Search

Research has been conducted on the versatile conditional-replication, integration and modular plasmids. Results indicate that these plasmids can be replicated at medium and high copy numbers in different hosts for creating gene or mutant libraries.

Escherichia coli reporter strains modeling the high-level type A and B vancomycin resistances of Enterococcus faecium BM4147 and Ent. faecalis have been developed to study the respective VanR-VanS two-component regulatory systems. [P.sub.vanH-], [P.sub.vanRa-], [P.sub.vanY-], and [P.sub.vanRb]-lacZ fusions report on expression from the vancomycin-resistant enterococci promoters of the type A vanRSHAXYZ and type B vanRSYWHBX gene clusters. These strains also express from single-copy chromosomal genes [vanR.sub.a], [vanR.sub.b], or [vanRS.sub.b] behind their respective promoter ([P.sub.vanRa] or [P.sub.vanRb]) or [vanS.sub.a] or [vanS.sub.b] behind the rhamnose-inducible [P.sub.rhaB]. Results show that activation (phosphorylation) of the response regulator [VanR.sub.a] by its sensor kinase [VanS.sub.a] leads to transcriptional activation of both [P.sub.vanH] and [P.sub.vanRa]. Additionally, [VanR.sub.b], activates its cognate promoters [P.sub.vanY] and [P.sub.vanRb], although this occurs only in the absence of [VanS.sub.b] and presumably is caused by [VanR.sub.b] phosphorylation by an unidentified endogenous E. coli kinase. Thus, [VanS.sub.b] interferes with activation of [VanR.sub.b] probably by acting as a phospho-[VanR.sub.b] phosphatase. Although both [VanR.sub.a] and [VanR.sub.b] activate their cognate promoters, neither activates the heterologous [P.sub.vanR], [P.sub.vanH], or [P.sub.vanY], arguing against the interchangeability of type A and B two-component regulatory switches in vancomycin-resistant enterococci. [VanR.sub.a] also is activated by the nonpartner kinase PhoR. Because this occurs in the absence of its inducing signal ([P.sub.i] limitation), PhoR autophosphorylation apparently is regulated in vivo. Furthermore, the activation of [VanR.sub.a] caused by cross talk from PhoR in the absence of a signal allows distinction of cross talk from cross-regulation as the latter, but not the former, responds to environmental cues.

Novel methods were used for constructing tightly regulated rhamnose promoter and arabinose fusions in researches involving Escherichia coli phosphate regulon. The approaches involved uncoupling phoR and phoB expression from the normal controls by putting them behind the rhamnose-controlled PrhaB or foreign, arabinose-controlled ParaB promoter. The results showed that the the strains exhibited almost normal Pi control of the Pho regulon after induction of the corresponding delta-phoR and delta-phoB mutant containing either rhamnose or arabinose.

The structure-based design of two mutant Escherichia coli purine repressor (PuR) proteins in which residue Arg190 is replaced by alanine or glutamine was examined using mutagenesis, analysis of in vivo function and in vitro corepressor binding measurements. Functional repressors in which the binding of guanine and hypoxanthine was retained resulted from the replacements of Arg190 with alanine or glutamine. These replacements also expanded the corepressor specificity of PuR to include adenine, guanine, hypoxanthine, as well as the R190A mutant 6-methylpurine.

A study of the regulation fo the Enterococcus faecium BM4147 vancomycin resistance gene cluster by the VanS-VanR two-component regulatory system in Escherichia coli was conducted. The research made use of appropriate strains for examining the in vivo activation of VanR by VanS, acetyl phosphate, nonpartner sensor kinase PhoR and mutant forms of VanS. Results indicated that the phospho-VanR is a transcriptional activator. Furthermore, VanS acts as a P-VanR phosphatase.

The DNA sequence of the gene encoding the early immunogenic protein P36 of Mycoplasma hyopneumoniae was described. It was shown that P36 isan L-lactate dehydrogenase. M. hyopneumoniae LDH, originally termed P36, is highly species-specific in its immunogenic reaction. The putative transcriptionpromoter of the M. hyopneumoniae ldh gene seems to be different from promoters found in other bacteria. Characteristic for the region upstream of the -10 boxes is a sequence rich in A + T.

Background: Diazabicyclooctanes (DBOs) are an increasingly important group of non β-lactam β-lactamase inhibitors, employed clinically in combinations such as ceftazidime/avibactam. The dose finding of such combinations is complicated using the traditional pharmacokinetic/pharmacodynamic (PK/PD) index approach, especially if the β-lactamase inhibitor has an antibiotic effect of its own.Objectives: To develop a novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model for ceftazidime/avibactam against Gram-negative pathogens, with the potential for combination dosage simulation.Methods: Four β-lactamase-producing Enterobacteriaceae, covering Ambler classes A, B and D, were exposed to ceftazidime and avibactam, alone and in combination, in static time-kill experiments. A PKPD model was developed and evaluated using internal and external evaluation, and combined with a population PK model and applied in dosage simulations.Results: The developed PKPD model included the effects of ceftazidime alone, avibactam alone and an 'enhancer' effect of avibactam on ceftazidime in addition to the β-lactamase inhibitory effect of avibactam. The model could describe an extensive external Pseudomonas aeruginosa data set with minor modifications to the enhancer effect, and the utility of the model for clinical dosage simulation was demonstrated by investigating the influence of the addition of avibactam.Conclusions: A novel mechanism-based PKPD model for the DBO/β-lactam combination ceftazidime/avibactam was developed that enables future comparison of the effect of avibactam with other DBO/β-lactam inhibitors in simulations, and may be an aid in translating PKPD results from in vitro to animals and humans. [ABSTRACT FROM AUTHOR]

Research reveals that a new enzymatic pathway involving three enzymes including ribose 1,5-bisphosphokinase, encoded by phnN gene, is required for the synthesis of 5-phospho-D-ribosyl alpha-1-diphosphate (PRPP). Furthermore, phnN gene product ribose 1,5-bisphosphokinase catalyzes phosphonae degradation. NAD biosynthesis requires PRPP in Escherichia coli.

Background: Diazabicyclooctanes (DBOs) are promising β-lactamase inhibitors. Some, including nacubactam (OP0595/RG6080), also bind PBP2 and have an enhancer effect, allowing activity against Enterobacteriaceae with MBLs, which DBOs do not inhibit. We tested the activity of nacubactam/β-lactam combinations against MBL-producing Enterobacteriaceae.Methods: Test panels comprised (i) 210 consecutive Enterobacteriaceae with NDM or VIM MBLs, as referred by UK diagnostic laboratories, and (ii) 99 supplementary MBL-producing Enterobacteriaceae, representing less prevalent phenotypes, species and enzymes. MICs were determined by CLSI agar dilution.Results: MICs of nacubactam alone were bimodal, clustering at 1-8 mg/L or >32 mg/L; >85% of values for Escherichia coli and Enterobacter spp. fell into the low MIC cluster, whereas Proteeae were universally resistant and the Klebsiella spp. were divided between the two groups. Depending on the prospective breakpoint (4 + 4 or 8 + 4 mg/L), and on whether all isolates were considered or solely the Consecutive Collection, meropenem/nacubactam and cefepime/nacubactam inhibited 80.3%-93.3% of MBL producers, with substantial gains over nacubactam alone. Against the most resistant isolates (comprising 57 organisms with MICs of nacubactam >32 mg/L, cefepime ≥128 mg/L and meropenem ≥128 mg/L), cefepime/nacubactam at 8 + 4 mg/L inhibited 63.2% and meropenem/nacubactam at 8 + 4 mg/L inhibited 43.9%. Aztreonam/nacubactam, incorporating an MBL-stable β-lactam partner, was almost universally active against the MBL producers and, unlike aztreonam/avibactam, had an enhancer effect.Conclusions: Nacubactam combinations, including those using MBL-labile β-lactams, e.g. meropenem and cefepime, can overcome most MBL-mediated resistance. This behaviour reflects nacubactam's direct antibacterial and enhancer activity. [ABSTRACT FROM AUTHOR]