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c Fifty-seven carbapenem-resistant Klebsiella pneumoniae isolates belonging to ST11 (50 isolates), ST423 (5 isolates), and two other sequence types were studied. All were positive for bla KPC-2 , bla TEM-1 , and bla CTX-M-14 . SDS-PAGE analysis of six representative isolates demonstrated varied porin expression. Nevertheless, when bla KPC-2 was deleted, carbapenem resistance was markedly reduced. Additionally, SHV-12, DHA-1, and/or VIM-1 appeared to contribute to accessory carbapenemase activity. In contrast, OmpK35 and/or OmpK36 deficiency seemed to serve only as a minor cooperative factor. Table S1 in the supplemental material for primer details). Standard multilocus sequence typing (MLST) protocols were utilized, with alternative primers for gapA, mdh, rpoB, and tonB used as required. Fifty isolates belonged to ST11, five belonged to ST423, and one each belonged to ST65 and a novel MLST type, ST977. The first carbapenem-resistant isolate, derived from a urine specimen obtained in August 2006, belonged to ST423. However, only four other ST423 isolates were detected over the study period. In contrast, ST11 remained endemic throughout this period. Intriguingly, isolates belonging to ST65 and ST977 appeared only once. Except for XJ-2 and XJ-4, which exhibited lower carbapenem MICs, all 57 isolates showed high-level resistance to ampicillin, cefotaxime, ceftazidime, imipenem, meropenem, and ertapenem. PCR analysis suggested that all but two isolates produced TEM-1, KPC-2, CTX-M-14, and SHV-12; bla was not detected in XJ-1 and XJ-4. In addition, all five ST423 isolates encoded DHA-1, and the XJ-5 (ST11) isolate uniquely produced VIM-1. CIsolates XJ-1, XJ-2, and XJ-3, representative of ST977, ST65, and ST423, respectively, were chosen for further analysis together with three ST11 isolates: XJ-4 because it had relatively low carbapenem MICs, XJ-5 because it had a supplementary bla VIM-1 gene, and XJ-6 because it was typical of most ST11 isolates. SDS-PAGE analysis of outer membrane proteins extracted as described previously (10) from cells grown overnight with shaking at 37°C in nutrient broth with or without 10 g/liter NaCl showed that XJ-1 expressed smaller quantities of OmpK36, while OmpK35 production was not detected for XJ-1, XJ-4, and XJ-5 (Fig. 1). Furthermore, as the likely OmpK36 protein bands of XJ-3 and XJ-6 were shifted upwards (see below for details), the status of OmpK35 bands in these strains could not be determined (Fig. 1).Although DNA mutations relative to that of NTUH-K2044 were detected in the ompK35 sequences of XJ-4, XJ-5, and XJ-6, no amino acid changes were predicted (Fig. 2). In contrast, the predicted OmpK35 of XJ-1 exhibited 25 amino acid substitutions and a single insertion, while the corresponding sequence of XJ-2 varied by a single amino acid substitution only. The OmpK35 protein of XJ-3 lacked a five-amino-acid string (EIYNK), which mapped to the B1 ␤-sheet. This string of amino acids was strictly conserved in the OmpK35 sequences of the remaining five clinical isolates, NTUH-K2044, a...

Agarases are generally classified into glycoside hydrolase families 16, 50, and 86 and are found to degrade agarose to frequently generate neoagarobiose, neoagarotetraose, or neoagarohexaose as the main products. In this study we have cloned a novel endo-type ␤-agarase gene, agaB, from marine Pseudoalteromonas sp. CY24. The novel agarase encoded by agaB gene has no significant sequence similarity with any known proteins including all glycoside hydrolases. It degrades agarose to generate neoagarooctaose and neoagarodecaose as the main end products. Based on the analyses of enzymatic kinetics and degradation patterns of different oligosaccharides, the agarase AgaB appears to have a large substrate binding cleft that accommodates 12 sugar units, with 8 sugar units toward the reducing end spanning subsites ؉1 to ؉8 and 4 sugar units toward the non-reducing end spanning subsites ؊4 to ؊1, and enzymatic cleavage taking place between subsites ؊1 and ؉1. In addition, 1 H NMR analysis shows that this enzyme hydrolyzes the glycosidic bond with inversion of anomeric configuration, in contrast to other known agarases that are retaining. Altogether, AgaB is structurally and functionally different from other known agarases and appears to represent a new family of glycoside hydrolase.

Crystallization, as a solid−liquid separation process, is employed to purify and isolate a great diversity of crystalline pharmaceutical products. In recent years, continuous crystallization has attracted increasing attention because of the product and process robustness as well as higher productivity. In this work, we review the use of novel continuous crystallizers or modified conventional continuous crystallizers for the preparation of polymorphs, chiral enantiomers, solvates/hydrates, cocrystals, and spherical crystals. In addition, the theoretical framework and verification of the model-based control approaches are demonstrated. The application of process analytical technology tools in classical feedback loop control strategies in continuous crystallization is also discussed. Despite all this, the application of continuous crystallization still remains challenging because of the existence of drawbacks such as fouling and blockages. Therefore, a systematic discussion should be done before continuous crystallization is more widely applied.

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This study aimed to clone and characterize a plasmid-mediated fosA subtype gene, fosA5, in a clinical strain of ESBL-producing Escherichia coli, which confers fosfomycin resistance. Detection of the fosA5 gene clarified the mechanism of fosfomycin resistance in a strain that was negative for known fosfomycin resistance genes. Monitoring and surveillance will be important to follow the changes in fosfomycin resistance and prevent further dissemination of fos genes.

BACKGROUND The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach. RESULTS Here, we successfully constructed a homozygous strain (Pxα6‐KO) with a 2‐nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non‐homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3–TM4 loop region. When compared to the background strain IPP‐S, the knockout strain Pxα6‐KO exhibited 229‐ and 1462‐fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3‐fold) effects on the toxicities of imidacloprid, abamectin, β‐cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2‐nt deletion mutation of nAChRα6 in the Pxα6‐KO strain. CONCLUSION In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6‐KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry