Isolation Procedure for CP E. coli from Caeca Samples under Review Towards an Increased Sensitivity

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Structural characterization of borneol dehydrogenase from Pseudomonas sp. TCU-HL1

During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor-degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein-expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X-ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C-terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition.

TRANSGLYCOSYLATION ACTIVITY AND CHARACTERIZATION OF RECOMBINANT SUCROSE PHOSPHORYLASE FROM LEUCONOSTOC MESENTEROIDES MBFWRS-3(1) EXPRESSED IN ESCHERICHIA COLI

Objective: Sucrose phosphorylase (SPase) is an enzyme that catalyzes the transfer of glucosyl to various acceptor molecules. Distinct types of SPaseshave been reported, and their transglycosylase activities have been shown to differ. In general, glycosylation is a process that is used to modifybioactive compounds. As such, glycosylation can increase the chemical stability of compounds and improve their characteristics such as reduce strongsmell and sour taste. We previously cloned recombinant SPase (SPaseWRS-3[1]) from Leuconostoc mesenteroides MBFWRS-3[1] in Escherichia coli.In the current study, we aimed to characterize SPaseWRS-3 and determine its transglycosylation activity using benzoic acid (BA), ascorbic acid, andkojic acid (KA).Methods: Expression analyses were conducted in lysogeny broth (LB) medium supplemented with tetracycline and expression was induced usingisopropyl-β-d-thiogalactopyranoside. The characteristics of the 56 kDa recombinant SPase (rec-SPase) were confirmed using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Rec-SPase activity was determined spectrophotometrically using sucrose as the substrate and NADPHas the end-product at 340 nm. Transglycosylation activity was evaluated using thin-layer chromatography (TLC) on silica gel plates.Results: Our results demonstrated that the rec-SPase had an activity of 98.52% relative to the reference SPase (ref-SPase). BA and KA were determinedto undergo glucosyl transfer by rec-SPase using ref-SPase, as observed with TLC. Our findings are consistent with those reported previously for theSPase isolated from L. mesenteroides.Conclusion: Recombinant SPase activity is comparable to reference SPase activity. Our study could be the initial study to deeply observe SPase activityin other substrates as well.

The Adaptive Morphology of Bacillus subtilis Biofilms: A Defense Mechanism against Bacterial Starvation

Biofilms are commonly defined as accumulations of microbes, embedded in a self-secreted, polysaccharide-rich extra-cellular matrix. This study aimed to characterize specific morphological changes that occur in Bacillus subtilis biofilms under nutrient-limiting growth conditions. Under varying levels of nutrient depletion, colony-type biofilms were found to exhibit different rates of spatial expansion and green fluorescent protein production. Specifically, colony-type biofilms grown on media with decreased lysogeny broth content exhibited increased spatial expansion and more stable GFP production over the entire growth period. By modeling the surface morphology of colony-type biofilms using confocal and multiphoton microscopy, we analyzed the appearance of distinctive folds or “wrinkles” that form as a result of lysogeny broth content reduction in the solid agar growth media. When subjected to varying nutritional conditions, the channel-like folds were shown to alter their morphology; growth on nutrient-depleted media was found to trigger the formation of large and straight wrinkles connecting the colony core to its periphery. To test a possible functional role of the formed channels, a fluorescent analogue of glucose was used to demonstrate preferential native uptake of the molecules into the channels’ interiors which supports their possible role in the transport of molecules throughout biofilm structures.

Simultaneous Quantification of L-arginine and Monosaccharides during Fermentation: An Advanced Chromatography Approach

Increasing demand for L-arginine by the food and pharmaceutical industries has sparked the search for sustainable ways of producing it. Microbial fermentation offers a suitable alternative; however, monitoring of arginine production and carbon source uptake during fermentation, requires simple and reliable quantitative methods compatible with the fermentation medium. Two methods for the simultaneous quantification of arginine and glucose or xylose are described here: high-performance anion-exchange chromatography coupled to integrated pulsed amperometric detection (HPAEC-IPAD) and reversed-phase ultra-high-performance liquid chromatography combined with charged aerosol detection (RP-UHPLC-CAD). Both were thoroughly validated in a lysogeny broth, a minimal medium, and a complex medium containing corn steep liquor. HPAEC-IPAD displayed an excellent specificity, accuracy, and precision for arginine, glucose, and xylose in minimal medium and lysogeny broth, whereas specificity and accuracy for arginine were somewhat lower in medium containing corn steep liquor. RP-UHPLC-CAD exhibited high accuracy and precision, and enabled successful monitoring of arginine and glucose or xylose in all media. The present study describes the first successful application of the above chromatographic methods for the determination and monitoring of L-arginine amounts during its fermentative production by a genetically modified Escherichia coli strain cultivated in various growth media.

Genome-wide mutational diversity inEscherichia colipopulation evolving in prolonged stationary-phase

Prolonged stationary-phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary-phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starvingE. colipopulations in lysogeny broth. A wide range of mutations accumulate over the course of one month in stationary-phase. SNPs constitute 64% of all mutations. A majority of these mutations are non-synonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency obtained by mutations in our experimental populations can not be explained by neutral drift. Moreover there is frequent genetic parallelism across populations suggesting that these mutations are under positive selection. Finally functional analysis of mutations suggests that regulatory mutations are frequent targets of selection.

Cranberry derivatives enhance biofilm formation and transiently impair swarming motility of the uropathogenProteus mirabilisHI4320

Proteus mirabilis is a major cause of catheter-associated urinary tract infection (CAUTI), emphasizing that novel strategies for targeting this bacterium are needed. Potential targets are P. mirabilis surface-associated swarming motility and the propensity of these bacteria to form biofilms that may lead to catheter blockage. We previously showed that the addition of cranberry powder (CP) to lysogeny broth (LB) medium resulted in impaired P. mirabilis swarming motility over short time periods (up to 16 h). Herein, we significantly expanded on those findings by exploring (i) the effects of cranberry derivatives on biofilm formation of P. mirabilis, (ii) whether swarming inhibition occurred transiently or over longer periods more relevant to real infections (∼3 days), (iii) whether swarming was also blocked by commercially available cranberry juices, (iv) whether CP or cranberry juices exhibited effects under natural urine conditions, and (v) the effects of cranberry on medium pH, which is an indirect indicator of urease activity. At short time scales (24 h), CP and commercially available pure cranberry juice impaired swarming motility and repelled actively swarming bacteria in LB medium. Over longer time periods more representative of infections (∼3 days), the capacity of the cranberry material to impair swarming diminished and bacteria would start to migrate across the surface, albeit by exhibiting a different motility phenotype to the regular “bull’s-eye” swarming phenotype of P. mirabilis. This bacterium did not swarm on urine agar or LB agar supplemented with urea, suggesting that any potential application of anti-swarming compounds may be better suited to settings external to the urine environment. Anti-swarming effects were confounded by the ability of cranberry products to enhance biofilm formation in both LB and urine conditions. These findings provide key insights into the long-term strategy of targeting P. mirabilis CAUTIs.