternative to molecular testing to classify subspecies of the M. abscessus complex. A total of 14 strains of the M. abscessus complex were obtained, and based on conventional analyses using housekeeping genes, 57% were determined to be M. abscessus subsp. abscessus, 43% were M. abscessus subsp. massiliense, and none were identified as M. abscessus subsp. bolletii. However, depending on the strain, it was not always possible to distinguish between the subspecies by mass spectrometry. Consequently, PCR products for the rrl and erm(41) genes were directly sequenced. Overall, 7.1% of the strains were identified to have a rrl mutation, and 92.9% carried a T at position 28 of erm(41). Results presented here suggest that the principal cause of treatment failure for M. abscessus complex infections is inducible macrolide resistance encoded by the erm(41) gene. From a strictly pragmatic standpoint, the phenotypic function of a putative erm(41) gene is the most important piece of information required by clinicians in order to prescribe an effective treatment. Although PCR amplification of erm(41) is not sufficient to differentiate between the M. abscessus complex subspecies, PCR can be easily and efficiently used to predict the sensitivity of members of the M. abscessus complex to clarithromycin.

SCCmec typing of PVL-positive community-acquired Staphylococcus aureus (CA-MRSA) at a Japanese hospital.

The epidemiology of Panton-Valentine leukocidin (PVL)-positive MRSA in community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was examined. Three hundred and forty-two CA-MRSA strains that were susceptible to imipenem and cefazolin were isolated from 1107 samples (intravenous catheter, blood, sputum, urine, skin, wound, and pharynx) from outpatients at Showa University Hospital in Japan between September 2009 and March 2017. The PVL gene was detected in 46 of 342 CA-MRSA strains, accounting for 13.5%. The type of SCCmec was determined by detection of each SCCmec-specific region, class complex, and ccr. SCCmec type IV comprised 33 strains, type V comprised 5 strains, type VII comprised 4 strains, and the unclassified type comprised 4 strains. Among the type IV strains, subtype IVa was dominant, comprising 23 of 33 strains, and the remaining 10 strains were of varying subtypes. The SCCmec type III-specific region, CZ049, was amplified in 2 type V strains, 4 type VII strains, and 4 unclassified strains. In 4 unclassified strains, CZ049 and ccr5 were detected, but neither the SCCmec-specific region nor class complex was detected. The PVL-positive rate was lower than that in Western countries. The SCCmec types of PVL-positive CA-MRSA strains were found to vary, indicating a diverse spreading route.

Bacteriuria With CTX-M-8 Extended-Spectrum ß-Lactamase-Producing Escherichia coli in a Patient With Incomplete Kawasaki Disease.

We report the case of a 2-month-old infant with incomplete Kawasaki disease that presented as an apparent urinary tract infection. The patient's fever persisted despite antibiotic treatment. Intravenous immunoglobulin and aspirin therapy cured both the incomplete Kawasaki disease and bacterial pyuria. Renal sonography, voiding cystourethrography, and renal parenchyma radionuclide scanning did not detect any abnormalities. Temporary dilation of the coronary artery was noted. In a urine specimen obtained through transurethral catheterization, the growth of 105 colony-forming units/mL of extended-spectrum ß-lactamase-producing Escherichia coli was detected. Polymerase chain reaction analysis revealed that the enzyme genotype was CTX-M-8, which is a rare type in Japan. In conclusion, attention should be paid to a misleading initial presentation of fever and pyuria, which might be interpreted as urinary tract infection in patients with Kawasaki disease. Furthermore, pediatricians should consider incomplete Kawasaki disease when patients present with fever and pyuria, which are consistent with urinary tract infection, but do not respond to antibiotic treatment.

Evaluation of Biological Activity of Mastic Extracts Based on Chemotherapeutic Indices.

BACKGROUND: Most previous mastic investigators have not considered its potent cytotoxicity that may significantly affect the interpretation of obtained data. In the present study, we re-evaluated several biological activities of mastic extracts, based on chemotherapeutic indexes. MATERIALS AND METHODS: Pulverized mastic gum was extracted with n-hexane and then with ethyl acetate or independently with methanol or n-butanol. Tumor specificity (TS) of the extracts was determined by their cytotoxicity against human malignant and non-malignant cells. Antibacterial activity was determined by their cytotoxicity against bacteria and normal oral cells. Antiviral activity was determined by their protection of viral infection and cytotoxic activity. Cytochrome P-450 (CYP) 3A4 activity was measured by ß-hydroxylation of testosterone. RESULTS: Ethyl acetate extract showed slightly higher tumor specificity (TS=2.6) and one order higher antibacterial activity (selectivity index (SI)=0.813) than other extracts (TS=1.4-2.5; SI=0.030-0.063). All extracts showed no anti-human immunodeficiency virus (HIV) activity, but some anti-herpes simplex virus (HSV) activity, which was masked by potent cytotoxicity. They showed strong inhibitory activity against CYP3A4. CONCLUSION: Ethyl acetate extraction following the removal of cytotoxic and CYP3A4 inhibitory substances by n-hexane can enhance antitumor and antibacterial activity of mastic.

Efficacy of non-carbapenem antibiotics for pediatric patients with first febrile urinary tract infection due to extended-spectrum beta-lactamase-producing Escherichia coli.

Although carbapenem is the recommended for urinary tract infection (UTI) caused by extended-spectrum beta-lactamase (ESBL)-producing organisms, non-carbapenems have been reported to be effective for adult patients with UTI caused by ESBL-producing organisms. The purpose of this study was to evaluate the efficacy of non-carbapenems for pediatric patients with UTI due to ESBL-producing Escherichia coli (E. coli) based on the microbiologic and clinical outcomes. Fifteen children, who were treated for first febrile UTI caused by ESBL-producing E. coli were enrolled in this study. Antimicrobial susceptibilities and ESBL production were determined according to the Clinical and Laboratory Standards Institute guidelines. To detect CTX-M genes, polymerase chain reaction was performed with specific primers for blaCTX-M detection. Of the 15 enrolled patients, 10 (66.7%) were boys and 5 (33.3%) were girls, with a median age of four months. VUR was detected in six patients (40%). For detection of blaCTX-M by PCR, CTX-M-3, CTX-M-8, CTX-M-14, and CTX-M-15 were detected in five, one, eight, and one patient, respectively. Overall, 14 of the 15 isolates (93.3%) were susceptible for fosfomycin (FOM), and all isolates were susceptible for cefmetazole (CMZ), flomoxef (FMOX), and imipenem/cilastatin (IPM/CS). Of the 15 patients, 12 (80%) clinically improved without the use of carbapenems. In conclusion, even if isolates of ESBL-producing E. coli are multidrug resistant based on MIC assessment, clinical susceptibility to non-carbapenems, such as CMZ, FMOX, and FOM, is possible. Accordingly, carbapenems may not be required all the time for treatment of pediatric UTI in clinical practice.

Antiviral and Antitumor Activity of Licorice Root Extracts.

BACKGROUND: In the search for anti-viral and antitumor substances from natural resources, antiviral and antitumor activities of licorice root extract and purified ingredients were investigated. MATERIALS AND METHODS: Viability of cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antiviral activity was quantified by the selectivity index, defined as the ratio of the 50% cytotoxic concentration (CC50) to the 50% effective concentration against human immunodeficiency virus (HIV) or herpes simplex virus (HSV)-infected cells (EC50). The tumor specificity was calculated by the ratio of CC50 against human normal oral cells to that against human oral squamous cell carcinoma cell lines. Licorice flavonoids and lower molecular polyphenols were subjected to quantitative structure-activity relationship analysis. RESULTS: Alkaline extract of licorice root had higher anti-HIV activity than did water extracts, confirming our previous reports. On the other hand, water extract, especially the flavonoid-rich fraction, had higher anti-HSV activity than did the alkaline extract. The flavonoid-rich fraction was more cytotoxic against human oral squamous cell carcinoma cell lines compared to normal oral cells, suggesting their tumor-specific cytotoxicity. CONCLUSION: The present study suggests that water and alkaline extracts of licorice root exert different mechanisms of actions against these two viruses. Physicochemical properties, rather than the category of compounds, may be important in determining their anti-HSV activity.

Analysis of Staphylococcal Toxins and Clinical Outcomes of Methicillin-Resistant Staphylococcus aureus Bacteremia.

It is well known that methicillin-resistant Staphylococcus aureus (MRSA) produces many virulence factors, such as hemolysins, leukocidins, proteases, enterotoxins, exfoliative toxins, and immune-modulatory factors. The aim of study was to identify staphylococcal pathogenicity that may affect the prognosis of patients with MRSA bacteremia. We obtained 149 MRSA strains from blood cultures between January 2009 and December 2014 in our institution. We collected information on patient characteristics, laboratory data, staphylococcal toxin genes, and susceptibility of the strain toward anti-MRSA agent and analyzed them as factors contributing to 30-d mortality. The "survival" and "dead" groups consisted of 103 and 46 patients, respectively. Multiple logistic regression analysis showed a four-fold increase in the risk of mortality in patients exhibiting isolated MRSA with staphylococcal enterotoxins (SEs) genes as well as toxic shock syndrome toxin-1 (TSST-1) genes [odds ratio: 3.89; 95% confidence interval: 1.20-12.60; p=0.024]. Kaplan-Meier analysis also showed significantly higher mortality in patient with isolated MRSA with SEs and TSST-1 genes. After adjusting for confounders, the coexistence of SEs and TSST-1 were independently associated with the 30-d mortality compared with treatment and susceptibility. The coexistence of superantigenic toxin genes greatly affects the clinical course and prognosis of patients with MRSA bacteremia.

Synergism of Alkaline Extract of the Leaves of Sasa senanensis Rehder and Antiviral Agents.

BACKGROUND: Previous studies have shown a much greater antiviral activity of alkaline extract of the leaves of Sasa senanensis Rehder (SE) against human immunodeficiency virus (HIV), compared to lignin precursors, tannins and flavonoids, suggesting its possible application to oral diseases. Systematic comparative study with herpes simplex virus (HSV) has been limited compared to that with HIV. In the present study, we investigated whether combination of SE with other popular antiviral agents further enhances their individual activity. MATERIALS AND METHODS: Cell viability of mock-infected, HIV-infected and HSV-infected cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The antiviral activity was evaluated by the selectivity index, defined as the ratio of 50% cytotoxic concentration to 50% effective concentration. Synergy between SE and antiviral agents was evaluated by MacSynerg and CompuSyn software. RESULTS: SE showed potent anti-HIV activity, although its activity was two-orders lower than that of azidothymidine, 2',3'-dideoxycytidine dextran sulfate and curdlan sulfate. Combination of SE with these antiviral agents produced synergistic effects. Using a newly established MTT assay system for anti-HSV activity, SE and acyclovir were found to have synergistic anti-HSV activity. CONCLUSION: The present study suggests the possible efficacy of the clinical application of SE combined with antiviral agents.

Validation of Cefazolin as Initial Antibiotic for First Upper Urinary Tract Infection in Children.

To validate the policy of administering cefazolin (CEZ) as a first-line antibiotic to children who are hospitalized with their first febrile urinary tract infection (UTI), we evaluated microbial susceptibility to CEZ and the efficacy of CEZ. The 75 enrolled children with febrile UTI were initially treated with CEZ. Switching CEZ was not required in 84% of the patients. The median fever duration, prevalence of bacteremia, prevalence of UTI caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli, and median duration of hospitalization were significantly higher in the CEZ-ineffective group. The risks of vesicoureteral reflux, indication of operation, and renal scarring are not increased, even when CEZ is ineffective as a first-line antibiotic. CEZ is effective in more than 80% of pediatric patients with their first febrile UTI, but it should be switched to appropriate antibiotics considering sepsis or the ESBL-producing Enterobacteriaceae pathogen, when fever does not improve within 72 hours.

Analysis of OXA23 and AmpCß-Lactamase Genes in Clinically Isolated Multidrug-Resistant Acinetobacter baumannii Clonal Complex 92.

Multidrug-resistant Acinetobacter baumannii, which is resistant to carbapenems, amino glycosides, and fluoroquinolones, was isolated from the wound of an outpatient. We performed Multi Locus Sequence Typing and analyzed the structures of the AmpC ß-lactamase and OXA23 carbapenemase genes. This strain was assigned as ST451, a member of clonal complex 92, by its MLST scheme. The structures of ISAba1-AmpC ß-lactamase and ISAba1-OXA23 carbapenemase were revealed and the major globally prevalent clone of carbapenem-resistant A. baumannii was identified.

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan.

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 µg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 µg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

More accurate measurement of vancomycin minimum inhibitory concentration indicates poor outcomes in meticillin-resistant Staphylococcus aureus bacteraemia.

Meticillin-resistant Staphylococcus aureus (MRSA) is an important pathogen associated with community-acquired and nosocomial infections. The aim of this study was to validate the vancomycin (VAN) minimum inhibitory concentration (MIC) and administration of VAN that may affect the prognosis of patients with MRSA bacteraemia. In total, 140 clinical MRSA strains from blood cultures were collected from January 2009 to December 2013 at a university hospital in Tokyo (Japan). Patient background, their clinical situation and the susceptibility of isolates to anti-MRSA agents in all cases were reviewed, and factors contributing to 30-day mortality were analysed. Susceptibility to anti-MRSA agents was measured by a microdilution susceptibility testing method. The VAN MIC was further evaluated at 0.25 µg/mL intervals from 0.5 µg/mL to 2.0 µg/mL. Multiple logistic regression analysis revealed a 4-fold increase in mortality of patients with a VAN MIC ≥1.5 µg/mL [odds ratio (OR)=3.952, 95% confidence interval (CI) 1.471-10.614; P=0.006]. A one-score increase in the Charlson co-morbidity index resulted in a 1.2-fold increase in the risk of death (OR=1.199, 95% CI 1.054-1.364; P=0.006). However, no significant difference was found in the ratio of the VAN 24-h area under the concentration-time curve to MIC between VAN MIC ≥1.5 µg/mL and <1.5 µg/mL. A significant increase in the MICs of teicoplanin and daptomycin was observed in strains with high VAN MICs. For patients with high VAN MICs, administration of these anti-MRSA antibiotics may have a poor outcome owing to cross-resistance.

Evaluation of new automated syphilis test reagents 'IMMUNOTICLES AUTO3' series: performance, biochemical reactivity, and clinical significance.

BACKGROUND: Automated nontreponemal and treponemal test reagents based on the latex agglutination method (immunoticles auto3 RPR: ITA3RPR and immunoticles auto3 TP: ITA3TP) have been developed to improve the issues of conventional manual methods such as their subjectivity, a massive amount of assays, and so on. We evaluated these reagents in regards to their performance, reactivity to antibody isotype, and their clinical significance. METHODS: ITA3RPR and ITA3TP were measured using a clinical chemistry analyzer. Reactivity to antibody isotype was examined by gel filtration analysis. RESULTS: ITA3RPR and ITA3TP showed reactivity to both IgM- and IgG-class antibodies and detected early infections. ITA3RPR was verified to show a higher reactivity to IgM-class antibodies than the conventional methods. ITA3RPR correlated with VDRL in the high titer range, and measurement values decreased with treatment. ITA3RPR showed a negative result earlier after treatment than conventional methods. ITA3TP showed high specificity and did not give any false-negative reaction. Significant differences in the measurement values of ITA3RPR between the infective and previous group were verified. CONCLUSIONS: The double test of ITA3RPR and ITA3TP enables efficient and objective judgment for syphilis diagnosis and treatments, achieving clinical availability.

Antibiotic susceptibility survey of blood-borne MRSA isolates in Japan from 2008 through 2011.

We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of ß-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan.

A molecular analysis of quinolone-resistant Haemophilus influenzae: validation of the mutations in Quinolone Resistance-Determining Regions.

The mechanism of quinolone-resistance is considered to be amino acid mutations in the type II topoisomerase. We validated the genetic mechanisms of quinolone resistance in Haemophilus influenzae. We obtained 29 H. influenzae strains from a nationwide surveillance program in Japan (including 11 quinolone-resistant strains [moxifloxacin: MFLX or levofloxacin MIC ≥2 µg/ml]). We analyzed the sequences of the Quinolone Resistance-Determining Regions (QRDRs) in GyrA, GyrB, ParC and ParE. Furthermore, we induced resistance in susceptible strains by exposing them to quinolone, and investigated the relationship between mutations in the QRDRs and the MICs. Five amino acid substitutions in GyrA (at Ser84 and Asp88) and ParC (at Gly82, Ser84 and Glu88) were found to be closely related to the MICs. The strains with a MFLX MIC of 0.125-1 and 2-4 µg/ml had one and two mutations, respectively. The strains with a MFLX MIC of ≥8 µg/ml had three or more mutations. The strains with induced resistance with MFLX MICs of 0.5-1 and ≥2 µg/ml also had one and two mutations, respectively. We confirmed that these five mutations strongly contribute to quinolone resistance and found that the degree of resistance is related to the number of the mutations. In addition, the three strains of 18 susceptible strains (16.7%) also had a single mutation. These strains may therefore be in the initial stage of quinolone resistance. Currently, the frequency of quinolone-resistant H. influenzae is still low. However, as has occurred with ß-lactams, an increase in quinolone use may lead to more quinolone-resistant strains.