Genomic and Functional Evaluation of Two Lacticaseibacillus paracasei and Two Lactiplantibacillus plantarum Strains, Isolated from a Rearing Tank of Rotifers (Brachionus plicatilis), as Probiotics for Aquaculture.

Aquaculture plays a crucial role in meeting the increasing global demand for food and protein sources. However, its expansion is followed by increasing challenges, such as infectious disease outbreaks and antibiotic misuse. The present study focuses on the genetic and functional analyses of two Lacticaseibacillus paracasei (BF3 and RT4) and two Lactiplantibacillus plantarum (BF12 and WT12) strains isolated from a rotifer cultivation tank used for turbot larviculture. Whole-genome sequencing (WGS) and bioinformatics analyses confirmed their probiotic potential, the absence of transferable antibiotic resistance genes, and the absence of virulence and pathogenicity factors. Bacteriocin mining identified a gene cluster encoding six plantaricins, suggesting their role in the antimicrobial activity exerted by these strains. In vitro cell-free protein synthesis (IV-CFPS) analyses was used to evaluate the expression of the plantaricin genes. The in vitro-synthesized class IIb (two-peptide bacteriocins) plantaricin E/F (PlnE/F) exerted antimicrobial activity against three indicator microorganisms, including the well-known ichthyopathogen Lactococcus garvieae. Furthermore, MALDI-TOF MS on colonies detected the presence of a major peptide that matches the dimeric form of plantaricins E (PlnE) and F (PlnF). This study emphasizes the importance of genome sequencing and bioinformatic analysis for evaluating aquaculture probiotic candidates. Moreover, it provides valuable insights into their genetic features and antimicrobial mechanisms, paving the way for their application as probiotics in larviculture, which is a major bottleneck in aquaculture.

Antibiotic Resistance Genes, Virulence Factors, and Biofilm Formation in Coagulase-Negative Staphylococcus spp. Isolates from European Hakes (Merluccius merluccius, L.) Caught in the Northeast Atlantic Ocean.

The indiscriminate use of antibiotics has contributed to the dissemination of multiresistant bacteria, which represents a public health concern. The aim of this work was to characterize 27 coagulase-negative staphylococci (CoNS) isolated from eight wild Northeast Atlantic hakes (Merluccius merluccius, L.) and taxonomically identified as Staphylococcus epidermidis (n = 16), Staphylococcus saprophyticus (n = 4), Staphylococcus hominis (n = 3), Staphylococcus pasteuri (n = 2), Staphylococcus edaphicus (n = 1), and Staphylococcus capitis (n = 1). Biofilm formation was evaluated with a microtiter assay, antibiotic susceptibility testing was performed using the disk diffusion method, and antibiotic resistance and virulence determinants were detected by PCR. Our results showed that all staphylococci produced biofilms and that 92.6% of the isolates were resistant to at least one antibiotic, mainly penicillin (88.8%), fusidic acid (40.7%), and erythromycin (37%). The penicillin resistance gene (blaZ) was detected in 66.6% (18) of the isolates, of which 10 also carried resistance genes to macrolides and lincosamides (mphC, msr(A/B), lnuA, or vgaA), 4 to fusidic acid (fusB), and 3 to trimethoprim-sulfamethoxazole (dfrA). At least one virulence gene (scn, hla, SCCmecIII, and/or SCCmecV) was detected in 48% of the isolates. This study suggests that wild European hake destined for human consumption could act as a vector of CoNS carrying antibiotic resistance genes and/or virulence factors.

Immunomodulatory effects of bacteriocinogenic and non-bacteriocinogenic Lactococcus cremoris of aquatic origin on rainbow trout (Oncorhynchus mykiss, Walbaum).

Lactic Acid Bacteria (LAB) are a group of bacteria frequently proposed as probiotics in aquaculture, as their administration has shown to confer positive effects on the growth, survival rate to pathogens and immunological status of the fish. In this respect, the production of antimicrobial peptides (referred to as bacteriocins) by LAB is a common trait thoroughly documented, being regarded as a key probiotic antimicrobial strategy. Although some studies have pointed to the direct immunomodulatory effects of these bacteriocins in mammals, this has been largely unexplored in fish. To this aim, in the current study, we have investigated the immunomodulatory effects of bacteriocins, by comparing the effects of a wild type nisin Z-expressing Lactococcus cremoris strain of aquatic origin to those exerted by a non-bacteriocinogenic isogenic mutant and a recombinant nisin Z, garvicin A and Q-producer multi-bacteriocinogenic strain. The transcriptional response elicited by the different strains in the rainbow trout intestinal epithelial cell line (RTgutGC) and in splenic leukocytes showed significant differences. Yet the adherence capacity to RTgutGC was similar for all strains. In splenocyte cultures, we also determined the effects of the different strains on the proliferation and survival of IgM+ B cells. Finally, while the different LAB elicited respiratory burst activity similarly, the bacteriocinogenic strains showed an increased ability to induce the production of nitric oxide (NO). The results obtained reveal a superior capacity of the bacteriocinogenic strains to modulate different immune functions, pointing to a direct immunomodulatory role of the bacteriocins, mainly nisin Z.

Design of Lactococcus lactis Strains Producing Garvicin A and/or Garvicin Q, Either Alone or Together with Nisin A or Nisin Z and High Antimicrobial Activity against Lactococcus garvieae.

Lactococcus garvieae is a main ichthyopathogen in rainbow trout (Oncorhynchus mykiss, Walbaum) farming, although bacteriocinogenic L. garvieae with antimicrobial activity against virulent strains of this species have also been identified. Some of the bacteriocins characterized, such as garvicin A (GarA) and garvicin Q (GarQ), may show potential for the control of the virulent L. garvieae in food, feed and other biotechnological applications. In this study, we report on the design of Lactococcus lactis strains that produce the bacteriocins GarA and/or GarQ, either alone or together with nisin A (NisA) or nisin Z (NisZ). Synthetic genes encoding the signal peptide of the lactococcal protein Usp45 (SPusp45), fused to mature GarA (lgnA) and/or mature GarQ (garQ) and their associated immunity genes (lgnI and garI, respectively), were cloned into the protein expression vectors pMG36c, which contains the P32 constitutive promoter, and pNZ8048c, which contains the inducible PnisA promoter. The transformation of recombinant vectors into lactococcal cells allowed for the production of GarA and/or GarQ by L. lactis subsp. cremoris NZ9000 and their co-production with NisA by Lactococcus lactis subsp. lactis DPC5598 and L. lactis subsp. lactis BB24. The strains L. lactis subsp. cremoris WA2-67 (pJFQI), a producer of GarQ and NisZ, and L. lactis subsp. cremoris WA2-67 (pJFQIAI), a producer of GarA, GarQ and NisZ, demonstrated the highest antimicrobial activity (5.1- to 10.7-fold and 17.3- to 68.2-fold, respectively) against virulent L. garvieae strains.

Draft Genome Sequence of Lactococcus lactis Subsp. cremoris WA2-67: A Promising Nisin-Producing Probiotic Strain Isolated from the Rearing Environment of a Spanish Rainbow Trout (Oncorhynchus mykiss, Walbaum) Farm.

Probiotics are a viable alternative to traditional chemotherapy agents to control infectious diseases in aquaculture. In this regard, Lactococcus lactis subsp. cremoris WA2-67 has previously demonstrated several probiotic features, such as a strong antimicrobial activity against ichthyopathogens, survival in freshwater, resistance to fish bile and low pH, and hydrophobicity. The aim of this manuscript is an in silico analysis of the whole-genome sequence (WGS) of this strain to gain deeper insights into its probiotic properties and their genetic basis. Genomic DNA was purified, and libraries prepared for Illumina sequencing. After trimming and assembly, resulting contigs were subjected to bioinformatic analyses. The draft genome of L. cremoris WA2-67 consists of 30 contigs (2,573,139 bp), and a total number of 2493 coding DNA sequences (CDSs). Via in silico analysis, the bacteriocinogenic genetic clusters encoding the lantibiotic nisin Z (NisZ) and two new bacteriocins were identified, in addition to several probiotic traits, such as the production of vitamins, amino acids, adhesion/aggregation, and stress resistance factors, as well as the absence of transferable antibiotic resistance determinants and genes encoding detrimental enzymatic activities and virulence factors. These results unveil diverse beneficial properties that support the use of L. cremoris WA2-67 as a probiotic for aquaculture.

Draft Genome Sequence of Weissella cibaria P71, a Promising Aquaculture Probiotic Strain Isolated from Common Octopus (Octopus vulgaris).

Weissella cibaria P71 is a lactic acid bacterium that was isolated from common octopus (Octopus vulgaris) and previously showed interesting probiotic properties for turbot (Scophthalmus maximus L.) farming. The draft genome sequence of this strain provides further data to support its potential as a probiotic for aquaculture.

Biochemical, genetic and transcriptional characterization of multibacteriocin production by the anti-pneumococcal dairy strain Streptococcus infantarius LP90.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Cloning and expression of synthetic genes encoding native, hybrid- and bacteriocin-derived chimeras from mature class IIa bacteriocins, by Pichia pastoris (syn. Komagataella spp.).

In this work, synthetic genes designed from (a), the native amino acid sequence of the class IIa bacteriocins enterocin HF (EntHF) and enterocin CRL35 (EntCRL35), (b) from hybrid bacteriocins derived from fusion of enterocin A (EntA) to itself and to EntHF and EntCRL35 through a tri-glycine peptide linker, and (c) from bacteriocin-derived chimeras devised from fusion of the N-terminal region of EntA and enterocin P (EntP) to the C-terminal end of EntHF and EntCRL35, were cloned in plasmid pPICZαA for expression by P. pastoris X-33. Synthetic genes encoding EntHF and EntCRL35 were also cloned in plasmid pP-αhSUMO3 for expression of the hSUMO3-fused bacteriocins by P. pastoris. Only recombinant P. pastoris expressing the bacteriocin-derived chimeras displayed a direct antimicrobial activity whereas P. pastoris X-33, producer of EntP::EntHF, showed the highest antimicrobial activity in their supernatants and in the multi-step chromatographic purified fractions. The MRM-ESI-LC-MS/MS (QTRAP) analysis of purified fractions from P. pastoris producers of hybrid- and bacteriocin-derived chimeras, permitted detection in the samples of peptides with the expected molecular mass of the bacteriocins produced. The antimicrobial activity of the EntP::EntHF chimera compared to that of the synthetic EntP::EntHF peptide, suggest that the biologically-produced bacteriocin-derived chimera shows a higher specific antimicrobial activity than its synthetic counterpart against different Listeria strains, including L. monocytogenes. More important, the N-terminal region of EntA and EntP seems to drive the production, processing and secretion of hybrid- and bacteriocin-derived chimeras, by P. pastoris X-33.

Effects of in-competitive season power-oriented and heavy resistance lower-body training on performance of elite female water polo players.

We examined the effect of 16 weeks of lower-body resistance and power-oriented training on key performance measures of elite female water polo players. Twenty-one players were randomly assigned to 2 groups: control group (C) who did in-water training only and a lower body strength (LBS) group, who performed resistance (full squat and split squat) and jump and power-oriented lower-body training (countermovement jump [CMJ] loaded and CMJ) sessions (twice per week) in addition to the same in-water training. In-water training was conducted 5 days per week for a total of 16 weeks. Twenty-meter maximal sprint swim (MSS), lower-body strength during 1 repetition maximum (1RM) full squat (FS), in-water boost and CMJ, and Throwing speed (ThS) were measured before and after the training. Pretraining results showed no statistically significant differences between the groups in any of the variables tested. After 16 weeks, no statistically significant improvement was found in any of the variables measured in the C group, however, significant improvement was found in the LBS group: in-water boost (4.6 cm, 12.02%, effect size [ES] = 1.02), CMJ (2.4 cm, 8.66%, ES = 0.85), FS (12.7 kg, 20.99%, ES = 2.41), and ThS (3.4 km·h, 6.86%, ES = 3.44). Lower-body resistance and power-oriented training in female water polo players for 16 weeks produced significant improvements in performance qualities highly specific to water polo performance. Therefore, we propose modifications to current training methodology for female water polo players to include resistance and power-oriented training during the competitive season in this sport.

Self-integrating inductive loop for measuring high frequency pulses.

High frequency pulses can be measured by means of inductive sensors. The main advantage of these sensors consists of non-contact measurements that isolate and protect measuring equipment. The objective of this paper is to present the implementation of an inductive sensor for measuring rapidly varying currents. It consists of a rectangular loop with a resistor at its terminals. The inductive loop gives the derivative of the current according to Faraday's law and the resistor connected to the loop modifies the sensor's frequency response to obtain an output proportional to the current pulse. The self-integrating inductive sensor was validated with two sensors, a non-inductive resistor and a commercial high frequency current transformer. The results were compared to determine the advantages and drawbacks of the probe as an adequate inductive transducer.