Júlia Miranda Trindade’s research while affiliated with Federal University of Minas Gerais and other places

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.

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 cut-off 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 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.

Lactococcus petauri is an emergent fish pathogen detected in mortality outbreaks in Nile tilapia and rainbow trout farms on the American continent. Diseased fish exhibits erratic swimming, eye disorders, dark skin pigmentation among other clinical signs. The multilocus sequence typing (MLST) is a well-established methodology for the study of bacterial genetic diversity, which makes it possible to evaluate the ancestry for different isolates. Therefore, this study performed the genetic investigation of L. petauri strains isolated from Brazilian native fish species through MLST approach. A total of 24 L. petauri strains were used in this study. The isolates were obtained from six species of native fish (Arapaima gigas, Brycon amazonicus, Colossoma macropomum, Pseudoplatystoma corruscans, Pseudoplatystoma fasciatum and a hybrid o f Pseudoplatystoma) reared between 2012 and 2023 on commercial farms in four Brazilian states (Amazonas, Bahia, Mato Grosso do Sul and Minas Gerais). These isolates were previously identified at the species level using gyrB gene sequencing. MLST was performed by sequencing the internal fragments from seven housekeeping genes (als, atpA, tuf, gapC, gyrB, rpoC and galP) according to Ferrario et al. (2013). Sequence types (ST) were defined by analyzing the seven sequences in the Lactococcus g a r v i e a e MLST database (https://pubmlst.org/organisms/lactococcus-garvieae). Phyloviz software was used to identify clonal complexes (CC) among L. petauri strains isolated from fish on the American continent and deposited on PubMLST database. The 24 isolates analyzed were grouped in fifteen different STs, three previously reported (ST25, n=1; ST29, n=8; and ST35, n=3) and twelve new STs (nST1, nST2, nST3, nST4, nST5, nST6, nST7, nST8, nST9, nST10, nST11 and nST12). The ST29, ST35, nST2, nST3, nST4, nST5, nST7, nST8 and nST9 were grouped into CC29. nST1 was grouped into CC27 with ST27 and ST61, whereas the nST6 grouped with ST47. The ST25, nST10, nST11 and nST12 were characterized as singletons. Brazilian L. petauri isolates obtained from Nile tilapia were previously classified as ST24 (n=29 isolates) and ST47 (n=1), whereas North American strains from rainbow trout were classified as ST14 (n=3) and ST145 (n=11). In conclusion, the Brazilian L. petauri strains from native fish species constitute a genetically diverse population, where, the majority of the isolates (75%) belong to the same clonal complex. However, the CC29 together with other five STs, except nST6, are unrelated to MLST lineages identified in Nile tilapia and rainbow trout outbreaks, suggesting that piscine American L. petauri populations evolved from distinct ancestors.

Piscine lactococcosis is a disease caused by Lactococcus garvieae, L. petauri and L. formosensis. It is considered to be an emerging disease and poses a risk to global aquaculture production. Studies of the genetic diversity of Lactococcus spp. strains have epidemiological importance since they provide information on the genetic relationships between isolates and indicate geographic and temporal distribution. The aim of this study was to evaluate the genetic diversity of Brazilian Lactococcus spp. isolates obtained from native fish species using PCR-based DNA fingerprinting techniques. For this, 36 isolates were evaluated by gyrB sequencing, molecular serotyping, REP-, BOX-, and RAPD-PCR approaches. The discriminatory power and the congruence between the typing methods were calculated. After the correct taxonomic identification of the isolate, a total of 14, 5 and 17 isolates were reclassified as L. garvieae, L. formosensis and L. petauri, respectively. Lactococcus garvieae and L. petauri are related to serotype I, while L. formosensis strains were classified as serotype II. The typing methods tested are useful for evaluating the genetic diversity of isolates and a comprehensive indication of the diversity was found. Heterogeneous and homogeneous populations were observed for L. garvieae and L. petauri, respectively. Among the genotyping methods evaluated, RAPD-PCR demonstrated a greater discriminatory power for L. formosensis and L. petauri, while REP-PCR showed better results for L. garvieae strains, thus these methodologies are recommended for genotyping these bacterial species. In conclusion, Brazilian isolates of Lactococcus spp. obtained from native fish species constitute a genetically diverse population and the techniques used in this study allowed a better genetic characterization of the isolates.

Piscine lactococcosis is a disease caused by Lactococcus garvieae, L. petauri and L. formosensis. It is considered to be an emerging disease and poses a risk to global aquaculture production. Studies of the genetic diversity of Lactococcus spp. strains have epidemiological importance since they provide information on the genetic relationships between isolates and indicate geographic and temporal distribution. The aim of this study was to evaluate the genetic diversity of Brazilian Lactococcus spp. isolates obtained from native fish species using PCR-based DNA fingerprinting techniques. For this, 36 isolates were selected and submitted to gyrB sequencing, molecular serotyping, REP-, BOX-, and RAPD-PCR approaches. The discriminatory power and the congruence between the typing methods were calculated. After the correct taxonomic discrimination of the isolate, a total of 14, 5 and 17 isolates were reclassified as L. garvieae, L. formosensis and L. petauri, respectively. L. garvieae and L. petauri are related to serotype I, while L. formosensis strains were classified as serotype II. The typing methods tested are useful for evaluating the genetic diversity of isolates and a comprehensive indication of the diversity was found. Heterogeneous and homogeneous populations were observed for L. garvieae and L. petauri, respectively. RAPD-PCR demonstrated a greater discriminatory power for L. formosensis and L. petauri, while REP-PCR showed better results for L. garvieae strains, thus these methodologies are recommended for genotyping these bacterial species. In conclusion, Brazilian isolates of Lactococcus spp. obtained from native fish species constitute a genetically diverse population and the techniques used in this study allowed a better genetic characterization of the isolates.