Search Results (9)

Background: The rise of multi-drug-resistant Gram-negative bacteria necessitates the development of new antimicrobial agents. Cefiderocol shows promising activity by exploiting bacterial iron transport systems to penetrate the outer membranes of resistant pathogens. Objectives: This study evaluates the efficacy of cefiderocol testing methods and trailing effect impact using a ComASP^® Cefiderocol panel, disk diffusion (DD), and MIC test strips (MTS) compared to iron-depleted broth microdilution (ID-BMD). Methods: A total of 131 Gram-negative strains from clinical samples was tested by commercial methods and the gold standard. Results were interpreted as per 2024 and 2023 EUCAST guidelines. Results: ID-BMD revealed high cefiderocol susceptibility among Enterobacterales and Pseudomonas aeruginosa, with one Klebsiella pneumoniae isolate being resistant. Acinetobacter baumannii exhibited higher MIC values, particularly considering trailing effects that complicated MIC readings. ComASP^® showed 97% categorical agreement (CA) and 66% essential agreement (EA) with ID-BMD for Enterobacterales but failed to detect the resistant K. pneumoniae. DD tests demonstrated variable CA (72% or 93%), and 38% or 34% of strains within the ATU according to EUCAST Breakpoint Tables v13.0 and 14.0, respectively, with major errors only. MTS for P. aeruginosa had 100% CA but 44% EA, and often underestimated MIC values. Conclusions: The study emphasizes the need for standardized criteria to address trailing effects and ATU and highlights the discrepancies between testing methods. While cefiderocol resistance remains rare, accurate susceptibility testing is crucial for its effective clinical use. The findings suggest that current commercial tests have limitations, necessitating careful interpretation and potential supplementary testing to guide appropriate antibiotic therapy. Full article

Neutral pullulanases, having a good application prospect in trehalose production, showed a limited expression level. In order to address this issue, two approaches were utilized to enhance the yield of a new neutral pullulanase variant (PulA3E) in B. subtilis. One involved using multiple copies of genome integration to increase its expression level and fermentation stability. The other focused on enhancing the PulA-type atypical secretion pathway to further improve the secretory expression of PulA3E. Several strains with different numbers of genome integrations, ranging from one to four copies, were constructed. The four-copy genome integration strain PD showed the highest extracellular pullulanase activity. Additionally, the integration sites ytxE, ytrF, and trpP were selected based on their ability to enhance the PulA-type atypical secretion pathway. Furthermore, overexpressing the predicated regulatory genes comEA and yvbW of the PulA-type atypical secretion pathway in PD further improved its extracellular expression. Three-liter fermenter scale-up production of PD and PD-ARY yielded extracellular pullulanase activity of 1767.1 U/mL at 54 h and 2465.1 U/mL at 78 h, respectively. Finally, supplementing PulA3E with 40 U/g maltodextrin in the multi-enzyme catalyzed system resulted in the highest trehalose production of 166 g/L and the substrate conversion rate of 83%, indicating its potential for industrial application. Full article

Cefiderocol is a siderophore-conjugated cephalosporin with potent activity against multidrug-resistant Gram-negative pathogens including Acinetobacter baumannii. The aim of this study was to evaluate cefiderocol testing methods on a relevant collection of 97 Acinetobacter spp. isolates. Commercialized broth microdilution methods (ComASP^®, Liofilchem and UMIC^®, Bruker), MIC test strips (Liofilchem) and disc diffusion using discs of three different brands (Mast Diagnostic, Liofilchem and Oxoid—Thermo Fisher Scientific) were compared with the broth microdilution reference method. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ±30%) but both BMD methods achieved acceptable categorical agreement rates (CA = 95.9% [93/97, 95% CI 89.9–98.4] and CA = 93.8% [91/97, 95% CI 87.2–97.1] for ComASP^® and UMIC^®, respectively) and bias < 30% (−7.2% and −25.2% for ComASP^® and UMIC^®, respectively). The use of MIC gradient testing is strongly discouraged due to misclassification of 55% (n = 23/42) of resistant strains. Finally, the disc diffusion method could be used to rapidly screen for susceptible strains by setting a critical diameter of 22 mm. Full article

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant-A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer’s guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii. Full article

Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of the transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of the histone-like nucleoid structuring protein, H-NS, in the regulation of the expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA significantly increased in a Δhns derivative of A. baumannii A118. The complementation of the mutant with a recombinant plasmid harboring hns restored the expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 Δhns strain was significantly higher than that of the wild-type. Similar, albeit not identical, there were consequences when hns was deleted from the hypervirulent A. baumannii AB5075 strain. In the AB5075 complemented strain, the reduction in gene expression in a few cases was not so pronounced that it reached wild-type levels, and the expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications were accompanied by an increase in the cells’ transformability. The results highlight a role of H-NS in A. baumannii’s natural competence. Full article

Historically, Micrococcus luteus was one of the first organisms used to study natural transformation, one of the main routes of horizontal gene transfer among prokaryotes. However, little is known about the molecular basis of competence development in M. luteus or any other representative of the phylum of high-GC Gram-positive bacteria (Actinobacteria), while this means of genetic exchange has been studied in great detail in Gram-negative and low-GC Gram-positive bacteria (Firmicutes). In order to identify new genetic elements involved in regulation of the comEA-comEC competence operon in M. luteus, we conducted random chemical mutagenesis of a reporter strain expressing lacZ under the control of the comEA-comEC promoter, followed by the screening of dysregulated mutants. Mutants with (i) upregulated com promoter under competence-repressing conditions and (ii) mutants with a repressed com promoter under competence-inducing conditions were isolated. After genotype and phenotype screening, the genomes of several mutant strains were sequenced. A selection of putative com-influencing mutations was reinserted into the genome of the M. luteus reporter strain as markerless single-nucleotide mutations to confirm their effect on com gene expression. This strategy revealed mutations affecting com gene expression at genetic loci different from previously known genes involved in natural transformation. Several of these mutations decreased transformation frequencies by several orders of magnitude, thus indicating significant roles in competence development or DNA acquisition in M. luteus. Among the identified loci, there was a new locus containing genes with similarity to genes of the tad clusters of M. luteus and other bacteria. Full article

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis. Full article

The literature has been notably less definitive in distinguishing between finite sample studies of seasonal stationarity than in seasonal unit root tests. Although the use of seasonal stationarity and unit root tests is advised to determine correctly the most appropriate form of the trend in a seasonal time series, such a use is rarely noted in the relevant studies on this topic. Recently, the seasonal KPSS test, with a null hypothesis of no seasonal unit roots, and based on quarterly data, has been introduced in the literature. The asymptotic theory of the seasonal KPSS test depends on whether data have been filtered by a preliminary regression. More specifically, one may proceed to extracting deterministic components, such as the mean and trend, from the data before testing. In this paper, we examine the effects of de-trending on the properties of the seasonal KPSS test in finite samples. A sketch of the test’s limit theory is subsequently provided. Moreover, a Monte Carlo study is conducted to analyze the behavior of the test for a monthly time series. The focus on this time-frequency is significant because, as we mentioned above, it was introduced for quarterly data. Overall, the results indicated that the seasonal KPSS test preserved its good size and power properties. Furthermore, our results corroborate those reported elsewhere in the literature for conventional stationarity tests. These subsequent results assumed that the nonparametric corrections of residual variances may lead to better in-sample properties of the seasonal KPSS test. Next, the seasonal KPSS test is applied to a monthly series of the United States (US) consumer price index. We were able to identify a number of seasonal unit roots in this time series. [1] [1] Table 1 in this paper is copyrighted and initially published by JMASM in 2012, Volume 11, Issue 1, pp. 69–77, ISSN: 1538–9472, JMASM Inc., PO Box 48023, Oak Park, MI 48237, USA, ea@jmasm.com. Full article

In the plant pathogenic bacterium, Pseudomonas syringae, the exopolysaccharide levan is synthesized by extracellular levansucrase (Lsc), which is encoded by two conserved 1,296-bp genes termed lscB and lscC in P. syringae strain PG4180. A third gene, lscA, is homologous to the 1,248-bp lsc gene of the bacterium Erwinia amylovora, causing fire blight. However, lscA is not expressed in P. syringae strain PG4180. Herein, PG4180 lscA was shown to be expressed from its native promoter in the Lsc-deficient E. amylovora mutant, Ea7/74-LS6, suggesting that lscA might be closely related to the E. amylovora lsc gene. Nucleotide sequence analysis revealed that lscB and lscC homologs in several P. syringae strains are part of a highly conserved 1.8-kb region containing the ORF, flanked by 450-452-bp and 49-51-bp up- and downstream sequences, respectively. Interestingly, the 450-452-bp upstream sequence, along with the initial 48-bp ORF sequence encoding for the N-terminal 16 amino acid residues of Lsc, were found to be highly similar to the respective sequence of a putatively prophage-borne glycosyl hydrolase-encoding gene in several P. syringae genomes. Minimal promoter regions of lscB and lscC were mapped in PG4180 by deletion analysis and were found to be located in similar positions upstream of lsc genes in three P. syringae genomes. Thus, a putative 498-500-bp promoter element was identified, which possesses the prophage-associated com gene and DNA encoding common N-terminal sequences of all 1,296-bp Lsc and two glycosyl hydrolases. Since the gene product of the non-expressed 1,248-bp lscA is lacking this conserved N-terminal region but is otherwise highly homologous to those of lscB and lscC, it was concluded that lscA might have been the ancestral lsc gene in E. amylovora and P. syringae. Our data indicated that its highly expressed paralogs in P. syringae are probably derived from subsequent recombination events initiated by insertion of the 498-500-bp promoter element, described herein, containing a translational start site. Full article