Identification of molecular determinants of antibiotic resistance in some fish farms of Ghana.

Antimicrobial resistance is a global health challenge caused by the ability of microorganisms including bacteria, fungi, protozoans and viruses to survive the effects of drugs that hitherto were effective against them. This study sought to investigate the presence of antibiotic-resistant bacteria and their corresponding molecular determinants in fish farms of the Central and Western Regions of Ghana. Management practices and antibiotic use at the fish farms were obtained through the administration of a questionnaire. Coliform and Gram-positive bacterial loads of catfish (Clarias gariepinus), tilapia (Oreochromis niloticus) intestinal microbiota, and pond water samples recovered on MacConkey Agar and Mannitol Salt Agar were determined. Bacterial isolates were identified using various biochemical assays. Antibiotic resistance profiles and possible responsible genes of bacterial isolates were determined using the disc diffusion and Polymerase Chain Reaction (PCR) methods respectively. The study revealed that none of the fish farm managers admitted using antibiotics for prevention and treatment of diseases and no major disease outbreak had ever been recorded. Bacterial loads of pond water exceeded the acceptable level of ≤100 E. coli and <10 coliforms per mL for wastewater recommended for use in fish farming. In all, 145 bacterial isolates comprising 99 Gram negative and 46 Gram-positive bacteria were stored and identified. Most isolates were resistant to at least an antibiotic. Both Gram-negative and Gram-positive bacteria were highly resistant to beta-lactam antibiotics with a corresponding high percentage detection of the bla TEM gene compared to other classes of antibiotics. This study has revealed the presence of various molecular determinants of antibiotic resistance including bla TEM, cmIA, qnrS, tetB and bla CTX-M, in multidrug-resistant bacteria at some fish farms in Ghana. There is the need to increase awareness about risks associated with the misuse and overuse of antibiotics by humans and the potential risk of spread of multi-drug resistant-bacteria in the environment.

Gene expression changes during short day induced terminal bud formation in Norway spruce.

The molecular basis for terminal bud formation in autumn is not well understood in conifers. By combining suppression subtractive hybridization and monitoring of gene expression by qRT-PCR analysis, we aimed to identify genes involved in photoperiodic control of growth cessation and bud set in Norway spruce. Close to 1400 ESTs were generated and their functional distribution differed between short day (SD-12 h photoperiod) and long day (LD-24 h photoperiod) libraries. Many genes with putative roles in protection against stress appeared differentially regulated under SD and LD, and also differed in transcript levels between 6 and 20 SDs. Of these, PaTFL1(TERMINAL FLOWER LIKE 1) showed strongly increased transcript levels at 6 SDs. PaCCCH(CCCH-TYPE ZINC FINGER) and PaCBF2&3(C-REPEAT BINDING FACTOR 2&3) showed a later response at 20 SDs, with increased and decreased transcript levels, respectively. For rhythmically expressed genes such as CBFs, such differences might represent a phase shift in peak expression, but might also suggest a putative role in response to SD. Multivariate analyses revealed strong differences in gene expression between LD, 6 SD and 20 SD. The robustness of the gene expression patterns was verified in 6 families differing in bud-set timing under natural light with gradually decreasing photoperiod.

Differential gene expression related to an epigenetic memory affecting climatic adaptation in Norway spruce.

In Norway spruce, the temperature during zygotic embryogenesis appears to adjust an adaptive epigenetic memory in the progeny that may regulate bud phenology and cold acclimation. Conditions colder than normal advance the timing whilst temperatures above normal delay the onset of these processes and altered performance is long lasting in progeny with identical genetic background. As a step toward unraveling the molecular mechanism behind an epigenetic memory, transcriptional analysis was performed on seedlings from seeds of six full-sib families produced at different embryogenesis temperature-cold (CE) vs warm (WE) under long and short day conditions. We prepared two suppressive subtracted cDNA libraries, forward and reverse, representing genes predominantly expressed in plants from seeds obtained after CE and WE embryogenesis following short day treatment (inducing bud set). Sequencing and annotation revealed considerable differences in the transcriptome of WE versus CE originated plants. By using qRT-PCR we studied the expression patterns of 32 selected candidate genes chosen from subtractive cDNA libraries analysis and nine siRNA pathways genes by a direct candidate approach. Eight genes, two transposons related genes, three with no match to Databases sequences and three genes from siRNA pathways (PaDCL1 and 2, PaSGS3) showed differential expression in progeny from CE and WE correlated with the family phenotypic differences. These findings may contribute to our understanding of the epigenetic mechanisms underlying adaptive changes acquired during embryogenesis.

Effect of bud burst forcing on transcript expression of selected genes in needles of Norway spruce during autumn.

Expression of selected genes in needles of Norway spruce (Picea abies [L.] Karst) was investigated by following their transcription levels during late autumn. Transcription was assessed in mature needles which likely serve as sensor of environmental cues that enable trees in the temperate and boreal regions to change between stages of growth, frost tolerance and bud dormancy. Samples were collected from grafts kept under outdoor conditions and after bud burst forcing in greenhouse at 20 degrees C (12 h darkness) for one week. Transcription was assayed with real-time RT-PCR. During the sampling period, chilling requirement was partially fulfilled, and time to bud burst after forcing was decreased. Of the 27 transcripts studied, expression of 16 was significantly affected either by forcing, sampling time, or interaction between them. PaSAP, PaACP, PaSGS3, PaWRKY, PaDIR9, PaCCCH and dehydrin genes responded drastically to forcing temperatures at all sampling points, showing no correlation with readiness for bud burst. Expression patterns of some vernalization pathway gene homologs PaVIN3, and also of PaMDC, PaLOV1 and PaDAL3 had a clear opposite trends between forcing and outdoor conditions, which could imply their role in chilling accumulation and bud burst regulation/cold acclimation. These genes could constitute putative candidates for further detailed study, whose regulation in needles may be involved in preparation towards bud burst and chilling accumulation sensing.

Dehydrins expression related to timing of bud burst in Norway spruce.

Cold deacclimation and preparation to flushing likely requires rehydration of meristems. Therefore, water stress related genes, such as dehydrins (DHN), might play an important role in providing protection during winter dormancy, deacclimation and bud burst timing processes. Here we report the sequence analysis of several Norway spruce DHN identified in late and early flushing suppressive subtraction hybridization cDNA libraries and in our Norway spruce EST database. We obtained 15 cDNAs, representing eight genes from three distinct types of DHN, and studied differential expression of these genes before and during bud burst in spring, using qRT-PCR. We found the visible reduction in transcript level of most DHN towards the bud burst, supported by a significant down-regulation of the DHN in needles during experimental induction of bud burst applied at three time points during autumn in Norway spruce grafts. For most of the DHN transcripts, their expression levels in late-flushing spruces were significantly higher than in the early flushing ones at the same calendar dates but were remarkably similar at the same bud developmental stage. From our results we may conclude that the difference between the early and the late families is in timing of the molecular processes leading to bud burst due to differences in their response to the increasing temperature in the spring. They are induced much earlier in the early flushing families.