The host microbiome is a promising source of probiotics for aquaculture species including Nile tilapia. In this study, the probiotic potential of autochthonous bacterial isolates from Nile tilapia and carp mid-intestines were screened in vitro. Two isolates (C61 and T70), closely related to Bacillus subtilis, exhibited antagonistic activity against multiple pathogen species and demonstrated multiple digestive enzyme activities. Their antagonistic activity in Aeromonas hydrophila assays remained even under simulated intestinal juice (SIJ) exposure. Subsequently, C61 (PT1) and T70 (PT2) were added to experimental diets at log 7 CFU/g of diet, and fed to Nile tilapia (5.32 ± 0.12 g) for 40 days. There were no significant differences observed in the growth performance across treatments. Despite limited Bacillus intestinal recovery levels, 16S rRNA gene metabarcoding revealed subtle shifts in the intestinal microbial community composition of the probiotic-fed groups. In addition, the PT1 group showed significantly longer mucosal fold length, elevated intestinal and skin goblet cell levels, and higher skin goblet cell coverage compared to the control. These results indicate the potential benefits of the isolates as functional feed additives for enhancing the mucosal health of Nile tilapia, but their benefits were likely achieved through transient activity given the low level of Bacillus recovery in the intestine.

Yeast-based feed additives have emerged as promising functional feed additives (FFAs) to promote sustainable aquaculture development through enhanced gut health and immune modulation in fish. The present study evaluated the impact of autolysed brewer’s yeast (ABY) and soluble dried yeast extract (SDYE) in improving the intestinal and skin histology, immune response, and intestinal microbiome of Atlantic salmon parr (Salmo salar) over a 9-week feeding trial. Three experimental diets were produced: a control diet, a diet supplemented with ABY at 2.5 g/kg, and a diet supplemented with SDYE at 2.5 g/kg. These diets were administered to triplicate tanks of Atlantic salmon. The yeast-supplemented diets, especially ABY, improved intestinal mucosal fold length, lamina propria width, microvilli density, and intestinal goblet cell counts, as well as skin goblet cell counts. The yeast additives had no detrimental effects on the fish haematology, with no significant differences in haemoglobin concentration, red blood cell counts, and white blood cell counts among the treatment groups. Gene expression analysis revealed upregulation of il-1β and muc-2 in fish fed the ABY diet, indicating enhanced immune function and potentially mucosal protection. Intestinal microbiota analysis revealed Firmicutes as the most dominant phylum in all groups, followed by Actinobacteriota. Distinct bacterial community shifts were observed between the treatment groups, with a significant increase in the relative abundance of taxa such as Staphylococcus in yeast-supplemented diets and a significant decrease in Streptococcus and Weissella. Collectively, these findings suggest that the yeast additives, especially ABY, enhance gut health and immune function without compromising growth performance.

Since its inception in the 1980s, advancements in PCR technology using improved thermal cyclers, engineered DNA polymerases and commercial master mixes, have led to increased PCR productivity. Despite these advancements, PCR cycling protocols have largely remained unchanged over the same period. This study aimed to systemically evaluate the effect of reduced PCR cycling parameters on amplicon production. The 1466bp fragment from the 16S rRNA gene present in low-, medium- and highCG bacteria was amplified using three commercially available PCR master mixes. The shortest cycling parameters required to successfully amplify the 16S fragment from all bacteria and master mixes comprised 30 -cycles of 5 s denaturation, 25 s annealing, and 25 s extension. While all produced an amplicon with sufficient yield to enable downstream sequence analysis, the PCRBIO Ultra Mix in conjunction with the shortened parameters was found to achieve the highest amplicon yield across low-, medium- and high CG bacteria. Comparing the run times to that of a typical 16S PCR protocol, the shortened cycling parameters reduced the program duration by 46 % and consumed 50 % less electricity, translating into increased productivity and helping to improve laboratory environmental sustainability. (c) 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

36 Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence 37 potential based on gene carriage and they are highly virulent in insect infection 38 models. However, strains of this lineage are reported in relatively low numbers in 39 many studies. ST127 strains are also usually widely susceptible to antibiotics and, 40 consequently, their true prevalence may be under-recognised, as they will be 41 eradicated during empiric therapy. A genuine concern is the possibility that members 42 of this highly virulent lineage will acquire resistance, leading to a more serious threat. 43 The aim of this study was to design and validate a PCR assay specific to ST127. 44 Genomic sequences obtained from various UPEC isolates from the leading clones 45 were used in comparative genomics to allow identification of highly discriminative 46 sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the 47 upaG (Autotransporter adhesin) gene were identified as meeting our criteria and 48 were used to develop a multiplex PCR assay. A total of 143 E. coli UPEC isolates 49 representing 99 different MLST clones from three locations (North West and South 50 West England and Riyadh, Saudi Arabia) were used to validate the PCR assay. The 51 multiplex PCR readily identified all 29 E. coli ST127 isolates, but equally importantly, 52 produced no false positives with representatives of any of the other 98 ST’s tested. 53 We report the design and validation of a specific multiplex PCR for the rapid and 54 reliable identification of ST127, which can be used for enhanced surveillance for this 55 high-risk clone. 56

Purpose. Members of the ST127 uropathogenic E. coli (UPEC) clone have a high virulence potential and are also highly virulent in insect infection models. However, strains of this lineage are reported in relatively low numbers in many studies. ST127 strains are also usually widely susceptible to antibiotics and, consequently, their true prevalence may be under-recognized as they will be eradicated during empirical therapy. A genuine concern is the possibility that members of this highly virulent lineage will acquire resistance, leading to a more serious threat. The aim of this study was to design and validate a PCR assay specific to ST127. Methodology. Genomic sequences obtained from various UPEC isolates from the leading clones were used in comparative genomic analyses to allow identification of highly discriminatory sequences specific to E. coli ST127. The fliC (flagellin) and a homologue of the upaG (autotransporter adhesin) gene were identified as meeting our criteria and were used to develop a multiplex PCR assay. A total of 143 UPEC isolates representing 99 different MLST clones from three locations (North West and South West England and Riyadh, Saudi Arabia) were used to validate the PCR assay. Results. The multiplex PCR readily identified all 29 E. coli ST127 isolates but, equally importantly, produced no false positives with representatives of any of the other 98 STs tested. Conclusion. We report the design and validation of a specific multiplex PCR for the rapid and reliable identification of ST127, which can be used for enhanced surveillance for this high-risk clone.

For historical reasons, much of the information available on the intestinal microbiota of fish is based on the use of conventional culture-dependent methods. This has consisted of sampling gut material and spreading gut homogenates on selective or general purpose agar, followed by incubation, colony counting and subsequent identification, typically by phenotypic/biochemical tests or during the last decade 16S rRNA sequencing. As is often the case with microbial communities from environmental samples, the gut microbiota of fish has been reported to be of low cultivability: cultivability using general purpose culture media has been reported to represent <0.1% of the total microbial community in the gastrointestinal (GI) tract of some fish species. A wide range of molecular ecology techniques are available based on the sequence variability of the 16S and 23S rRNA genes, and over the last 10-12 years such approaches have become more commonly used to investigate the gut microbiomes of fish species. The molecular based approaches used have depended on the aim of the studies: (1) clone libraries have been used to identify the microbiota composition; (2) fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) and temporal temperature gradient electrophoresis (TTGE) have been used to analyse microbial community structure and diversity; (3) quantitative real-time PCR (qPCR) or fluorescent in situ hybridization (FISH) have been used to determine the abundance of particular taxa or total microbial levels; and (4) FISH and immunohistochemistry have been used to assess bacterial-host interactions at the mucosal brush border. In a limited number of recent studies, next-generation sequencing (NGS) approaches have been used to generate extensive libraries to extend our knowledge of the 'rare biosphere'. This review provides an overview of the techniques which have been used in evaluations of fish gut microbiota, the techniques with most relevance to future studies, and discusses the benefits and drawbacks of each approach with reference to the microbial populations present in the GI tract of fish.