The distribution characteristics of global blaOXA-carrying Klebsiella pneumoniae.

OBJECTIVE: To analyze the distribution of blaOXA among global Klebsiella pneumoniae and the characteristics of blaOXA-carrying K. pneumoniae. MATERIALS AND METHODS: The genomes of global K. pneumoniae were downloaded from NCBI by Aspera software. After quality check, the distribution of blaOXA among the qualified genomes was investigated by annotation with the resistant determinant database. The phylogenetic tree was constructed for the blaOXA variants based on the single nucleotide polymorphism (SNP) to explore the evolutionary relationship between these variants. The MLST (multi-locus sequence type) website and blastn tools were utilized to determine the sequence types (STs) of these blaOXA-carrying strains. and sample resource, isolation country, date and host were extracted by perl program for analyzing the characteristics of these strains. RESULTS: A total of 12,356 K. pneumoniae genomes were downloaded and 11,429 ones were qualified. Among them, 4386 strains were found to carry 5610 blaOXA variants which belonged to 27 varieties of blaOXAs, blaOXA-1 (n = 2891, 51.5%) and blaOXA-9 (n = 969, 17.3%) were the most prevalent blaOXA variants, followed by blaOXA-48 (n = 800, 14.3%) and blaOXA-232 (n = 480, 8.6%). The phylogenetic tree displayed 8 clades, three of them were composed of carbapenem-hydrolyzing oxacillinase (CHO). Totally, 300 distinct STs were identified among 4386 strains with ST11 (n = 477, 10.9%) being the most predominant one followed by ST258 (n = 410, 9.4%). Homo sapiens (2696/4386, 61.5%) was the main host for blaOXA-carrying K. pneumoniae isolates. The blaOXA-9-carrying K. pneumoniae strains were mostly found in the United States and blaOXA-48-carrying K. pneumoniae strains were mainly distributed in Europe and Asia. CONCLUSION: Among the global K. pneumoniae, numerous blaOXA variants were identified with blaOXA-1, blaOXA-9, blaOXA-48 and blaOXA-232 being the most prevalent ones, indicating that blaOXA rapidly evolved under the selective pressure of antimicrobial agents. ST11 and ST258 were the main clones for blaOXA-carrying K. pneumoniae.

Identification of atmospheric emerging contaminants from industrial emissions: A case study of halogenated hydrocarbons emitted by the pharmaceutical industry.

With the development of the pharmaceutical industry, halogenated hydrocarbons, which are the main raw materials and emissions of the pharmaceutical industry, may be defined as atmospheric emerging contaminants due to toxicity and low oxidation of the atmosphere. This study analyzed the volatile organic compounds (VOCs) emissions from four pharmaceutical companies located in the Yangtze River Delta. Samples were taken three times at each of the selected fixed and fugitive sampling sites in each company. Through testing, 141 VOCs were identified. The mean concentration and proportion of halogenated hydrocarbons from the four pharmaceutical companies were the highest of all the industries in the industrial park. They reached 18.9 ppm and 28.8 %, respectively. Fixed emissions of the companies exhibited the mean maximum concentration of dichloromethane and chlorobenzene, which are 11.4 ppm and 250.67 ppb. The mean concentration of fugitive emission of dichloromethane from the four companies in this study is lower than that of pharmaceutical companies in other studies. Newly detected halogenated hydrocarbons, such as 1,1-dichloropropanone and dichloronitromethane, present potential non-cancer and cancer risks to workers. Chlorobenzene was identified as a key potential cancer risk halogenated hydrocarbon the value of which reaches 0.00965. 2,6-dichloropyridine could be a potential emerging contaminant due to its lower MIR value and higher potential cancer risk. The study suggests that relevant pharmaceutical companies focus on the emissions of chlorobenzene and dichloromethane, which may be the atmospheric emerging contaminants for the pharmaceutical industry and focus on improve the treatment of waste gases in workshops and sewage stations.

Stability assessment of housekeeping genes for qRT-PCR in Yersinia enterocolitica cultured at 22°C and 37°C.

Yersinia enterocolitica, a species within the genus Yersinia, thrives optimally at 22-25°C but can also grow at the mammalian core body temperature of 37°C. This dual temperature adaptability necessitates establishing both temperature conditions in research to examine the effects on various biological processes. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the lack of alternatives and information often leads to the default use of the 16S rRNA gene despite potential limitations. This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 Y. enterocolitica strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. After acquiring Cq values, gene stability was evaluated using four established algorithms-ΔCq, geNorm, NormFinder, and BestKeeper-and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method. Our study suggests that the genes glnS, nuoB, glmS, gyrB, dnaK, and thrS maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use of 16S rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.IMPORTANCEIn our study, we focused on identifying stable reference genes for quantitative real-time PCR (qRT-PCR) experiments on Y. enterocolitica cultured at different temperatures (22°C and 37°C). After thoroughly evaluating 16 candidate genes, we identified six genes-glnS, nuoB, glmS, gyrB, dnaK, and thrS-as exhibiting stable expression across these temperature conditions, making them ideal reference genes for Y. enterocolitica studies. This discovery is crucial for ensuring the accuracy and reliability of qRT-PCR data, as the choice of appropriate reference genes is key to normalizing expression data and minimizing experimental variability. Importantly, our research extended beyond bioinformatics analysis by incorporating validation with clinical strains, bridging the gap between theoretical predictions and practical application. This approach not only underscores the robustness and reliability of our findings but also directly addresses the critical need for experimental validation in the field. By providing a set of validated, stably expressed reference genes, our work offers valuable guidance for designing experiments involving Y. enterocolitica, enhancing the reliability of research outcomes, and advancing our understanding of this significant pathogen.

Draft genome sequence of yeast Vanrija sp. strain TS01, isolated from leukemia patient's urine.

Vanrija sp. strain TS01 was isolated from urine sample from a leukemia patient in Nanjing, China. The closest known relative strain is Vanrija humicola with average nucleotide identity value of 93.1. The draft genome comprises 22.0 Mb in 52 contigs, with G + C content of 62.57%.

Housekeeping gene stability in Pesudomonas aeruginosa PAO1 under the pressure of commonly used antibiotics in molecular microbiology assays.

Pseudomonas aeruginosa is an opportunistic human pathogen notorious for its remarkable capacity of multi-drug resistance, and has become one of the most important model bacteria in clinical bacteriology research. Quantitative real-time PCR is a reliable method widely used in gene expression analysis, for which the selection of a set of appropriate housekeeping genes is a key prerequisite for the accuracy of the results. However, it is easy to overlook that the expression level of housekeeping gene may vary in different conditions, especially in the condition of molecular microbiology assays, where tested strains are generally cultured under the pre-set antibiotic selection pressures, and how this affects the stability of commonly used housekeeping genes remains unclear. In this study, the expression stability of ten classic housekeeping genes (algD, gyrA, anr, nadB, recA, fabD, proC, ampC, rpoS, and rpsL) under the pressure of eight laboratory commonly used antibiotics (kanamycin, gentamycin, tetracycline, chloramphenicol, hygromycin B, apramycin, tellurite, and zeocin) were tested. Results showed that the stability of housekeeping gene expression was indeed affected by the types of antibiotics added, and of course the best reference gene set varied for different antibiotics. This study provides a comprehensive summary of the effects of laboratory antibiotics on the stability of housekeeping genes in P. aeruginosa, highlighting the necessity to select housekeeping genes according to the type of antibiotics used in the initial stage of experiment.

Correction to: The H741D mutation in Tac1p contributes to the upregulation of CDR1 and CDR2 expression in Candida albicans.

Unfortunately, an error occurred in the author affiliations.

The discovery of potential phosphopantetheinyl transferase Ppt2 inhibitors against drug-resistant Candida albicans.

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.

The H741D mutation in Tac1p contributes to the upregulation of CDR1 and CDR2 expression in Candida albicans.

The wide use of antifungal agents has led to the development of resistance in the pathogenic yeast strain Candida albicans. Gain-of-function mutations in transcription factors such as Tac1p demonstrated their ability to control expression of the ABC transporter genes CDR1 and CDR2, and mediation of azole resistance. Previously, we obtained a series of azole-resistant isolates with high-level expression of CDR1 or/and CDR2, and identified the novel H741D mutation in Tac1p. In the present study, the TAC1 alleles from isolate C13 were introduced into tac1Δ/Δ mutant. The H741D change was seen in TAC1C13 in addition to several other amino acid differences. Hyperactive alleles TAC1C13 exhibited higher minimum inhibitory concentrations (MICs) of fluconazole and itraconazole than that observed in SN152 containing the wild-type TAC1 allele. And alleles TAC1C13 conferred constitutively high levels of Cdr1p and Cdr2p. Moreover, the importance of H741D in conferring hyperactivity to TAC1 was also confirmed by site-directed mutagenesis. Compared with SN152, the presence of H741D resulted in > 2-fold increase in CDR1 and CDR2 gene and protein expression, > 4-fold increase in fluconazole and itraconazole MICs and higher rates of Rhodamine 6G efflux by 43.24%.

Expression of fluconazole resistance-associated genes in biofilm from 23 clinical isolates of Candida albicans.

This study aimed to establish the influence of biofilm from clinical isolates of Candida albicans on fluconazole resistance, focusing on efflux pumps and azole-targeted enzymes. Twenty-three C. albicans clinical isolates were collected from two hospitals in Shanghai, China. Antifungal susceptibility tests were performed on biofilm and planktonic cells. A crystal violet assay was used to monitor biofilm growth. Real-time RT-PCR was performed to quantify the expression of the transporter-related genes MDR1, CDR1, and CDR2 as well as ERG11, a gene encoding an enzyme targeted by antifungal drugs. Fluconazole resistance was shown to increase in biofilm in a time-dependent manner. No significant differences were observed between different strains of C. albicans. Genes encoding efflux pumps were overexpressed in early stages of biofilm formation and could also be induced by fluconazole. While ERG11 was not upregulated in biofilm, it was overexpressed upon the addition of fluconazole to biofilm and planktonic cells. Gene expression also appeared to be related to the original genotype of the strain. The upregulation of genes encoding efflux pumps demonstrates their role in the development of fluconazole resistance during the early stages of C. albicans biofilm formation.

FLO8 deletion leads to azole resistance by upregulating CDR1 and CDR2 in Candida albicans.

Candida albicans has the ability to switch reversibly between budding yeast, filamentous, pseudohypha, and hyphal forms, a process in which the transcription factor Flo8 plays an important role. This ability is important for the virulence and pathogenicity of C. albicans. To determine whether Flo8 plays a role in the regulation of drug sensitivity, we constructed a FLO8 null mutant flo8/flo8 from the parental strain SN152 and a Flo8-overexpressing strain, flo8/flo8::FLO8. The susceptibility of the isolates to antifungal agents was then evaluated using the agar dilution and broth microdilution methods. Expression of drug resistance-related genes by the isolates was investigated by real-time PCR. The flo8/flo8 mutation isolates exhibited increased resistance to fluconazole, voriconazole, and itraconazole compared with the wild-type and drug sensitivity was restored by FLO8 overexpression (flo8/flo8∷FLO8). Of seven drug resistance-related genes, the FLO8 null mutation resulted in increased CDR1 and CDR2 expression (1.60-fold and 5.27-fold, respectively) compared with SN152, while FLO8 overexpression resulted in decreased CDR1 expression (0.63-fold). These results suggest that Flo8 is involved in the susceptibility of C. albicans to antifungal azoles, with FLO8 deletion leading to constitutive overexpression of CDR1 and CDR2 and resistance to antifungal azoles.