In Vitro Derivation of Fluoroquinolone-Resistant Mutants from Multiple Lineages of Haemophilus influenzae and Identification of Mutations Associated with Fluoroquinolone Resistance.

Haemophilus influenzae is a pathogenic bacterium that causes respiratory and otolaryngological infections. The increasing prevalence of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) is a clinical concern. Fluoroquinolones are alternative agents to ß-lactams. However, the emergence and increasing prevalence of fluoroquinolone-resistant H. influenzae have been reported. The current risk of fluoroquinolone resistance in H. influenzae (especially in high-BLNAR) has not yet been evaluated. Here, we examined the development of fluoroquinolone resistance in fluoroquinolone-susceptible clinical H. influenzae isolates in vitro during passaging in the presence of moxifloxacin (from 0.03 to 128 mg/liter). Twenty-nine isolates were examined. Seventeen isolates (58.6%) showed reduced moxifloxacin susceptibility, and 10 of these 17 isolates (34.5% of all isolates) exceeded the Clinical and Laboratory Standards Institute breakpoint for moxifloxacin (MIC of >1 mg/liter) after repeat cultivation on moxifloxacin-containing agar. Seven of these ten isolates were high-BLNAR and represented multiple lineages. We identified 56 novel mutations in 45 genes induced during the development of fluoroquinolone resistance, except the defined quinolone resistance-determining regions (Ser84Leu and Asp88Tyr/Gly/Asn in GyrA and Gly82Asp, Ser84Arg, and Glu88Lys in ParC). Glu153Leu and ΔGlu606 in GyrA, Ser467Tyr and Glu469Asp in GyrB, and ompP2 mutations were novel mutations contributing to fluoroquinolone resistance in H. influenzae In conclusion, H. influenzae clinical isolates from multiple lineages can acquire fluoroquinolone resistance by multiple novel mutations. The higher rate of derivation of fluoroquinolone-resistant H. influenzae from high-BLNAR than ß-lactamase-negative ampicillin-susceptible isolates (P = 0.01) raises the possibility of the emergence and spread of fluoroquinolone-resistant high-BLNAR in the clinical setting.

Emergence of vancomycin- and teicoplanin-resistant Enterococcus faecium via vanD5-harbouring large genomic island.

BACKGROUND: Treatment of VRE is of clinical concern. While certain numbers of vanD-type VRE have been isolated, only two vanD5-harbouring Enterococcus faecium isolates have been reported in Canada and Japan. METHODS: We report the isolation of vanD5-type E. faecium and the first ever determination of the whole-genome sequence to investigate the possible mechanisms of the acquisition of the vanD5 gene cluster in E. faecium. RESULTS: Two vanD5-harbouring vancomycin-resistant E. faecium were isolated from the skin (SMVRE19) and faeces (SMVRE20) of a patient with a skin ulcer in Japan. The isolates exhibited vancomycin and teicoplanin MIC values of 128 mg/L, whilst the previous isolates of vanD5-harbouring E. faecium were only resistant to vancomycin. SMVRE19 and SMVRE20 were clones related to ST18, which is also seen in vanA- and vanB-type VRE. These isolates harboured an insertion element, ISEfm1, in the ddl gene, similar to a previously described teicoplanin-resistant vanD3-type E. faecium. The vanD5 gene cluster was integrated into the SMVRE20 chromosome as a part of a large genomic island (approximately 127 kb), similar to other recently spreading vanD variants in the Netherlands. The genomic island shared the greatest similarity with a part of the Blautia coccoides genome sequence, except for the region surrounding the vanD gene cluster. CONCLUSIONS: This study reports that emergence of vancomycin- and teicoplanin-resistant vanD5-type E. faecium occurred via acquisition of the vanD5 cluster and ISEfm1 insertion into ddl. Considering the genetic similarity between the various VRE strains, the current study should serve as a warning against the spread of vanD5-type VRE.

Clonality investigation of clinical Escherichia coli isolates by polymerase chain reaction-based open-reading frame typing method.

Escherichia coli (E. coli) causes urinary tract infections, pneumonia, surgical site infections, and bloodstream infections and is the important pathogen for both community-acquired and healthcare-associated infections. To investigate the clonality of E. coli is important for infection control and prevention. We aimed to investigate the clonality of clinical E. coli isolates using Cica Geneus E. coli polymerase chain reaction (PCR)-based open-reading frame typing (POT) KIT and clarify the clinical usefulness of this kit. About 124 E. coli isolates obtained from inpatients at Sapporo Medical University Hospital were used. The POT method was used to classify 124 clinical isolates into 87 POT numbers. In addition to the clonality, it was possible to obtain additional information that 20 of the 124 isolates were extended-spectrum ß-lactamase (ESBL) producing E. coli (5 isolates of CTX-M-1 group and 15 isolates of CTX-M-9 group) and 13 were sequence type (ST) 131 clone. Furthermore, when these ESBL-producing 20 isolates were compared with pulsed-field gel electrophoresis (PFGE) or multilocus sequence typing (MLST), Simpson's index of diversity was 0.968 in POT method, 0.979 in PFGE, and 0.584 in MLST. POT method had an analytical power similar to that of PFGE. In conclusion, attention should be paid to the difference in the interpretation of the results between the POT method and the PFGE, but POT method may be useful to timely monitor the spread of E. coli in medical facilities.

Prevalence and characterization of Clostridioides difficile isolates from retail food products (vegetables and meats) in Japan.

The present study aimed to elucidate the prevalence of Clostridioides difficile in Japanese retail food products. For this purpose, retail food samples (242 fresh vegetables and 266 retail meat samples: 89 chicken meat; 28 chicken liver; 200 pork meat; 24 pig liver; 127 beef meat) were collected from 14 supermarkets between 2015 and 2019. C. difficile was isolated from eight (3.3%) fresh vegetable, six (6.7%) chicken meat, one (3.6%) chicken liver, one (0.5%) pork meat, and two (1.6%) beef meat samples; it was not isolated from pig liver. Of these isolates, 35% were toxigenic. All isolates were typable by PCR ribotyping and were resolved into 12 PCR ribotypes. Among these isolates, ribotype 014, which is distributed worldwide including in Japanese clinical cases, was detected among vegetable isolates. Therefore, although the C. difficile contamination rate in Japanese retail foods was low, these sources can be contaminated and could transmit these bacteria to humans.

Multiclonal Expansion and High Prevalence of ß-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones.

ß-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were ß-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

Contribution of Novel Amino Acid Alterations in PmrA or PmrB to Colistin Resistance in mcr-Negative Escherichia coli Clinical Isolates, Including Major Multidrug-Resistant Lineages O25b:H4-ST131-H30Rx and Non-x.

Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance (mcr) gene-negative colistin-resistant Escherichia coli clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-H30Rx (isolates SRE34 and SRE44; MIC for colistin = 16 mg/liter), non-x (SME296; MIC = 8 mg/liter), and O18-ST416 (SME222; MIC = 4 mg/liter). In this study, we investigated the colistin resistance mechanism and identified novel amino acid substitutions or deletions in the PmrAB two-component system that activates eptA (encoding a phosphoethanolamine transferase) and arnT (encoding an undecaprenyl phosphate-alpha-4-amino-4-deoxy-l-arabinose arabinosyl transferase) in all colistin-resistant isolates. SRE34 possessed deletion Δ27-45 (LISVFWLWHESTEQIQLFE) in PmrB, SRE44 possessed substitution L105P in PmrA, and both SME222 and SME296 included substitution G206D in PmrB. Matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed that lipid A is modified with phosphoethanolamine in all four isolates. Deletion of pmrAB decreased colistin MICs to 0.5 mg/liter and lowered eptA and arnT expression. Chromosomal replacement of mutated pmrA or pmrB in colistin-susceptible O25b:H4-ST131 strain SME98 (colistin MIC = 0.5 mg/liter) increased the colistin MIC to that of the respective parent colistin-resistant isolate. In addition, SME98 mutants in which pmrAB was replaced with mutated pmrAB showed no significant differences in bacterial growth and competition culture from the parent strain, except for the mutant with L105P in PmrA, whose growth was significantly suppressed in the presence of the parent strain. In conclusion, some O25b:H4-ST131 strains appear to acquire colistin resistance via phosphoethanolamine modification of lipid A through amino acid changes in PmrAB, and the amino acid changes in PmrB do not influence bacterial growth.

Inoculum effect of high concentrations of methicillin-susceptible Staphylococcus aureus on the efficacy of cefazolin and other beta-lactams.

The existence of a cefazolin inoculum effect (InE) of methicillin-susceptible Staphylococcus aureus (MSSA), which is speculated to be a reason for cefazolin treatment failure in MSSA infections, is controversial. In Japan, although cefazolin is one of the therapeutic choices for patients with MSSA infection, there are few reports of this effect. Additionally, the association between InE and blaZ type in beta-lactams other than cefazolin has not been well documented. In this study, we confirmed an MSSA InE in several beta-lactams, including cefazolin, and its relationship with blaZ, using 52 MSSA isolates from blood cultures. Three isolates (5.8%) that possessed type A blaZ showed a pronounced cefazolin InE. Five isolates (9.6%) showed pronounced InE with sulbactam/ampicillin; four isolates had type C blaZ and one had type A blaZ. However, we confirmed InE in MSSA isolates with blaZ not only type A and C but also B and D. For cefotaxime, ceftriaxone, imipenem, and meropenem, regardless of the presence of blaZ, we did not observe a significant increase in MICs at a high inoculum of MSSA. Hence, our results suggest that the above four beta-lactams are good alternatives to cefazolin if InE leads to treatment failure in a patient.

Tigecycline Nonsusceptibility Occurs Exclusively in Fluoroquinolone-Resistant Escherichia coli Clinical Isolates, Including the Major Multidrug-Resistant Lineages O25b:H4-ST131-H30R and O1-ST648.

Tigecycline (TGC) is a last-line drug for multidrug-resistant Enterobacteriaceae We investigated the mechanism(s) underlying TGC nonsusceptibility (TGC resistant/intermediate) in Escherichia coli clinical isolates. The MIC of TGC was determined for 277 fluoroquinolone-susceptible isolates (ciprofloxacin [CIP] MIC, <0.125 mg/liter) and 194 fluoroquinolone-resistant isolates (CIP MIC, >2 mg/liter). The MIC50 and MIC90 for TGC in fluoroquinolone-resistant isolates were 2-fold higher than those in fluoroquinolone-susceptible isolates (MIC50, 0.5 mg/liter versus 0.25 mg/liter; MIC90, 1 mg/liter versus 0.5 mg/liter, respectively). Two fluoroquinolone-resistant isolates (O25b:H4-ST131-H30R and O125:H37-ST48) were TGC resistant (MICs of 4 and 16 mg/liter, respectively), and four other isolates of O25b:H4-ST131-H30R and an isolate of O1-ST648 showed an intermediate interpretation (MIC, 2 mg/liter). No TGC-resistant/intermediate strains were found among the fluoroquinolone-susceptible isolates. The TGC-resistant/intermediate isolates expressed higher levels of acrA and acrB and had lower intracellular TGC concentrations than susceptible isolates, and they possessed mutations in acrR and/or marR The MICs of acrAB-deficient mutants were markedly lower (0.25 mg/liter) than those of the parental strain. After continuous stepwise exposure to CIP in vitro, six of eight TGC-susceptible isolates had reduced TGC susceptibility. Two of them acquired TGC resistance (TGC MIC, 4 mg/liter) and exhibited expression of acrA and acrB and mutations in acrR and/or marR In conclusion, a population of fluoroquinolone-resistant E. coli isolates, including major extraintestinal pathogenic lineages O25b:H4-ST131-H30R and O1-ST648, showed reduced susceptibility to TGC due to overexpression of the efflux pump AcrAB-TolC, leading to decreased intracellular concentrations of the antibiotics that may be associated with the development of fluoroquinolone resistance.

The efficacy of faropenem for patients with acute cystitis caused by extended spectrum ß-lactamase producing Escherichia coli.

The number of patients with acute cystitis caused by extended spectrum ß lactamase (ESBL)-producing Escherichia coli (E. coli) is increasing gradually. Although it is reported that ESBL-producing E. coli are sensitive to faropenem (FRPM), there are few clinical studies on the efficiency of FRPM against acute cystitis caused by the bacteria. Therefore, we retrospectively reviewed the medical charts of patients with acute cystitis caused by ESBL-producing E. coli who were treated with the oral antimicrobial agent faropenem (FRPM) in our institution from June 2011 to May 2015. Ten patients with acute cystitis caused by ESBL producing E. coli were treated with FRPM. Although clinical cure was achieved in 9 of them, it reoccurred in 3. This study revealed that the treatment regimen with FRPM for patients with acute cystitis caused by ESBL-producing E. coli is promising. However, a non-negligible number of recurrences were caused by ESBL-producing E. coli because of the nature of underlying diseases or pathologies in the urinary tract.

[Four Cases of Bacteremia Caused by Helicobacter cinaedi in the Urological Ward at About the Same Time].

Here we report the outbreak of bacteremia caused by Helicobacter cinaedi (H. cinaedi) in the urology ward. Case 1 was a man in his seventies with prostate cancer. Bacteremia caused by H. cinaedi developed 6 days after robot-assisted radical prostatectomy. Case 2 was a man in his sixties with small cell carcinoma of the prostate. Bacteremia developed at 5 days of docetaxel therapy. Case 3 was a man in his fifties with left renal pelvis carcinoma. Bacteremia developed 3 days after laparoscopic left nephroureterectomy. Case 4 was a man in his seventies with right renal pelvic carcinoma and bladder cancer. Bacteremia developed 22 days after laparoscopic right nephroureterectomy and laparoscopic radical cystectomy. Each bacteremia occurred almost simultaneously. Fortunately, all 4 cases were treated by antibiotics successfully and there were no cases of recurrence. Whole environmental inspection of the ward did not reveal H. cinaedi. However, multilocus sequence typing proved the strains in cases 3 and 4 to be the same. Therefore, cross-infection was suspected. H. cinaedi can develop to a pathogen of immunocompromised infection. This report clarified that this pathogen can cause bacteremia in the urology ward.

Identification of a bacteriolysis-associated virulence factor against lung epithelial cells in Pseudomonas aeruginosa PAO-1 cell lysate.

The precise identities of the virulence factors of Pseudomonas aeruginosa after bacteriolysis are still unknown. In the present study, we identified PA0423 protein, which was isolated from the Pseudomonas PAO-1 strain, as the factor responsible for cytotoxicity in lung epithelial cells. Whole bacterial cell lysate of P. aeruginosa PAO-1 caused cytotoxicity in A549 lung epithelial cells. This cytotoxic factor could be partially purified via gel-filtration and anion-exchange column chromatography, and its activity was attenuated by proteinase K treatment. The cytotoxic fraction increased caspase-3/7 activity in A549 cells, suggesting the induction of apoptosis. This fraction was then subjected to amino-acid sequence analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, resulting in the identification of 7 matches, 4 of which were with known proteins (PA0122, PA2687, PA3406, and PA0423). Deletion mutant analysis of these 7 candidates revealed that only the PA0423 mutation led to reduced cytotoxicity, indicating that this protein is the virulence factor. Furthermore, PA0423 recombinant protein was constructed, purified, and refolded. Transduction of recombinant PA0423, but not PA0122, into A549 cells engendered a dose-dependent cytotoxic effect. These results show the first evidence that specific bacteriolysis-induced virulence factor PA0423 from Pseudomonas is toxic to lung epithelial cells.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2012].

The nationwide surveillance of antibacterial susceptibility to meropenem (MEPM) and other parenteral antibiotics against clinical isolates during 2012 in Japan was conducted. A total of 2985 strains including 955 strains of Gram-positive bacteria, 1782 strains of Gram-negative bacteria, and 248 strains of anaerobic bacteria obtained from 31 medical institutions were examined. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). 2. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous studies in 2009 or 2006. Therefore, the tendency to increase in antimicrobial resistance rates was not observed. 3. MEPM resistance against Pseudomonas aeruginosa was 17.8% (56/315 strains). Compared to our previous results, it was the lowest than that in 2006 and 2009. 4. Carbapenem-resistant Klebsiella pneumoniae, and multi-drug-resistant Acinetobacter species, which emerged in worldwide, were not observed. 5. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 6.2% (59/951 strains) in enterobacteriaceae, which increased compared with that of our previous studies in 2009 or before. Whereas, the proportion of metallo-beta-lactamase strains was 1.6% (5/315 strains) in P. aeruginosa, which was stable. In conclusion, the results from this surveillance suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 17 years passed after available for commercial use in Japan.

Optimal left ventricular diameter measurement in subjects with sigmoid septum: comparison with three-dimensional left ventricular volume.

BACKGROUND: The purpose of this study was to determine the most appropriate measurement of left ventricular (LV) end-diastolic diameter for subjects with the sigmoid septum (SS) by measuring the LV end-diastolic diameter at the base and mid-ventricle and by examining the relationship between these measurements and the three-dimensional (3D) echocardiographic LV end-diastolic volume. METHODS: In 91 patients who underwent echocardiography for screening cardiovascular abnormalities, the aorto-septal angle (ASA) was measured as an index of the sigmoid septum. LV end-diastolic diameter was measured at the base and mid-ventricular level (DDbase and DDmid, respectively), and their average value was calculated (DDavg). By using 3D echocardiography, LV end-diastolic volume (EDV3D) was measured. RESULTS: Among 91 patients, 48 patients had narrow ASA (< 120 degrees) and were divided into the sigmoid septum (SS) group, and the remaining 43 patients were divided into the non-SS group. In the SS group, all DDbase, DDmid, and DDavg were significantly correlated with EDV3D (r = 0.59, 0.80, and 0.76, respectively), and the correlation coefficient between DDbase and EDV3D was significantly lower than that between DDmid and EDV3D (p < 0.01). On the other hand, in the non-SS group, all DDbase, DDmid, and DDavg were significantly correlated with EDV3D (r = 0.77, 0.85, and 0.84, respectively), and the correlation coefficient between DDbase and EDV3D was statistically comparable to that between DDmid and EDV3D (p = 0.12). ASA was significantly correlated with the difference of DDmid minus DDbase (r = - 0.71, p < 0.001). CONCLUSIONS: In patients with SS, DDmid and DDavg were well reflected the 3D echocardiographic LV end-diastolic volume.

[Nationwide surveillance of parenteral antibiotics containing meropenem activities against clinically isolated strains in 2009].

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 2655 strains including 810 strains of Gram-positive bacteria, 1635 strains of Gram-negative bacteria, and 210 strains of anaerobic bacteria obtained from 30 medical institutions during 2009 was examined. The results were as follows; (1) MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multidrug resistant strains including methicillin-resistant Staphylococcus aureus (MRSA). (2) MEPM maintained potent and stable antibacterial activity against Pseudomonas aeruginosa. The proportion of MEPM-resistant strains to ciprofloxacin-resistant strains or imipenem-resistant strains were 53.1% and 58.0% respectively. (3) The proportion of extended-spectrum beta-lactamase (ESBL) strains was 3.1% (26 strains) in enterobacteriaceae. And the proportion of metallo-beta-lactamase strains was 2.0% (6 strains) in P. aeruginosa. (4) Of all species tested, there were no species except for Bacteroides fragilis group, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem for serious infections treatment at present, 14 years passed after available for commercial use in Japan.

Prevalence of Various Vaccine Candidate Proteins in Clinical Isolates of Streptococcus pneumoniae: Characterization of the Novel Pht Fusion Proteins PhtA/B and PhtA/D.

Pneumococcal proteins unrelated to serotypes are considered to be candidates of antigens in next-generation vaccines. In the present study, the prevalence of vaccine candidate protein genes, along with serotypes and antimicrobial resistance determinants, was investigated in a total of 57 isolates obtained from a tertiary care hospital in Japan. All of the pediatric isolates and 76.6% of the adult isolates did not belong to PCV13 (a 13-valent pneumococcal conjugate vaccine) serotypes, and 70.2% of all isolates showed multidrug resistance. All of the isolates had ply, pavA, nanA, and nanB, and high prevalence was noted for the pspA and pspC genes (96.5% and 78.9%, respectively). Detection rates for the pneumococcal histidine triad protein (Pht) genes phtA, phtB, phtD, and phtE were 49.1%, 26.3%, 61.4%, and 100%, respectively. Two fusion-type genes, phtA/B and phtA/D, were identified, with a prevalence of 36.9% and 14.0%, respectively. These fusion types showed 78.1-90.0% nucleotide sequence identity with phtA, phtB, and phtD. The most prevalent pht profile was phtA + phtD + phtE (26.3%), followed by phtA/B + phtE (19.3%) and phtA/B + phtD + phtE (17.5%), while pht profiles including phtD and/or phtA/phtD were found in 71.9% of isolates. The present study revealed the presence of two fusion types of Pht and their unexpectedly high prevalence. These fusion types, as well as PhtA and PhtB, contained sequences similar to the B cell epitopes that have been previously reported for PhtD.

Clonal Diversity and Genetic Characteristics of Methicillin-Resistant Staphylococcus aureus Isolates from a Tertiary Care Hospital in Japan.

Molecular epidemiological characteristics were investigated for 1,041 isolates of methicillin-resistant Staphylococcus aureus (MRSA) collected in a tertiary care hospital in northern Japan for a 4-year period (2011-2014). Genotypes (staphylococcal cassette chromosome mec [SCCmec], sequence type, spa, coa, etc.) and the presence of drug resistance/virulence factor genes in the isolates were analyzed by multiplex/uniplex PCR, and PCR-direct sequencing as needed. Among these MRSA, predominant SCCmec type was IIa (87.2%), followed by IV (10.1%) and V (1.2%). The SCCmec IIa-MRSA belonged to coagulase genotype (coa) IIa and ST5/ST764, which are known as major health care-associated-MRSA (HA-MRSA) in Japan (New York/Japan clone) and its variant. Panton-Valentine leucocidine (PVL) genes were detected in only five isolates (0.5%) with genotypes ST8-SCCmec IVa/spa-t008/coa-IIIa (USA300 clone), ST6-SCCmec IVb, and ST59-SCCmec V (Taiwanese clone). Arginine catabolic mobile element (ACME) type I and II' were identified in three and five isolates of ST8-SCCmec IVa and ST764-SCCmec IIa MRSA, respectively. PVL-/ACME- isolates were classified into various STs/clonal complexes (CCs), with CC1, CC5, CC8, CC89, and CC121 being common. It was notable that SCCmec IVl was the most common among SCCmec IV subtypes, and was carried by almost half of coa-IIIa isolates (47%, 34/72) without PVL genes, which represented the novel ST8 MRSA clone spreading in Japan (i.e., "ST8/CA-MRSA/J"). Uncommon MRSA clones in Japan, ST72-SCCmec IV (South Korean clone), ST398 livestock-associated clone, and ST20 bovine-associated MRSA, were identified. Furthermore, we isolated PVL-negative ST8-SCCmec I/coa-IIIa and ST81-SCCmec V/coa-VIIa MRSA, which were considered presumptive novel clones. The present study revealed the genetic diversity of HA-MRSA, including potentially emerging clones of putative different origins.

Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles.

Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with ß-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

Release of large amounts of lipopolysaccharides from Pseudomonas aeruginosa cells reduces their susceptibility to colistin.

Pseudomonas aeruginosa is an important etiological agent of opportunistic infections. Injectable colistin is available as a last-line treatment option for multidrug-resistant P. aeruginosa infections. When cells were inoculated at a high number, colistin-susceptible P. aeruginosa grew on agar medium containing colistin at a concentration 10-fold higher than the minimum inhibitory concentration without acquiring colistin resistance. This study examined the responsible mechanism for growth in the presence of a high concentration of colistin. Cell wash fluid derived from P. aeruginosa efficiently reduced colistin antimicrobial activity. This reduction was mediated by lipopolysaccharide (LPS) in the wash fluid. Extracellular LPS inhibited colistin activity more effectively than cell-bound LPS in fixed cells. Cell wash fluids from Escherichia coli and Acinetobacter baumannii also reduced colistin activity; however, they were less potent than those from P. aeruginosa. The amount of LPS in cell wash fluid from P. aeruginosa was approximately 10-fold higher than that in fluid from E. coli or A. baumannii. In conclusion, cell-free LPS derived from bacterial cells inhibited the antimicrobial activity of colistin, and this effect was greatest for P. aeruginosa. Thus, large amounts of broken and dead cells of P. aeruginosa at infection foci will reduce the effectiveness of colistin, even against cells that have not yet acquired resistance.

Contribution of Type II Topoisomerase Mutations to Fluoroquinolone Resistance in Enterococcus faecium from Japanese Clinical Setting.

High-level fluoroquinolone resistance is conferred by the mutation of conserved serine and acidic amino acids in the quinolone resistance-determining region (QRDR) of the A subunits of the type II topoisomerases, DNA gyrase (GyrA) and topoisomerase IV (ParC). In Japan, fluoroquinolone-resistant Enterococcus faecium continues to emerge in clinical settings. We analyzed 131 Japanese E. faecium clinical isolates for susceptibility to levofloxacin (LVFX), and QRDR mutational status. The bacterial collection had a high percentage of resistance (79%) and showed elevated drug minimal inhibitory concentrations (MICs). Eighty-three isolates had single or combined mutations in gyrA and/or parC; all were resistant to LVFX. A strong correlation was evident between log-transformed MICs and the total number of QRDR mutations (r = 0.7899), confirming the involvement of QRDR mutations in drug resistance, as previously described. Three-dimensional modeling indicated that the amino acid change(s) in QRDR could disrupt the interaction between the enzymes and drugs: the most common cause of quinolone resistance. Interestingly, eight isolates had a single mutation on gyrA and exhibited significantly reduced susceptibility. These data imply that either DNA gyrase or topoisomerase IV can be the primary target of fluoroquinolones, although topoisomerase IV is commonly thought to be the primary target in gram-positive bacteria.