Isolation of Serratia marcescens mutants which could overproduce and excrete Escherichia coli alkaline phosphatase and beta-lactamase

Serratia marcescens mutants, which excrete Escherichia coli alkaline phosphatase (APase) encoded by the plasmid-bearing phoA gene, were isolated after mutagenesis by N-methyl--nitro-N-nitrosoguanidine. These mutants produced two to four times as much APase as did the parent strain under a phosphate-limiting condition, and more than 70% of the enzyme was released into the culture medium. In addition, overproduction and excretion of beta-lactamase was observed in these mutants.     

Utilization of vegetable oil in the production of clavulanic acid by <Emphasis Type="Italic">Streptomyces clavuligerus</Emphasis>ATCC 27064

Production of clavulanic acid (CA) by Streptomyces clavuligerus ATCC 27064 in shake-flask culture (28 °C, 250 rev min^–1) was evaluated, with media containing different types and concentrations of edible vegetable oil. Firstly, four media based on those reported in the literature were examined. The medium containing soybean oil and starch as carbon and energy source gave the best production results. This medium, with the starch replaced by glycerol, and with various soybean oil concentrations (16, 23 and 30 g l^–1) was utilized to further investigate CA production. Medium containing 23 g l^–1 led to the highest CA productivity (722 mg l^–1 in 120 h) and that one containing 30 g l^–1 gave the highest CA titre (753 mg l^–1 in 130 h). Also, substitution of corn and sunflower edible oils furnished similarly good results in terms of CA titre and productivity. It can be concluded that easily available vegetable oil is a very promising substrate for CA production, since it is converted slowly to glycerol and fatty acids, which are the main carbon and energy source for the microorganism.     

产β-内酰胺酶金黄色葡萄球菌的耐药分析

目的对产β-内酰胺酶金黄色葡萄球菌的标本分布和药物敏感试验进行分析,为临床使用抗生素提供依据。方法按照《全国临床检验操作规程》的要求接种．对分离出的220株金黄色葡萄球菌（Sta phylococcs aureus,Sau）,用头孢菌素显色法测定其β-内酰胺酶。结果220株金黄色葡萄球菌中产β-内酰胺酶171株,产酶率为77．7％,产酶株除对万古霉素、替考拉宁、利福平及复方新诺明敏感外,对其它的抗生素均呈现不同程度耐药,其中青霉素G、氨苄西林耐药率均为99．4％,红霉素为86．1％。结论产β-内酰胺酶株对青霉素类、大环内酯类抗生素呈现高度耐药,临床治疗不宜使用此类抗生素,提示临床选择药物时,应在药敏试验的指导下合理选择抗生素。     

Boronic acid inhibitors of the class A β-lactamase KPC-2

The rapid rise of antimicrobial resistance is one of the greatest challenges currently facing medical science. The most common cause of resistance to β-lactam antibiotics is the expression of β-lactamase enzymes, such as KPC-2. As such the development of novel inhibitors of KPC-2 and related enzymes is of the upmost importance. We report the design and synthesis of novel boronic acid transition state analogs containing a 1,4-substituted 1,2,3-triazole linker based on the known inhibitor 3-nitrophenyl boronic acid and demonstrate that they are promising scaffolds for the development inhibitors of KPC-2 with the ability to recover sensitivity to the antibiotic cefotaxime.     

Serendipitous discovery of aryl boronic acids as β-lactamase inhibitors

High throughput screening for β-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).     

Bis(benzoyl) phosphate inactivators of beta-lactamase C from Mtb

The class A β-lactamase BlaC is a cell surface expressed serine hydrolase of Mycobacterium tuberculosis (Mtb), one of the causative agents for Tuberculosis in humans. Mtb has demonstrated increased susceptibility to β-lactam antibiotics upon inactivation of BlaC; thus, making BlaC a rational enzyme target for therapeutic agents. Herein, we present the synthesis and structure-activity-relationship data for the 1st-generation library of bis(benzoyl) phosphates (1–10). Substituent effects ranged from σ_p = −0.27 to 0.78 for electronic and π = −0.41 to 1.98 for hydrophobic parameters. Compounds 1, 4 and 5 demonstrated the greatest inhibitory potency against BlaC in a time-dependent manner (k_obs = 0.212, 0.324, and 0.450 mn⁻¹ respectively). Combined crystal structure data and mass spectrometric analysis of a tryptic digest for BlaC inactivated with 4 provided evidence that the mechanism of inactivation by this bis(benzoyl) phosphate scaffold occurs via phosphorylation of the active-site Ser-70, ultimately leading to an aged form of the enzyme.     

A computational model of antibiotic-resistance mechanisms in methicillin-resistant Staphylococcus aureus (MRSA)

An agent-based model of bacteria-antibiotic interactions has been developed that incorporates the antibiotic-resistance mechanisms of Methicillin-Resistant Staphylococcus aureus (MRSA). The model, called the Micro-Gen Bacterial Simulator, uses information about the cell biology of bacteria to produce global information about population growth in different environmental conditions. It facilitates a detailed systems-level investigation of the dynamics involved in bacteria-antibiotic interactions and a means to relate this information to traditional high-level properties such as the Minimum Inhibitory Concentration (MIC) of an antibiotic. The two main resistance strategies against β-lactam antibiotics employed by MRSA were incorporated into the model: β-lactamase enzymes, which hydrolytically cleave antibiotic molecules, and penicillin-binding proteins (PBP2a) with reduced binding affinities for antibiotics. Initial tests with three common antibiotics (penicillin, ampicillin and cephalothin) indicate that the model can be used to generate quantitatively accurate predictions of MICs for antibiotics against different strains of MRSA from basic cellular and biochemical information. Furthermore, by varying key parameters in the model, the relative impact of different kinetic parameters associated with the two resistance mechanisms to β-lactam antibiotics on cell survival in the presence of antibiotics was investigated.     

212株产β-内酰胺酶葡萄球菌的菌种分布及耐药分析

目的对212株产β-内酰胺酶葡萄球菌的菌种分布和耐药性进行分析,为临床使用抗生素提供依据。方法用Phoenix-100全自动微生物鉴定/药敏系统鉴定分离到的葡萄球菌,并检测其产β-内酰胺酶情况及对20种药物的最低抑菌浓度。用WHONET5.4软件进行数据分析。结果 251株葡萄球菌中产β-内酰胺酶212株,产酶率为84.5%,以金黄色葡萄球菌131株(61.8%),表皮葡萄球菌32株(15.1%),溶血葡萄球菌30株(14.2%)为主。以上菌株除对万古霉素、利奈唑烷、替考拉宁、利福平、呋喃妥因、喹努普汀/达福普汀、甲氧苄胺嘧啶和莫匹罗星敏感外,对其他抗菌素均呈现不同程度耐药,其中青霉素、氨苄西林耐药率均为100%,红霉素为65.1%,苯唑西林53.3%,克林霉素为50.9%,四环素为47.6%。结论临床应积极进行病原学及耐药监测,并在药敏试验结果指导下合理选择抗生素;一旦出现耐甲氧西林金黄色葡萄球菌(MRSA),必须迅速采取控制措施,避免引起医院内感染的暴发流行。     

Thiophenyl oxime-derived phosphonates as nano-molar class C beta-lactamase inhibitors reducing MIC of imipenem against Pseudomonas aeruginosa and Acinetobacter baumannii

The preparation and characterization of a series of thiophenyl oxime phosphonate beta-lactamase inhibitors is described. A number of these analogs were potent and selective inhibitors of class C beta-lactamases from Pseudomonas aeruginosa and Enterobacter cloacae. Compounds 3b and 7 reduced the MIC of imipenem against an AmpC expressing strain of imipenem-resistant P. aeruginosa. A number of the title compounds retained micromolar potency against the class D OXA-40 beta-lactamase from Acinetobacter baumannii and at high concentrations compound 3b was shown to reduce the MIC of imipenem against a highly imipenem-resistant strain of A. baumanii expressing the OXA-40 beta-lactamase. In mice compound 3b exhibited phamacokinetics similar to imipenem.     

Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity

Antibody phage display provides a powerful and efficient tool for the discovery and development of monoclonal antibodies for therapeutic and other applications. Antibody clones from synthetic libraries with optimized design features have several distinct advantages that include high stability, high levels of expression, and ease of downstream optimization and engineering. In this study, a fully synthetic human scFv library with six diversified CDRs was constructed by polymerase chain reaction assembly of overlapping oligonucleotides. In order to maximize the functional diversity of the library, a β-lactamase selection strategy was employed in which the assembled scFv gene repertoire was fused to the 5′-end of the β-lactamase gene, and in-frame scFv clones were enriched by carbenicillin selection. A final library with an estimated total diversity of 7.6 × 10⁹, greater than 70% functional diversity, and diversification of all six CDRs was obtained after insertion of fully randomized CDR-H3 sequences into this proofread repertoire. The performance of the library was validated using a number of target antigens, against which multiple unique scFv sequences with dissociation constants in the nanomolar range were isolated.