Increased gene expression and copy number of mutated blaKPC lead to high-level ceftazidime/avibactam resistance in Klebsiella pneumoniae

Abstract Background Resistance to ceftazidime-avibactam was reported, and it is important to investigate the mechanisms of ceftazidime/avibactam resistance in K. pneumoniae with mutations in blaKPC. Results We report the mutated blaKPC is not the only mechanism related to CZA resistance, and investigate the role of outer porin defects, efflux pump, and relative gene expression and copy number of blaKPC and ompk35/36. Four ceftazidime/avibactam-sensitive isolates detected wild type blaKPC-2, while 4 ceftazidime/avibactam-resistant isolates detected mutated blaKPC (blaKPC-51, blaKPC-52, and blaKPC-33). Compared with other ceftazidime/avibactam-resistant isolates with the minimal inhibitory concentration of ceftazidime/avibactam ranging 128–256 mg/L, the relative gene expression and copy number of blaKPC was increased in the isolate which carried blaKPC-51 and also showed the highest minimal inhibitory concentration of ceftazidime/avibactam at 2048 mg/L. The truncated Ompk35 contributes rare to ceftazidime/avibactam resistance in our isolates. No significant difference in minimal inhibitory concentration of ceftazidime/avibactam was observed after the addition of PABN. Conclusions Increased gene expression and copy number of mutated blaKPC can cause high-level ceftazidime/avibactam resistance.

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A 4-Gene Signature of CDKN1, FDXR, SESN1 and PCNA Radiation Biomarkers for Prediction of Patient Radiosensitivity

International Journal of Molecular Sciences ◽

10.3390/ijms221910607 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10607

Author(s):

Orla Howe ◽

Lisa White ◽

Daniel Cullen ◽

Grainne O’Brien ◽

Laura Shields ◽

...

Keyword(s):

Gene Expression ◽

Prostate Cancer ◽

Healthy Donor ◽

Gene Signature ◽

Sampling Time ◽

Relative Gene Expression ◽

Significant Difference ◽

Healthy Control ◽

Radiation Induced ◽

Relative Gene

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

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Expression of the qepA1 gene is induced under antibiotic exposure

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkab045 ◽

2021 ◽

Author(s):

Gerrit Brandis ◽

Jonas Gockel ◽

Linnéa Garoff ◽

Lionel Guy ◽

Diarmaid Hughes

Keyword(s):

Gene Expression ◽

Efflux Pump ◽

Class I ◽

Upstream Region ◽

Antibiotic Exposure ◽

Negative Effects ◽

Integrase Gene ◽

Bacterial Fitness ◽

High Level

Abstract Background The qepA1 gene encodes an efflux pump that reduces susceptibility to ciprofloxacin. Little is known about the regulation of qepA1 expression. Objectives To assess the potential role of ciprofloxacin and other antibiotics in the regulation of qepA1 gene expression. To identify the promoter that drives qepA1 expression and other factors involved in expression regulation. To assess whether the identified features are universal among qepA alleles. Methods A translational qepA1-yfp fusion under the control of the qepA1 upstream region was cloned into the Escherichia coli chromosome. Expression of the fusion protein was measured in the presence of various antibiotics. Deletions within the upstream region were introduced to identify regions involved in gene expression and regulation. The qepA1 coding sequence and upstream region were compared with all available qepA sequences. Results Cellular stress caused by the presence of various antibiotics can induce qepA1 expression. The qepA1 gene is fused to a class I integron and gene expression is driven by the Pc promoter within the integrase gene. A segment within the integron belonging to a truncated dfrB4 gene is essential for the regulation of qepA1 expression. This genetic context is universal among all sequenced qepA alleles. Conclusions The fusion of the qepA1 gene to a class I integron has created a novel regulatory unit that enables qepA1 expression to be under the control of antibiotic exposure. This setup mitigates potential negative effects of QepA1 production on bacterial fitness by restricting high-level expression to environmental conditions in which QepA1 is beneficial.

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Effect of Vitrification on Folliculogenesis-Related Genes in Ovarian Follicles of Bubalus bubalis

Indian Journal of Animal Research ◽

10.18805/ijar.b-4245 ◽

2020 ◽

Author(s):

D. Dua ◽

A. Alam ◽

M. S. Chauhan ◽

P. Palta ◽

M. K. Singh

Keyword(s):

Gene Expression ◽

Mtt Assay ◽

Bubalus Bubalis ◽

Relative Gene Expression ◽

Follicular Cells ◽

Significant Difference ◽

Hematoxylin And Eosin ◽

Hematoxylin And Eosin Staining ◽

Morphological Differences ◽

Relative Gene

Background: Many years have been devoted to preserve fertility, but the effect of cryopreservation on gene functionality in primary, secondary and tertiary follicular stages; is still unclear. The present study was designed to assess the effect of vitrification on the histological structures and expression of follicular cells related genes. Methods: The buffalo ovarian cortical tissues were vitrified in two-steps. The sliced cortical tissues were incubated with VS1 (8.5% DMSO and 8.5% EG) for 10 min and sequentially incubated and stored in liquid nitrogen in VS2 (16.5% DMSO, 16.5% EG and 0.1M Sucrose). Morphological differences were assessed by hematoxylin and eosin staining, which indicated similar structures in both groups. Further, functionality of these follicles were evaluated by the relative gene expression of folliculogenesis-related genes; FOXO3, NLRP5, WNT4, SF1, VEGFA and HAS2. No significant difference was observed between the vitrified groups as compared to control. Moreover, toxicity in follicular cells during vitrification was rectified by MTT assay which also showed no significant difference. Conclusion: Present study can be considered as the key work that helps in filling the gaps regarding the growth of follicles after cryopreservation in buffalo species, as this imparts nonsignificant injury on follicular functionality and development.

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Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

Applied Sciences ◽

10.3390/app11157144 ◽

2021 ◽

Vol 11 (15) ◽

pp. 7144

Author(s):

Muriel C. Bischof ◽

Sonja Häckel ◽

Andrea Oberli ◽

Andreas S. Croft ◽

Katharina A. C. Oswald ◽

...

Keyword(s):

Gene Expression ◽

Intervertebral Disc ◽

Metabolic Activity ◽

Cell Metabolism ◽

Trauma Patients ◽

Low Back ◽

Relative Gene Expression ◽

Progenitor Cell Population ◽

Ivd Degeneration ◽

Relative Gene

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

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Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-021-00439-6 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Saeed Sharifi ◽

Bita Bakhshi ◽

Shahin Najar-peerayeh

Keyword(s):

Gene Expression ◽

Point Mutation ◽

Campylobacter Jejuni ◽

Significant Contribution ◽

Efflux Pump ◽

Campylobacter Coli ◽

Rapd Pcr ◽

Resistant Strains ◽

High Level ◽

Level Resistance

Abstract Background Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. Methods A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. Results Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. Conclusion Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

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