Molecular characteristics and genetic analysis of the genes related to carbapenemase, efflux pump, and outer membrane proteins in carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a life-threatening pathogen that has not been fully investigated on a molecular basis. Therefore, the molecular mechanisms of carbapenem resistance in CRKP collected from medical institutions in Hyogo Prefecture has been analyzed. Antimicrobial susceptibilities and the presence of carbapenemase along with epidemiological analyzes using multilocus sequence typing (MLST) have been investigated. The relative expression of efflux pump genes and mutations of ompK35 and ompK36, encoding the outer membrane porin, were also assessed for their relationship with carbapenem resistance. Most of the collected 22 CRKP isolates were non-susceptible to imipenem (68.2%), meropenem (90.9%), and ertapenem (81.8%), but all 22 strains were susceptible to colistin. Twelve strains (54.5%) were detected for carbapenemase genes such as blaIMP-6. Sequence type 37 was detected by MLST in 10 strains (45.5%). Non-carbapenemase-producing strains had high resistance rates for three carbapenems, and the main cause of resistance was ompK35 mutation. In conclusion, the main cause of resistance was imipenemase metallo-ß-lactamase (IMP-6) production in carbapenemase-producing strains, and ompK35 mutation in non-carbapenemase-producing strains. Susceptibility to carbapenem did not differ in CRKP regardless of carbapenemase production, except for imipenem susceptibility. This result contributes to a more insightful understanding of the mechanisms of CRKP in Japan.

Comparative genetic analysis of the antimicrobial susceptibilities and virulence of hypermucoviscous and non-hypermucoviscous ESBL-producing Klebsiella pneumoniae in Japan.

BACKGROUND: Hypermucoviscous (HMV) Klebsiella pneumoniae produces large amounts of capsular polysaccharides, leading to high mortality. Since extended spectrum beta-lactamase (ESBL)-producing HMV K. pneumoniae strains have increased in Japan, we investigated and compared the antimicrobial susceptibilities and genetic characteristics of HMV and non-HMV ESBL-producing K. pneumoniae. METHODS: We investigated 291 ESBL-producing K. pneumoniae collected between 2012 and 2018, and in them 54 HMV strains were identified and comparable 53 non-HMV strains were selected. Then, ESBL gene detection, plasmid replicon typing, and virulence gene detection were done by PCR amplification. RESULTS: Almost all of the HMV K. pneumoniae strains possessed uge (98.1%), wabG (96.3%), rmpA (94.4%), iucA (79.6%), fimH (70.4%), iroB (70.4%), and peg-344 (70.4%). These genes were found less frequently in non-HMV strains (uge 20.8%, wabG 83.0%, rmpA 7.5%, iucA 3.8%, fimH 9.4%, iroB 5.7%, and peg-344 1.9%). K2 capsule type (40.7%) was most common in HMV strains. HMV strains showed higher resistance to cefepime (p = 0.001) and piperacillin/tazobactam (p = 0.005) than non-HMV strains. CTX-M-15 (75.9%, 60.4%) was the dominant ESBL type in both HMV and non-HMV strains, and the most common plasmid replicon type was IncFII (52.1%) in CTX-M-15-producing strains. CONCLUSIONS: We found that HMV strains had more virulence genes and showed higher resistance to antibiotics than non-HMV strains. The most common capsule type was K2. CTX-M-15 was the most common type of ESBL gene in both HMV and non-HMV strains in Japan. The FII plasmid might be related to the spread of CTX-M-15 among K. pneumoniae strains.

Impact on quinolone resistance of plasmid-mediated quinolone resistance gene and mutations in quinolone resistance-determining regions in extended spectrum beta lactamase-producing Klebsiella pneumoniae isolated from urinary tract infection patients.

Klebsiella pneumoniae is a typical pathogen in urinary tract infections (UTI), and the emergence of extended spectrum beta-lactamase (ESBL)-producing strains has been frequently reported, accompanied by higher quinolone resistance rates. There are two major mechanisms of quinolone resistance, mutations in quinolone resistance-determining regions (QRDR) and the presence of the plasmid-mediated quinolone resistance (PMQR) genes. This study aimed to investigate quinolone resistance among 105 ESBL-producing K. pneumoniae specimens isolated from UTI patients in Indonesia. These were characterized for antimicrobial resistance to nalidixic acid, ciprofloxacin, and levofloxacin, QRDR mutations in gyrA and parC and the presence of PMQR genes. We found that 84.8% of the collected isolates were resistant to at least one of the quinolones. QRDR mutation in gyrA was observed in 49.5% of these strains and parC mutations in 61.0%. PMQR genes were identified in 84.8% of strains. The QRDR mutations clearly had a greater effect on resistance than the PMQR genes. In conclusion, we found high quinolone resistance rates in Indonesian ESBL-producing K. pneumoniae, in which QRDR mutation played a major role.

Genetic characteristics of azithromycin-resistant Neisseria gonorrhoeae collected in Hyogo, Japan during 2015-2019.

Introduction. Azithromycin (AZM) is a therapeutic drug for sexually transmitted infections and is used for Neisseria gonorrhoeae when first- and second-line drugs are not available. Recently, the susceptibility of N. gonorrhoeae against AZM has been decreasing worldwide.Hypothesis/Gap Statement. Azithromycin-resistance (AZM-R) rates among N. gonorrhoeae in Japan are increasing, and the gene mutations and epidemiological characteristics of AZM-R in N. gonorrhoeae have not been fully investigated.Aim. We determined the susceptibility to AZM and its correlation with genetic characteristics of N. gonorrhoeae.Methodology. We investigated the susceptibility to AZM and genetic characteristics of N. gonorrhoeae. Mutations in domain V of the 23S rRNA gene and mtrR were examined in 93 isolates, including 13 AZM-R isolates. Spread and clonality were examined using sequence types (STs) of multi-antigen sequence typing for N. gonorrhoeae (NG-MAST), and whole genome analysis (WGA) to identify single nucleotide polymorphisms.Results. The number of AZM-R isolates increased gradually from 2015 to 2019 in Hyogo (P=0.008). C2599T mutations in 23S rRNA significantly increased in AZM-R isolates (P<0.001). NG-MAST ST4207 and ST6762 were frequently detected in AZM-R isolates, and they had higher MICs to AZM from 6 to 24 µg/ml. The phylogenic tree-based WGA showed that all isolates with ST4207 were contained in the same clade, and isolates with ST6762 were divided into two clades, AZM-S isolates and AZM-R isolates, which were different from the cluster containing ST1407.Conclusion. Our study showed yearly increases in AZM-R rates in N. gonorrhoeae. NG-MAST ST4207 and ST6762 were not detected in our previous study in 2015 and were frequently identified in isolates with higher MICs to AZM. WGA confirmed that isolates with these STs are closely related to each other. Continued surveillance is needed to detect the emergence and confirm the spread of NG-MAST ST4207 and ST6762.

Correlation Between Changes in Syphilis Treatment and Jarisch-Herxheimer Reaction.

The number of syphilis patients has significantly increased recently in Japan and worldwide. Previous reports, even in large institutions, may not accurately reflect the current situation in urological clinics. We therefore collected data from 11 urological clinics in Hyogo Prefecture, Japan over a 2-year period subdivided into 1) August 2016 to July 2017 and 2) August 2017 to July 2018 to compare changes in syphilis consults. We analyzed the patient data including a rapid plasma reagin test (RPR), Treponema pallidum (TP) antibody, clinical stage, therapy, and presence of Jarisch-Herxheimer reaction. In total, 45 patients presented for a first consultation, 22 in the first year and 23 in the second year. Almost all patients were male. Initial consolidation and hard chancre were the major symptoms. RPR values and TP antibody values did not change. The treatment period with amoxicillin was significantly longer in the first year (p = 0.006). A Jarisch-Herxheimer reaction was seen in 13.6% in the first year and 60.9% in the second year (p = 0.001). The duration of antibiotic treatments was more likely to be based on the guidelines for antibiotic use in the second year, but Jarisch-Herxheimer reactions increased. Further follow-up including recurrent patients is necessary to draw definitive conclusions.

Comparison between antimicrobial stewardship program and intervention by infection control team for managing antibiotic use in neurogenic bladder-related urinary tract infection patients: A retrospective chart audit.

BACKGROUND: Antimicrobial prescriptions are relatively common in urologic outpatients. Therefore, it is necessary to investigate the impact of antimicrobial stewardship program (ASP) interventions. METHODS: In urology outpatients, antimicrobial use density (AUD), antimicrobial agent costs, isolation of urinary tract infection (UTI)-causing organisms and their antimicrobial susceptibilities were compared between intervention by infection control team (ICT) era (pre-2014) and ASP era (post-2014) in 2739 patients with lower urinary tract symptoms, including neurogenic bladder patients with UTI or suspected UTI, from 2011 to 2020. RESULTS: In the ASP, overall AUD (P<.001), cefotiam (CTM) (P=.0013), 2nd-generation cephalosporins (P=.026), cefdinir (CFDN) (P<.001), levofloxacin (LVFX) (P<.001), sitafloxacin (STFX) (P=.0016), and tosufloxacin (TFLX) (P=.0044) showed a significant decrease, but cefaclor (P=.019) showed a significant increase. Regarding antimicrobial agent costs, overall (P=.016), CTM (P=.021), 2nd-generation cephalosporins (P=.033), CFDN (P=.016), LVFX (P=.016), STFX (P=.033), and TFLX (P=.033) showed a significant decrease in the ASP. UTI-causing antimicrobial susceptibilities, CTM (P=.035), LVFX (P=.026) and sulfamethoxazole/trimethoprim (P=.048) in E. coli, and minocycline (P=.026) in K. pneumoniae showed a significant improve in the ASP. CONCLUSION: ASP contributed to decrease AUD and antimicrobial agent costs, and to improve antimicrobial susceptibilities of E. coli and K. pneumoniae to several antibiotics, compared to ICT. Further prospective studies are necessary for definitive conclusions.

Differential effects of chromosome and plasmid blaCTX-M-15 genes on antibiotic susceptibilities in extended-spectrum beta-lactamase-producing Escherichia coli isolates from patients with urinary tract infection.

OBJECTIVES: To compare antibiotic susceptibilities between chromosomal and plasmid blaCTX-M-15 locations in urinary tract infection-causing extended-spectrum ß-lactamases-producing Escherichia coli blaCTX-M-15 isolated in Indonesia. METHODS: A total of 84 strains identified as extended-spectrum ß-lactamases-producing E. coli were isolated from patients with urinary tract infection in Indonesia in 2015. Antimicrobial susceptibility tests were performed on these strains using 18 antibiotics, and extended-spectrum ß-lactamase bla genes were detected by polymerase chain reaction. Gene localization of blaCTX-M-15 -positive strains was confirmed by Southern blot hybridization, and epidemiological typing was conducted using multilocus sequence typing. RESULTS: Of 54 strains harboring the blaCTX-M-15 gene, 27 showed localization on chromosome, 20 on plasmid, and seven on chromosome and plasmid. Most multilocus sequence typing sequence types of the 27 strains with chromosomal blaCTX-M-15 were ST405 (25.9%) and ST131 (22.2%) strains, whereas the 20 strains with plasmid-blaCTX-M-15 were mostly ST410 (55.0%). CONCLUSIONS: Extended-spectrum ß-lactamases-producing E. coli blaCTX-M-15 with plasmid genes show significantly higher resistant rates against piperacillin-tazobactam but lower resistant rates against chloramphenicol compared to chromosomal strains in Indonesian patients with urinary tract infection. Mechanistic investigations will be necessary to advance our knowledge of antimicrobial resistance in urinary tract infection.

The Antimicrobial Resistance Characteristics of Imipenem-Non-Susceptible, Imipenemase-6-Producing Escherichia coli.

Imipenemase-6 (IMP-6) type carbapenemase-producing Enterobacteriaceae is regarded as dangerous due to its unique lack of antimicrobial susceptibility. It is resistant to meropenem (MEPM) but susceptible to imipenem (IPM). In addition to carbapenemase, outer membrane porins and efflux pumps also play roles in carbapenem resistance by reducing the antimicrobial concentration inside cells. Extended-spectrum ß-lactamase (ESBL) is transmitted with IMP-6 by the plasmid and broadens the spectrum of antimicrobial resistance. We collected 42 strains of IMP-6-producing Escherichia coli and conducted a molecular analysis of carbapenemase, ESBL, porin, efflux, and epidemiological characteristics using plasmid replicon typing. Among the 42 isolates, 21 strains were susceptible to IPM (50.0%) and 1 (2.4%) to MEPM. Seventeen strains (40.5%) co-produced CTX-M-2 type ESBL. We found that the relative expression of ompC and ompF significantly correlated with the MIC of IPM (p = 0.01 and p = 0.03, respectively). Sixty-eight% of CTX-M-2-non-producing strains had IncI1, which was significantly different from CTX-M-2-producing strains (p < 0.001). In conclusion, 50.0% of our IMP-6-producing strains were non-susceptible to IPM, which is different from the typical pattern and can be attributed to decreased porin expression. Further studies investigating other types of carbapenemase are warranted.