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Quantitative PCR standard curves for (A) diluted P6 primer amplification products from L. petauri UCD-JR1 and (B) spiked water samples with gDNA from the same isolate. Spiked water samples were run in quadruplicate in three separate experiments. The figure is a composite of the three trials. PCR product dilutions were run once in triplicate.
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Piscine lactococcosis is a significant threat to cultured and wild fish populations worldwide. The disease typically presents as a per-acute to acute hemorrhagic septicemia causing high morbidity and mortality, recalcitrant to antimicrobial treatment or management interventions. Historically, the disease was attributed to the gram-positive pathogen...
Citations
... The precise microbiological diagnosis of L. garvieae is challenging because of the close homology of the species belonging to the genus Lactococcus. Importantly, L. garvieae, L. petauri, and L. formosensis cannot be discriminated using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), 16S sequencing, or biochemical tests [3,7]. In recent years, some relatively new approaches have been developed to identify these species: whole-genome sequencing, intergenic spacer 16S-23S sequencing, gyrB gene sequencing, multiplex PCR, and serotype analysis using PCR [7][8][9][10]. ...
... Importantly, L. garvieae, L. petauri, and L. formosensis cannot be discriminated using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), 16S sequencing, or biochemical tests [3,7]. In recent years, some relatively new approaches have been developed to identify these species: whole-genome sequencing, intergenic spacer 16S-23S sequencing, gyrB gene sequencing, multiplex PCR, and serotype analysis using PCR [7][8][9][10]. ...
... In recent years, new species closely related to Lactococcus garvieae have been described that cannot be discriminated using MALDI-TOF, 16S sequencing, or biochemical tests [3,7]. This is why new approaches have been developed to identify these species: whole-genome sequencing, intergenic spacer 16S-23S sequencing, gyrB gene sequencing, multiplex PCR, and serotype analysis using PCR [7][8][9][10]. ...
Lactococcosis caused by Lactococcus garvieae is a bacterial infection affecting fish with a considerable economic impact. Recently, L. garvieae has established itself as an opportunistic pathogen in humans. The aim of the current study was to test classical and molecular-biological methods for the identification of L. garvieae and examine antimicrobial susceptibility and capsule production, an important virulence factor. Additionally, tests for differentiation from closely related species, as well as epidemiological typing, were performed. In a period of 18 years (2002–2019), 24 isolates presumptively identified as L. garvieae were collected from Oncorhynchus mykiss and Salmo salar fish obtained either from retail stores or fish farms. In order to confirm the species, optimized PCR-based protocols were used. As a result, 21 of the tested strains were proved to be L. garvieae (n = 21). The remaining three isolates were Lactococcus lactis, Streptococcus iniae, and Enterococcus faecalis. Epidemiological typing by randomly amplified polymorphic DNA was performed. Except for a single KG+ isolate, all other strains belonged to the European capsular serotype KG−. All L. garvieae isolates showed susceptibility to all tested antibiotics with the exception of clindamycin, which was a diagnostic sign. A thorough optimization of diagnostic methods is essential to determining the etiology of specific infections affecting the personnel at risk in fish farms, the food industry, or within the broader community.
... This is because most of the articles, including some recent ones, did not perform the correct taxonomic classification of the isolates, which is currently recommended [47]. Only three studies assessed the antimicrobial resistance profile after correct species identification, using disk diffusion [10,48] and broth dilution [18,49]. Furthermore, Öztürk et al. [18] suggest that this heterogeneity is related to the overuse or misuse of antimicrobials at the farm level and the lack of established susceptibility cutoff values for each of the three species that cause piscine lactococcosis. ...
Lactococcus spp. has emerged as a pathogen that is affecting global aquaculture, with L. garvieae, L. petauri, and L. formosensis causing piscine lactococcosis. While antimicrobials are commonly used to treat diseases in aquaculture, reports of antimicrobial resistance in fish isolates are increasing. However, little is known about the susceptibility patterns of Lactococcus spp. strains isolated from native fish species in Brazil. This study aimed to assess the antimicrobial susceptibility of these strains and establish a provisional epidemiological cutoff value for L. garvieae using the normalized resistance interpretation approach. A total of 47 isolates were tested: 17 L. garvieae, 24 L. petauri, and 6 L. formosensis. The isolates were classified as wild-type (WT) or non-wild-type (NWT) based on inhibition zone diameters. Isolates classified as NWT for three or more antimicrobial classes were considered multidrug-resistant, and the multiple antibiotic resistance (MAR) index was calculated. The results revealed heterogeneity in antimicrobial resistance profiles, with higher resistance to trimethoprim/sulfamethoxazole and norfloxacin. Resistance to other antimicrobials, including florfenicol and oxytetracycline (approved for use in Brazil), varied according to the bacterial species. Lactococcus petauri (87.5%) and L. formosensis (66.7%) showed the highest multidrug resistance, compared to L. garvieae (11.7%), along with higher MAR index values. These findings suggest that multidrug-resistant strains could pose future challenges in the production of native species, underscoring the need for ongoing monitoring of antimicrobial resistance and responsible use of antimicrobials in aquaculture.
... It is important to highlight that some lactic acid bacteria can be potentially pathogenic for fish. For example, Enterococcus spp. is known fish pathogens from the order Lactobacillales, and members of the Streptococcaceae (Lactococcus garvieae, L. petauri, and Streptococcus spp.) have been implicated in significant losses in cultured fish (Abdelsalam et al. 2021;Swaminathan et al. 2021;Egger et al. 2023;Saticioglu et al. 2023;Heckman et al. 2024). Unfortunately, the methods used to characterize the bacterial microbiota in the present study do not provide the proper resolution to discriminate among species (Mignard and Flandrois 2006;Winand et al. 2020). ...
This study evaluated the supplementation of iron and copper nanoparticles in channel catfish diets and their influences on growth and health. A comparative feeding trial was carried out for 9 weeks to evaluate combinations of iron and copper nanoparticles: only iron nanoparticles (IronNP), only copper nanoparticles (CopperNP), CopperNP + IronNP, and a control diet supplemented with inorganic iron and copper (FeSO4 and CuSO4). After a 9-week feeding trial, growth performance, hematological parameters, whole-body proximate composition, and intestinal microbiota were evaluated, and fish were subjected to a bacterial challenge against Edwardsiella ictaluri to evaluate the contribution of the experimental treatments to fish health status. No statistical differences were detected for catfish fed the various diets in terms of production performance or survival after bacterial challenge. The hematocrit and RBC counts from fish fed the diet containing copper nanoparticles were significantly lower than the control group. A higher relative abundance of gram-positive bacteria was found in the digesta of catfish fed diets containing copper nanoparticles. Furthermore, in the context of hematology, iron nanoparticles did not impact the blood parameters of channel catfish; however, reduced hematocrits were observed in fish fed the copper nanoparticle diet, which lacked supplemental dietary iron, thus reinforcing the importance of dietary iron to catfish hematopoiesis. Nonetheless, additional studies are needed to investigate the effects of dietary copper nanoparticle supplementation in catfish diets to better illuminate its effects on the intestinal microbiota.
... Among them, L. garvieae is a significant global bacterial pathogen of both marine and freshwater environments, especially at higher water temperatures (≥ 15℃) [2,3] that causes hyperacute systemic infection [4]. Conventional diagnostic tools i.e. bacteriology, biochemical tests, mass spectrometry, 16s rRNA gene sequencing are insufficient for the differentiation at the species level, and hence, the use of species-specific gene amplification or sequencing of multiple conserved regions provides a holistic approach for species-level identification of bacteria [5]. The bacterium was formerly called Enterococcus seriolicida, a Gram-positive, facultatively anaerobic, short chains forming, alpha-haemolytic and non-motile ovoid cocci [6]. ...
Macrobrachium rosenbergii is a commercially important freshwater prawn cultured on a large scale mostly in south and south east Asian countries. Diseases are one of the bottlenecks for the successful culture and production of this important species. Lactococcus garvieae is a Gram-positive coccus commonly found in aquatic environments causing fish and shellfish diseases. In the present investigation, we have isolated and characterized L. garvieae, as etiological agent of white muscle disease in freshwater prawn juveniles. The infected prawn samples showing clinical signs of opaque and whitish muscles, sluggishness and mortality were collected and processed. The isolated bacterium was identified using biochemical methods and 16s rDNA sequencing and species-specific PCR. The sequence obtained revealed > 99% identity with L. garvieae reported elsewhere. Koch’s postulate was experimentally established through intramuscular challenge and the infected prawn muscle revealed massive coagulative necrosis with the presence of cocci. The isolate was found to be resistant to antibiotics namely clindamycin, cefoxitin, amoxicillin and co-trimoxazole. The heavy metal tolerance assay revealed the isolate to be tolerant to Cu2+ and Cr6+ and less tolerant to Hg2+. Class I integron was also identified in L. garvieae isolate. Further, the isolate was screened for several virulence genes and found to have hemolysins 1, 2, and 3, adhesin PsaA, adhesin Pav, enolase, LPxTG 1, 3, and 4, adhesin clusters 1 and 2, and adhesin in the PCR assay. It is the first report of L. garvieae infection in freshwater prawns in India and will pave the way for developing suitable preventive measures for future sustainable culture and production of this important aquaculture species.
Lactococcosis, primarily caused by Lactococcus garvieae , is prevalent in various freshwater fish species globally. Yet, its significance in Thai fish aquaculture remains poorly understood. This study investigated Lactococcosis‐associated diseases across four red tilapia ( Oreochromis sp.) and one Asian sea bass ( Lates calcarifer ) farm in Thailand in 2022. Chronically diseased fish displaying clinical signs such as exophthalmia or corneal opacity, erratic swimming, scale loss and skin haemorrhage, as well as apparently healthy specimens, were studied. Bacterial isolation was performed from the fish internal organs. Gram‐positive bacterial isolates were selected and later identified as L. lactis ( n = 4), L. taiwanensis ( n = 1), L. garvieae ( n = 2) and L. petauri ( n = 2) based on bacterial 16S rDNA and gyrB partial sequences. Sub‐adults of Nile tilapia were intracoelomic (IC) injected with representatives of each of the three Lactococcus species ( L. garvieae , L. lactis and L. taiwanensis ) at varying doses (10 ³ –10 ⁷ CFU/fish), while juvenile Asian sea bass were IC injected with L. petauri at doses ranging from 10 ² to 10 ⁸ CFU/fish. Control groups received culture medium alone. By the end of the 21‐day test, mortality rates from all challenge groups ranged from 0% to 14.82%. Some clinical signs were observed in groups challenged with higher doses of the bacteria. Enzyme‐linked immunosorbent assay (ELISA) showed that survivor fish exposed to high bacterial doses elicited specific antibodies IgM. In summary, this study identifies the presence of Lactococcus species in farmed fishes, which exhibit low virulence in Nile tilapia and Asian sea bass under laboratory‐controlled challenges. These bacterial isolates appear to be opportunistic rather than pathogenic, potentially indicating underlying health challenges within the affected farms.
Lactococcus petauri is an emerging pathogen causing piscine lactococcosis in various aquatic species. This disease has been increasingly reported as a cause of mortality in aquaculture. Due to the indiscriminate use of antimicrobials and to promote viable alternatives for their replacement, bioactive peptides with antimicrobial properties can be explored as tools against bacterial infections. Thus, the objective of this study was to identify the pathogen responsible for a mortality outbreak in cage-cultured Nile tilapia (Oreochromis niloticus), assess its pathogenicity, and evaluate the therapeutic efficacy of a commercial bioactive peptide and florfenicol against L. petauri. The bacterium was isolated from affected Nile tilapia exhibiting external and internal hemorrhages, ocular opacity, anorexia, and ascites. Koch’s postulates were fulfilled by inoculating healthy fish with 3.6 × 107 CFU/mL of the bacterium, which induced clinical signs of generalized hemorrhage, ascites, and melanosis. Histopathological analysis revealed severe lesions in the liver, spleen, kidney, intestine, and heart, confirming the pathogen's high virulence in Nile tilapia. To evaluate potential treatments, fish were divided into six groups: G1) inoculated with L. petauri and treated with 15 mg/kg of bioactive peptide via gavage; G2) inoculated and treated with 15 mg/kg of florfenicol via gavage; G3) inoculated and treated with intraperitoneal injection of bioactive peptide (15 mg/kg); G4) inoculated and left untreated; G5) treated with PBS via gavage; and G6) inoculated with PBS. After 30 days, survival rates were G1: 36.85%, G2: 16.79%, G3: 26.44%, and G4: 6.7%. Significantly higher survival was observed in groups G1, G2, and G3 compared to G4. Moreover, bacterial persistence was only absent in groups G1 and G3. The study demonstrates the effectiveness of the bioactive peptide in treating L. petauri infections and preventing bacterial persistence in Nile tilapia, suggesting it is a viable alternative to traditional antimicrobials.
Lactococcus garvieae has been considered for a long time the only causal agent of lactococcosis. In recent years, different papers reported the involvement of other two bacterial species: Lactococcus petauri and Lactococcus formosensis. A different host tropism has been described for these species where L. garvieae and L. petauri are predominant species in fish and humans’ infections, while L. formosensis in bovine. L. garvieae has been reported as rare infectious agent in dog. This paper represents the first isolation of L. petauri in two domesticated dog cases from urine and skin samples, respectively. The recovered L. petauri has been identified using PCR and sequencing based on Internal Transcribe Spacer (ITS) and phylogenetic analysis showed that it belongs to the L. petauri cluster with a 100% of identity with sequences previously reported from fish isolates while there were differences with L. petauri isolated from urinary tract infection from humans. L. petauri in human infection has been considered not necessarily deriving from the ingestion of contaminated food but rather as an opportunistic pathogen colonization intestinal tract. Differences among virulotypes have been reported for humans and dogs, and a comparison was also made between the virulotyping of L. petauri and L. garvieae in dogs. The antimicrobial pattern showed susceptibility for the election treatment molecules. These data contribute to our understanding of the host trophism of this species which was misclassified for long time and provide new data on its virulence factors and antimicrobial resistance.
