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Colistin is a last-line drug for multidrug-resistant Gram-negative bacteria. We previously reported four plasmid-mediated colistin resistance () gene-negative colistin-resistant clinical isolates, including the major pathogenic and fluoroquinolone-resistant strains O25b:H4-ST131-30Rx (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 (encoding a phosphoethanolamine transferase) and (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 decreased colistin MICs to 0.5 mg/liter and lowered and expression. Chromosomal replacement of mutated or 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 was replaced with mutated 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.

We investigated the contribution of quinolone resistance-determining region (QRDR) mutations to fluoroquinolone (enrofloxa-cin, orbifloxacin and danofloxacin) susceptibility in 58 Mycoplasma bovis isolates from dairy cattle in Japan. Fluoroquinolone non-resistant isolates (fluoroquinolone-MICs≤2 µg/ml) possessed no QRDR mutations (19 isolates) or Ser83Leu in GyrA (32 isolates). Fluoroquinolone-resistant isolates (fluoroquinolone-MICs≥4 µg/ml) possessed Ser83Leu in GyrA and Ser81Pro in ParC (3 isolates) or Ser83Phe in GyrA and Ser80Ile in ParC (4 isolates). Laboratory-derived fluoroquinolone-resistant mutants selected from 2 isolates (possessing Ser83Leu in GyrA) had an amino acid substitution in ParC at the same position (Ser80Ile or Ser81Tyr) as fluoroquinolone-resistant isolates, suggesting a concurrent amino acid substitution in ParC (Ser80 or Ser81) is important in fluoroquinolone resistance in M. bovis isolates. Mycoplasma bovis (Mb), a causative agent of bovine respiratory disease, causes high morbidity, and has an economic impact on the cattle industry [3]. Antimicrobials, such as fluoroquinolones, macrolides and tetracyclines, are important for treating Mb infections and preventing its spread in dairy calves. Fluoroquinolones are broad-spectrum anti-microbials, and enrofloxacin (ENR), orbifloxacin (ORB) and danofloxacin (DAN) have been approved for use in cattle in Japan. Fluoroquinolone minimum inhibitory concentrations (MICs) against Mb have previously been investigated [6, 8]. Mb isolated from cattle in Japan showed various ENR MICs (range≤0.125-4 µg/ml) [8]. Ten of eleven ENR-resistant (ENR MIC≥2 µg/ml) Mb isolates derived from cattle in Israel carried 2 missense mutations within quinolone resistance-determining regions (QRDRs), causing the amino acid substitutions (AASs) Ser83Phe in GyrA and Asp84Asn in ParC [6]. Although studies have suggested that QRDR mutations contribute to elevated ENR MICs, the association between QRDR mutations and fluoroquinolone MICs has yet to be confirmed in Mb [6]. We investigated QRDR mutations in Mb isolates and in vitro fluoroquinolone-selected mutants obtained from Mb isolates with various ENR, ORB and DAN MICs to clarify the effect of AAS in GyrA and ParC on fluoroquinolone resistance in Mb isolates from dairy calves in Japan. Fifty-eight Mb isolates were derived from nasal swabs of asymptomatic dairy calves from 51 farms located throughout Hokkaido during 2010-2011. These isolates were provided by 7 branches of the Hokkaido Agricultural Insurance Scheme (Dounan, Iburi-Hidaka, Ishikari-Sorachi, Kitami-Ohotsuku, Kushiro-Nemuro, Souya-Rumoi-Kamikawa and Tokachi) and were cultured in 1 ml NK broth (Kanto Chemical , Tokyo, Japan). After culture on modified Hayflick agar (35.5 g/l of PPLO, 15% equine serum, 2.5% yeast extract and 0.0024% deoxyribonucleic acid; Kanto Chemical) at 37°C in 5% CO 2 for 3 days to 1 week, a single colony was picked, inoculated into 1 ml of NK broth and further cultured at 37°C in 5% CO 2 for 3 days. ENR, ORB and DAN MICs were determined using ...

BackgroundMost patients with multiple myeloma (MM) are considered to be incurable, and relapse owing to minimal residual disease (MRD) is the main cause of death among these patients. Therefore, new technologies to assess deeper response are required.Patients and methodsWe retrospectively analyzed 125 patients with MM who underwent high-dose melphalan plus autologous stem-cell transplantation (ASCT) to detect MRD in autograft/bone marrow (BM) cells using a next-generation sequencing (NGS)-based method and allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR).ResultsNGS-based method was applicable to 90% and this method had at least one to two logs greater sensitivity compared to ASO-PCR. MRD negative by NGS [MRDNGS(−)] (defined as <10−6) in post-ASCT BM cases (n = 26) showed a significantly better progression-free survival (PFS) (96% at 4 years, P < 0.001) and overall survival (OS) (100% at 4 years, P =0.04) than MRDNGS(+) in post-ASCT BM cases (n = 25). When restricting the analysis to the 39 complete response cases, patients who were MRDNGS(−) (n = 24) showed a significantly better PFS than those that were MRDNGS(+) (n = 15) (P =0.02). Moreover, MRDNGS(−) in post-ASCT BM cases (n = 12) showed significantly a better PFS than MRDNGS(+) cases (n = 7) where MRD was not detected by ASO-PCR (P = 0.001). Patients whose autografts were negative by NGS-based MRD assessment (<10−7) (n = 19) had 92% PFS and 100% OS at 4 years post-ASCT. Conversely, the NGS-based MRD positive patients who received post-ASCT treatment using novel agents (n = 49) had a significantly better PFS (P = 0.001) and tended to have a better OS (P= 0.214) than those that were untreated (n = 33).ConclusionsLow level MRD detected by NGS-based platform but not ASO-PCR has significant prognostic value when assessing either the autograft product or BM cells post-ASCT.

Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.