Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Meng L

PROVIDER: S-EPMC10040570 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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

Meng Lingning L Cao Xiaoli X Li Chuchu C Li Jia J Xie Hui H Shi Jiping J Han Mei M Shen Han H Liu Chang C

Frontiers in microbiology 20230313

<i>Pseudomonas aeruginosa</i> 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 housekee ...[more]

PMID: 36992935

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Project description:BackgroundGene expression analysis using real-time RT-PCR (qRT-PCR) is increasingly important in biological research due to the high-throughput and accuracy of qRT-PCR. For accurate and reliable gene expression analysis, normalization of gene expression data against housekeeping genes or internal control genes is required. The stability of reference genes has a tremendous effect on the results of relative quantification of gene expression by qRT-PCR. The expression stability of reference genes could vary according to tissues, age of individuals and experimental conditions. In the pig however, very little information is available on the expression stability of reference genes. The aim of this research was therefore to develop a new set of reference genes which can be used for normalization of mRNA expression data of genes expressed in varieties of porcine tissues at different ages.ResultsThe mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined in varieties of tissues collected from newborn, young and adult pigs. geNorm, NormFinder and BestKeeper software were used to rank the genes according to their stability. geNorm software revealed that RPL4, PPIA and YWHAZ showed high stability in newborn and adult pigs, while B2M, YWHAZ and SDHA showed high stability in young pigs. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that TBP was the most stable gene in newborn and young pigs, while PPIA was most stable in adult pigs. Moreover, geNorm software suggested that the geometric mean of three most stable gene would be the suitable combination for accurate normalization of gene expression study.ConclusionsAlthough, there was discrepancy in the ranking order of reference genes obtained by different analysing software methods, the geometric mean of the RPL4, PPIA and YWHAZ seems to be the most appropriate combination of housekeeping genes for accurate normalization of gene expression data in different porcine tissues at different ages.

Project description:Bacteria frequently encounter selection by both phages and antibiotics. However, our knowledge on the evolutionary interactions between phages and antibiotics are still limited. Here, we characterized a phage-resistant Pseudomonas aeruginosa variant PAO1-R1 that shows increased sensitivity to gentamicin and polymyxin B. Using whole genome sequencing, significant genome differences were observed between the reference P. aeruginosa PAO1 and PAO1-R1. Compared to PAO1, 64 gene-encoding proteins with nonsynonymous single nucleotide polymorphisms (SNPs) and 31 genes with insertion/deletion (indel) mutations were found in PAO1-R1. We observed a significant reduction in phage adsorption rate for both phage vB_Pae_QDWS and vB_Pae_W3 against PAO1-R1 and proposed that disruption of phage adsorption is likely the main cause for evolving resistance. Because the majority of spontaneous mutations are closely related to membrane components, alterations in the cell envelope may explain the antibiotic-sensitive phenotype of PAO1-R1. Collectively, we demonstrate that the evolution of phage resistance comes with fitness defects resulting in antibiotic sensitization. Our finding provides new insights into the evolutionary interactions between resistance to the phage and sensitivity to antibiotics, which may have implications for the future clinical use of steering in phage therapies. IMPORTANCE Bacteria frequently encounter the selection pressure from both antibiotics and lytic phages. Little is known about the evolutionary interactions between antibiotics and phages. Our study provides new insights into the trade-off mechanism between resistance to the phage and sensitivity to antibiotics. This evolutionary trade-off is not dependent on the outer membrane proteins (OMPs) of the multidrug efflux pumps. The disruption of phage adsorption that induced phage resistance and the changes in structure or composition of membranes are presumably one of the major causes for antibiotic sensitivity. Our finding may fill some gaps in the field of phage-host interplay and have implications for the future clinical use of steering in phage therapies.

Project description:ObjectivesThe study aimed at identifying the commonly used non-prescribed antibiotics (NPAs) and the main health conditions leading to the practices of self-medication with antibiotics (SMAs) in Maputo city, Mozambique.DesignCross-sectional qualitative study based on individual and group interviews.SettingThe study was conducted in nine pharmacies of three socioeconomic areas of Maputo city, from October 2018 to March 2019.ParticipantsThe study included 32 pharmacy clients and 17 pharmacists. The pharmacy clients included men 10 (31%) and women 22 (69%) ranging from 19 to 67 years while the pharmacists included men 6 (35,3%) and women 11 (64,7%) with ages ranging from 24 to 47 years.FindingsThe majority of the pharmacy clients 30 (93.75%) admitted frequent use of NPAs, 15 (88.2%) out of the 17 pharmacists admitted dispensing NPAs. While the majority of the participants (16) mentioned the use of amoxicillin, also known as 'two colours medicine', 14 mentioned the use of cotrimoxazole and seven mentioned amoxicillin with clavulanic acid. Two to five participants also used tetracycline, ciprofloxacin, azithromycin, doxycycline, erythromycin, metronidazole and phenoxymethylpenicillin. The above mentioned NPAs were used to treat self-perceived sore throat, fever, pain, cough, vaginal discharge, eye problems, the common influenza, urinary infections, respiratory tract infections, wounds and toothaches.ConclusionsAntibiotics are perceived as essential medical resources to manage health and illnesses. While taking an active role in their health-disease process, participants commonly used amoxicillin, 'two colours', cotrimoxazole and amoxicillin with clavulanic acid to manage their health and that of their families. In this sense, the practices of SMAs were perceived as part of the self-care process and not necessarily as misuse of antibiotics. A wideunderstanding of health-seeking beliefs and behaviours regarding the utilisation of antibiotics is needed to inform public health experts, health policymakers and other stake-holders in designing and implementing public health education and health promotion programsat all levels in Mozambique.

Project description:BACKGROUND: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS). RESULTS: The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were RPL13A and YWHAZ (M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were RPL13A and SDHA (M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were B2M and TBP (M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were SDHA and YWHAZ (K6) or SDHA and HMBS (DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression. CONCLUSION: The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.

Dataset Information

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

Publications

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

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