Ser253Leu substitution in PmrB contributes to colistin resistance in clinical Acinetobacter nosocomialis.

Infections caused by extensively drug-resistant (XDR) Acinetobacter nosocomialis have become a challenging problem. The frequent use of colistin as the last resort drug for XDR bacteria has led to the emergence of colistin-resistant A. nosocomialis (ColRAN) in hospitals. The mechanism of colistin resistance in A. nosocomialis remains unclear. This study aimed to investigate the mechanisms underlying colistin resistance in clinical ColRAN isolates. We collected 36 A. nosocomialis isolates from clinical blood cultures, including 24 ColRAN and 12 colistin-susceptible A. nosocomialis (ColSAN). The 24 ColRAN isolates clustered with ST1272 (13), ST433 (eight), ST1275 (two), and ST410 (one) by multilocus sequence typing. There was a positive relationship between pmrCAB operon expression and colistin resistance. Further analysis showed that colistin resistance was related to an amino acid substitution, Ser253Leu in PmrB. By introducing a series of recombinant PmrB constructs into a PmrB knockout strain and protein structural model analyses, we demonstrated that the association between Ser253Leu and Leu244 in PmrB was coupled with colistin resistance in ColRAN. To the best of our knowledge, this is the first study demonstrating that the key amino acid Ser253Leu in PmrB is associated with overexpression of the pmrCAB operon and hence colistin resistance. This study provides insight into the mechanism of colistin resistance in A. nosocomialis.

Rapid typing of carbapenem-resistant Acinetobacter baumannii and Acinetobacter nosocomialis by multiplex Pan- and OXA-PCR assays.

Introduction. Outbreaks of carbapenem-resistant A. baumannii and A. nosocomialis have occurred worldwide in healthcare settings. Rapid and reliable molecular typing of bacterial isolates is vital for the effective surveillance of institutional outbreaks. The Pan-PCR and OXA-PCR assays are two multiplex PCR-based assays for the molecular typing of Acinetobacter species.Gap statement. However, few studies have investigated the discriminatory power of two multiplex PCR assays in in the genotyping of Acinetobacter species.Aim. We aimed to evaluate the efficacies of the Pan-PCR and OXA-PCR assays for molecular typing of A. baumannii and A. nosocomialis.Methodology. A total of 105 carbapenem-resistant A. baumannii isolates (CRABs) and 93 carbapenem-resistant A. nosocomialis isolates (CRANs) obtained from blood cultures were used for molecular typing by the Pan-PCR and OXA-PCR assays and two multilocus sequence typing (MLST) schemes.Results. The isolates were individually divided into 12 and 21 different sequence types via the Pasteur and Oxford MLST schemes, respectively. Additionally, these isolates were distinguished into 18 different types by the Pan-PCR and OXA-PCR assays. The results of the Pan-PCR and OXA-PCR assays distinguished CRABs and CRANs with a sensitivity of 98.13 % and a specificity of 100 %.Conclusion. The Pan-PCR and OXA-PCR assays are promising alternative methods for rapid molecular typing of CRABs and CRANs in a routine laboratory setting.

AdeABC Efflux Pump Controlled by AdeRS Two Component System Conferring Resistance to Tigecycline, Omadacycline and Eravacycline in Clinical Carbapenem Resistant Acinetobacter nosocomialis.

Carbapenem-resistant Acinetobacter nosocomialis (CRAn) is a significant public health concern. Tigecycline non-susceptible CRAn (Tn-CRAn) isolates have emerged worldwide. Tigecycline resistance is mainly related to the overexpression of AdeABC efflux pump controlled by AdeRS two-component system (TCS). Two novel tetracycline derivatives, omadacycline and eravacycline, may present a treatment option for CRAn. This study investigated the in vitro antimicrobial activity of tigecycline, omadacycline and eravacycline against clinical CRAn isolates and the contribution of efflux pumps in their resistance. Eighty-nine clinical CRAn isolates, including 57 Tn-CRAn isolates were evaluated for minimum inhibitory concentrations (MICs) by the broth microdilution. The relationship between the antimicrobial resistance and efflux pump expression was assessed by their responses to the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). The contribution of the AdeABC efflux pump in their resistance was determined by the complementation of the AdeRS two-component system in wild-type, adeRS operon and adeB gene knockout strains. Among the 89 isolates, omadacycline and eravacycline MICs were correlated closely with those of tigecycline. They demonstrated improved potency, based on MIC90 values, by showing a 4 to 8-fold greater potency than tigecycline. The synergetic effects of tigecycline, omadacycline and eravacycline with NMP were observed in 57 (100%), 13 (22.8%), and 51 (89.5%) of Tn-CRAn isolates, respectively. Further analysis showed that the laboratory strain carrying the Type 1 adeRS operon increased the tigecycline, omadacycline and eravacycline MICs by 4-8-folds, respectively. Eravacycline demonstrated improved potency over tigecycline against populations of CRAn, including Tn-CRAn isolates. The over-expression of AdeABC efflux pumps was directly activated by the AdeRS two-component system and simultaneously reduced the susceptibilities of tigecycline, eravacycline, and omadacycline. Omadacycline and eravacycline MICs were correlated closely with those of eravacycline.

Production and Characterization of Neutralizing Antibodies against Bungarus multicinctus Snake Venom.

The venom of the banded krait (Bungarus multicinctus), one of the major venomous species in Taiwan, contains neurotoxic venom proteins (B. multicinctus proteins) that pose a serious medical problem in tropical and subtropical countries. Even though horse-derived serum is an efficient therapy against snake venom, it is associated with a high cost and side effects. Therefore, developing a more cost-effective alternative treatment option is highly envisaged. In this study, chickens were immunized with B. multicinctus proteins, and polyclonal immunoglobulin Y (IgY) antibodies were purified from eggs. IgY showed a binding activity to B. multicinctus proteins that was similar to horse antivenin, and its titer in chickens lasted for at least 6 months. We constructed two antibody libraries by phage display antibody technology, which contain 1.0 × 107 and 2.9 × 108 transformants, respectively. After biopanning, a phage-based enzyme-linked immunosorbent assay (ELISA) indicated that specific clones were enriched. Thirty randomly selected clones expressing monoclonal single-chain variable-fragment (scFv) antibodies were classified into four groups with a short linker and two with a long linker. These selected scFv antibodies showed specific binding activities to B. multicinctus proteins but not to the venomous proteins of other snakes. Most importantly, polyclonal IgY demonstrated a similar neutralization efficiency as did horse-derived antivenin in mice that were injected with a minimum lethal dosage (MLD) of venom proteins. A mixture of several monoclonal anti-B. multicinctus scFv antibodies was also able to partially inhibit the lethal effect on mice. We profoundly believe that IgY and scFv antibodies can be applied in developing diagnostic agents for wound exudates and as an alternative treatment for snakebite envenomation in the future.IMPORTANCE Snake envenomation is one of the global medical issues of concern. Horse-derived antivenin is an effective way to treat snakebites, but it is costly and occasionally causes severe side effects. In this study, we first generated and characterized IgY antibodies with neutralization activity in chickens. Subsequently, we generated a panel of monoclonal scFv antibodies using phage display antibody technology. A mixture of scFv antibodies was able to partially inhibit the lethal effect in mice that were injected with lethal dosages of venom proteins and prolong their survival time. We believe that chicken-derived IgY and scFv antibodies have great potential for the development of diagnostic agents for wound exudates and therapeutic agents against snake envenomation in the future.