Hydroxyhexylitaconic acids as potent IMP-type metallo-ß-lactamase inhibitors for controlling carbapenem resistance in Enterobacterales.

Metallo-ß-lactamases (MBLs) represent one of the main causes of carbapenem resistance in the order Enterobacterales. To combat MBL-producing carbapenem-resistant Enterobacterales, the development of MBL inhibitors can restore carbapenem efficacy for such resistant bacteria. Microbial natural products are a promising source of attractive seed compounds for the development of antimicrobial agents. Here, we report that hydroxyhexylitaconic acids (HHIAs) produced by a member of the genus Aspergillus can suppress carbapenem resistance conferred by MBLs, particularly IMP (imipenemase)-type MBLs. HHIAs were found to be competitive inhibitors with micromolar orders of magnitude against IMP-1 and showed weak inhibitory activity toward VIM-2, while no inhibitory activity against NDM-1 was observed despite the high dosage. The elongated methylene chains of HHIAs seem to play a crucial role in exerting inhibitory activity because itaconic acid, a structural analog without long methylene chains, did not show inhibitory activity against IMP-1. The addition of HHIAs restored meropenem and imipenem efficacy to satisfactory clinical levels against IMP-type MBL-producing Escherichia coli and Klebsiella pneumoniae clinical isolates. Unlike EDTA and Aspergillomarasmine A, HHIAs did not cause the loss of zinc ions from the active site, resulting in the structural instability of MBLs. X-ray crystallography and in silico docking simulation analyses revealed that two neighboring carboxylates of HHIAs coordinated with two zinc ions in the active sites of VIM-2 and IMP-1, which formed a key interaction observed in MBL inhibitors. Our results indicated that HHIAs are promising for initiating the design of potent inhibitors of IMP-type MBLs.IMPORTANCEThe number and type of metallo-ß-lactamase (MΒL) are increasing over time. Carbapenem resistance conferred by MΒL is a significant threat to our antibiotic regimen, and the development of MΒL inhibitors is urgently required to restore carbapenem efficacy. Microbial natural products have served as important sources for developing antimicrobial agents targeting pathogenic bacteria since the discovery of antibiotics in the mid-20th century. MΒL inhibitors derived from microbial natural products are still rare compared to those derived from chemical compound libraries. Hydroxyhexylitaconic acids (HHIAs) produced by members of the genus Aspergillus have potent inhibitory activity against clinically relevant IMP-type MBL. HHIAs may be good lead compounds for the development of MBL inhibitors applicable for controlling carbapenem resistance in IMP-type MBL-producing Enterobacterales.

Spread of seb-Positive Methicillin-Resistant Staphylococcus aureus SCCmec Type II-ST764 Among Elderly Japanese in Nonacute Care Settings.

We investigated the prevalence and molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) among 356 residents of nine long-term care facilities (LTCFs) in Japan during 2015 and 2017. In total, 800 specimens were tested and 39 MRSA isolates were recovered from 31 (8.71%) residents. PCR-based open reading frame typing (POT) and pulsed-field gel electrophoresis typing were performed for the 39 MRSA isolates; five of them showing identical pulsotypes, and POT scores were excluded in further analysis. Staphylococcal cassette chromosome mec (SCCmec) typing, multilocus sequence typing, and toxin gene detection were performed for one representative MRSA isolate per resident. Among the 34 unrelated MRSA isolates, 15 (44.1%) and 19 (55.9%) were of SCCmec types II and IV, respectively, and belonged to seven sequence types (STs). Among the 15 SCCmec II isolates, 11 (73.3%), 3, and 1 belonged to ST764 (clonal complex [CC]5), ST5 (CC5), and ST630 (CC8), respectively. Among the 19 SCCmec IV isolates, 13 (68.4%), 3, 2, and 1 belonged to ST1 (CC1), ST474 (CC1), ST8 (CC8), and ST380 (CC8), respectively. Among the 14 CC5 lineage-SCCmec II isolates, one ST5 isolate and 7 of the 11 ST764 isolates (63.6%) carried seb gene, and 14 (87.5%) of 16 CC1 lineage-SCCmec IV isolates had sea gene (p < 0.05). The results indicate that the seb-positive SCCmec type II-ST764 clone has spread in Japanese LTCF environments. As LTCF residents have multiple comorbidities and increased susceptibility to infections, it is necessary to monitor MRSA colonization in LTCFs through periodic screening to prevent dissemination.

Predominance of Serogroup 19 CC320/271 among Penicillin-Nonsusceptible Streptococcus pneumoniae Isolates after Introduction of the PCV7 Vaccine in Several Regions of Japan.

Multidrug-resistant Streptococcus pneumoniae serogroup 19, including serotypes 19A and 19F, associated with clonal complex 320/271 (CC320/271), has been previously shown to be predominant in many countries after introduction of a 7-valent pneumococcal conjugate vaccine (PCV7). However, in Japan there has been no epidemiological research focused on penicillin-nonsusceptible isolates after this event. Therefore, we aimed to characterize penicillin-nonsusceptible S. pneumoniae (PNSSP; penicillin minimum inhibitory concentration [MIC] ≥ 4.0 µg/ml) after the introduction of PCV7 in Japan. Throughout Japan, we collected 1,057 pneumococcal isolates from 2010 to 2014. We then evaluated MICs and performed serotyping, multilocus sequence typing, and sequencing of penicillin-binding protein genes in 51 isolates (penicillin MIC ≥ 2.0 µg/ml). Twenty-three isolates (2.2%) showed penicillin nonsusceptibility (penicillin MIC ≥ 4.0 µg/ml). Serotypes 19F (14 isolates, 60.9%) and 23F (4 isolates, 17.4%), which are covered by the vaccine, were predominant among PNSSP strains. Only 3 isolates belonged to nonvaccine serotype 19A. Among the PNSSP isolates, CC320/271 (16/23 strains, 69.6%) was the most prevalent clone. Moreover, CC320/271 clones showed high MIC values of a third-generation cephalosporin. Thus, we demonstrated clonal predominance of serogroup 19 CC320/271 with strong resistance to ß-lactams including a third-generation cephalosporin among PNSSP isolates.

In vitro reduction of antibacterial activity of tigecycline against multidrug-resistant Acinetobacter baumannii with host stress hormone norepinephrine.

The host stress hormone norepinephrine (NE), also called noradrenaline, is reported to augment bacterial growth and pathogenicity, but few studies have focused on the effect of NE on the activity of antimicrobials. The aim of this study was to clarify whether NE affects antimicrobial activity against multidrug-resistant Acinetobacter baumannii (MDR-AB). Time-kill studies of tigecycline (TIG) and colistin (COL) against MDR-AB as well as assays for factors contributing to antibiotic resistance were performed using MDR-AB clinical strains both in the presence and absence of 10 µM NE. In addition, expression of three efflux pump genes (adeB, adeJ and adeG) in the presence and absence of NE was analysed by quantitative reverse transcription PCR. Viable bacterial cell counts in TIG-supplemented medium containing NE were significantly increased compared with those in medium without NE. In contrast, NE had little influence on viable bacterial cell counts in the presence of COL. NE-supplemented medium resulted in an ca. 2 log increase in growth and in bacterial cell numbers adhering on polyurethane, silicone and polyvinylchloride surfaces. Amounts of biofilm in the presence of NE were ca. 3-fold higher than without NE. Expression of the adeG gene was upregulated 4-6-fold in the presence of NE. In conclusion, NE augmented factors contributing to antibiotic resistance and markedly reduced the in vitro antibacterial activity of TIG against MDR-AB. These findings suggest that NE treatment may contribute to the failure of TIG therapy in patients with MDR-AB infections.

Characterization of Piperacillin/Tazobactam-Resistant Klebsiella oxytoca Recovered from a Nosocomial Outbreak.

We characterized 12 clinical isolates of Klebsiella oxytoca with the extended-spectrum ß-lactamase (ESBL) phenotype (high minimum inhibitory concentration [MIC] values of ceftriaxone) recovered over 9 months at a university hospital in Japan. To determine the clonality of the isolates, we used pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and PCR analyses to detect blaRBI, which encodes the ß-lactamase RbiA, OXY-2-4 with overproduce-type promoter. Moreover, we performed the isoelectric focusing (IEF) of ß-lactamases, and the determination of the MICs of ß-lactams including piperacillin/tazobactam for 12 clinical isolates and E. coli HB101 with pKOB23, which contains blaRBI, by the agar dilution method. Finally, we performed the initial screening and phenotypic confirmatory tests for ESBLs. Each of the 12 clinical isolates had an identical PFGE pulsotype and MLST sequence type (ST9). All 12 clinical isolates harbored identical blaRBI. The IEF revealed that the clinical isolate produced only one ß-lactamase. E. coli HB101 (pKOB23) and all 12 isolates demonstrated equally resistance to piperacillin/tazobactam (MICs, >128 µg/ml). The phenotypic confirmatory test after the initial screening test for ESBLs can discriminate ß-lactamase RbiA-producing K. oxytoca from ß-lactamase CTX-M-producing K. oxytoca. Twelve clinical isolates of K. oxytoca, which were recovered from an outbreak at one university hospital, had identical genotypes and produced ß-lactamase RbiA that conferred resistance to piperacillin/tazobactam. In order to detect K. oxytoca isolates that produce RbiA to promote research concerning ß-lactamase RbiA-producing K. oxytoca, the phenotypic confirmatory test after the initial screening test for ESBLs would be useful.