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f Carbapenem-resistant Acinetobacter baumannii (CRAb) shelter cohabiting carbapenem-susceptible bacteria from carbapenem killing via extracellular release of carbapenem-hydrolyzing class D ␤-lactamases, including OXA-58. However, the mechanism of the extracellular release of OXA-58 has not been elucidated. In silico analysis predicted OXA-58 to be translocated to the periplasm via the Sec system. Using cell fractionation and Western blotting, OXA-58 with the signal peptide and C terminus deleted was not detected in the periplasmic and extracellular fractions. Overexpression of enhanced green fluorescent protein fused to the OXA-58 signal peptide led to its periplasmic translocation but not extracellular release, suggesting that OXA-58 is selectively released. The majority of the extracellular OXA-58 was associated with outer membrane vesicles (OMVs). The OMV-associated OXA-58 was detected only in a strain overexpressing OXA-58. The presence of OXA-58 in OMVs was confirmed by a carbapenem inactivation bioassay, proteomic analysis, and transmission electron microscopy. Imipenem treatment increased OMV formation and caused cell lysis, resulting in an increase in the OMV-associated and OMV-independent release of extracellular OXA-58. OMV-independent OXA-58 hydrolyzed nitrocefin more rapidly than OMV-associated OXA-58 but was more susceptible to proteinase K degradation. Rose bengal, an SecA inhibitor, inhibited the periplasmic translocation and OMV-associated release of OXA-58 and abolished the sheltering effect of CRAb. This study demonstrated that the majority of the extracellular OXA-58 is selectively released via OMVs after Sec-dependent periplasmic translocation. Addition of imipenem increased both OMV-associated and OMV-independent OXA-58, which may have different biological roles. SecA inhibitor could abolish the carbapenem-sheltering effect of CRAb.A cinetobacter baumannii is a major cause of nosocomial infections worldwide. The rapid emergence of carbapenem-resistant isolates has severely reduced therapeutic options (1, 2). Recently, we demonstrated that carbapenem-resistant A. baumannii (CRAb) sheltered coexisting carbapenem-susceptible bacteria, preventing them from being killed by carbapenem and, thereby, leading to polymicrobial infections with enhanced pathogenicity compared to that of monomicrobial infection (3). This sheltering effect is clinically relevant because 20 to 50% of A. baumannii infections have been found to be polymicrobial (4-6).The primary mechanism of carbapenem resistance in A. baumannii is high-level production of carbapenemases, especially carbapenem-hydrolyzing class D ␤-lactamases (CHDLs), which include the OXA-23, -40, -51, -58, and -143 classes (7). We demonstrated that the extracellular release of CHDLs contributed to the sheltering effect (3), but this was seen only when CHDLs were expressed at high levels using a strong promoter. At the time of the earlier study, the mechanism for the extracellular release of CHDLs had not been elucidated.In this study, we determined that ex...

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Heat curing of edible whey protein isolate (WPI) films was studied as a means of improving mechanical and water vapor barrier properties. Curing temperature and relative humidity (RH) effects on the rate of change of maximum tensile stress (TS), elongation at break (E), Young's Modulus (Y m ), and water vapor permeability (WVP) were investigated. Cure time linearly affected TS and Y m , while influencing E and WVP exponentially. Increased cure temperature and reduced RH accelerated TS increase, E and Y m decrease, and increased improvement in WVP barrier properties. These data support the hypothesis that heat curing may elicit additional crosslinking of protein, yielding increased TS and decreased E and WVP; however, further work is required to confirm this hypothesis.

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