Bacterial pathogens isolated from turbot ( Scophthalmus maximus ) in Türkiye: Preliminary findings from a one-year study

Lactococcosis, a disease caused by Lactococcus garvieae, L. formosensis, and L. petauri, has been continuously documented to inflict significant economic losses on cultured fish species globally. This study reported the first identification of Lactococcus petauri from reared turbot (Scophthalmus maximus) using biochemical tests, 16S rRNA gene sequence, and multiplex PCR assay with species-specific primers. The 16S rRNA partial sequence generated in this study has been deposited in the GenBank database under accession number PQ669659 and compared with available sequences in the GenBank database. To our knowledge, this study reports a L. petauri infection for the first time in flat fish aquaculture, offering valuable contributions to understanding lactococcosis systematics and molecular epidemiology both in Türkiye and globally. This first report provides valuable insights into this species’ ecological distribution, pathogenicity, and adaptability, laying the groundwork for future fish pathology and aquatic microbiology studies.

There has been a tremendous increase in the number and types of various industries, with all industrial operations generating wastes in one form or the other. Electroplating sector contributes a major part in deteriorating the local environment at a massive scale due to the persistent accumulation of heavy metals in the environment. Nature of the microbial biodiversity of industrial effluents, in particular, the effluent from the electroplating industry remains largely uncharacterized. In this study, a unique set of chemo-heterotrophic organisms comprising of nine bacteria and eleven fungi were isolated from the effluents of a gold electroplating industry effluent, located in Mumbai, India. The culture isolates were identified by biochemical tests and partial 16S rRNA and 18S rRNA gene sequence matches. Among the cultures isolated and identified, four were novel and hitherto unreported species of bacteria and ten were new strains of fungi. The sequence data of novel isolates obtained were submitted at NCBI, GenBank to acquire unique accession numbers subsequent to which new strain designations were given to them by the authors. The bacterial isolates were designated as Staphylococcus sp. ss-1, Achromobacter sp. ss-2, Macrococcus sp. ss-4 and Bacillus sp. ss-6. New strain designations were assigned to the fungal isolates as Talaromyces marneffei strain GEF-1, Penicillium pinophilum strain GEF-2, Curvularia lunata strain GEF-3, Aspergillus tamarii strain GEF-4, Aspergillus tamarii strain GEF-5, Aspergillus sydowii strain GEF-6, Aspergillus flavus strain GEF-7, Aspergillus niger strain GEF-8, Aspergillus awamori strain GEF-9 and Cladosporium sphaerospermum strain GEF-11. The study highlights the presence of new isolates endowed with metal resistance genes possibly for multiple heavy metal resistance to be able to survive in several heavy metal polluted environment. Indigenous microorganisms isolated from the electroplating effluents may be studied to examine their potential to produce enzymes, and bioactive compounds for application in agricultural, pharmaceutical, industrial, environmental and medical sciences.

Unlike phenotypic methods of bacterial identification, 16S rRNA gene sequencing is an innovative DNA-based technique for the rapid identification of foodborne pathogens. In this study, the ability of both partial 16S rRNA gene sequencing and the API Rapid20E, for identification of 48 different bacterial strains isolated from meat products, plus 2 control bacterial strains, was compared. Genotypically, the partial (770 bp) 16S rRNA gene sequencing was able to identify 38 (76%) isolates to the species level and 11 (22%) isolates to the genus level, whereas only one (2%) isolate could not be correctly identified. Phenotypically, the API Rapid20E could identify 24 (48%) isolates to the specie level, and 14 (28%) isolates to the genus level, while 12 (24%) isolates could not be discriminated at any taxonomic level. The results revealed that partial 16S rRNA gene sequencing was useful in ascertaining the relevance of tested strains; hence, it can be used in food microbiology laboratory for correct and rapid identification of foodborne pathogens.

Unlike phenotypic methods of bacterial identification, 16S rRNA gene sequencing is an innovative DNA-based technique for the rapid identification of foodborne pathogens. In this study, the ability of both partial 16S rRNA gene sequencing and the API Rapid20E, for identification of 48 different bacterial strains isolated from meat products, plus 2 control bacterial strains, was compared. Genotypically, the partial (770 bp) 16S rRNA gene sequencing was able to identify 38 (76%) isolates to the species level and 11 (22%) isolates to the genus level, whereas only one (2%) isolate could not be correctly identified. Phenotypically, the API Rapid20E could identify 24 (48%) isolates to the specie level, and 14 (28%) isolates to the genus level, while 12 (24%) isolates could not be discriminated at any taxonomic level. The results revealed that partial 16S rRNA gene sequencing was useful in ascertaining the relevance of tested strains; hence, it can be used in food microbiology laboratory for correct and rapid identification of foodborne pathogens.

Transcriptome analysis of turbot (Scophthalmus maximus) kidney responses to inactivated bivalent vaccine against Aeromonas salmonicida and Edwardsiella tarda

BackgroundShiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen, that is transmitted from a variety of animals, especially cattle to humans via contaminated food, water, feaces or contact with infected environment or animals. The ability of STEC strains to cause gastrointestinal complications in human is due to the production of Shiga toxins (sxt). However, the transmission of multidrug-resistance STEC strains are linked with a severity of disease outcomes and horizontal spread of resistance genes in other pathogens. The result of this has emerged as a significant threat to public health, animal health, food safety, and the environment. Therefore, the purpose of this study is to investigate the antibiogram profile of enteric E. coli O157 isolated from food products and cattle faeces samples in Zagazig City, Al-Sharkia, Egypt, and to reveal the presence of Shiga toxin genes stx1 and stx2 as virulence factors in multidrug-resistant isolates. In addition to this, the partial 16S rRNA sequencing was used for the identification and genetic recoding of the obtained STEC isolates.ResultsThere was a total of sixty-five samples collected from different geographical regions at Zagazig City, Al-Sharkia—Egypt, which were divided into: 15 chicken meat (C), 10 luncheon (L), 10 hamburgers (H), and 30 cattle faeces (CF). From the sixty-five samples, only 10 samples (one from H, and 9 from CF) were identified as suspicious E. coli O157 with colourless colonies on sorbitol MacConkey agar media with Cefixime- Telurite supplement at the last step of most probable number (MPN) technique. Eight isolates (all from CF) were identified as multidrug-resistant (MDR) as they showed resistance to three antibiotics with multiple antibiotic resistance (MAR) index ≥ 0.23, which were assessed by standard Kirby-Bauer disc diffusion method. These eight isolates demonstrated complete resistance (100%) against amoxicillin/clavulanic acid, and high frequencies of resistance (90%, 70%, 60%,60%, and 40%) against cefoxitin, polymixin, erythromycin, ceftazidime, and piperacillin, respectively. Those eight MDR E. coli O157 underwent serological assay to confirm their serotype. Only two isolates (CF8, and CF13), both from CF, were showed strong agglutination with antisera O157 and H7, as well as resistance against 8 out of 13 of the used antibiotics with the highest MAR index (0.62). The presence of virulence genes Shiga toxins (stx1 and stx2) was assessed by PCR technique. CF8 was confirmed for carrying stx2, while CF13 was carrying both genes stx1, and stx2. Both isolates were identified by partial molecular 16S rRNA sequencing and have an accession number (Acc. No.) of LC666912, and LC666913 on gene bank. Phylogenetic analysis showed that CF8, and CF13 were highly homologous (98%) to E. coli H7 strain, and (100%) to E. coli DH7, respectively.ConclusionThe results of this study provides evidence for the occurrence of E. coli O157:H7 that carries Shiga toxins stx1 and/or stx2, with a high frequency of resistance to antibiotics commonly used in human and veterinary medicine, in Zagazig City, Al-Sharkia, Egypt. This has a high extent of public health risk posed by animal reservoirs and food products with respect to easy transmission causing outbreaks and transfer resistance genes to other pathogens in animal, human, and plants. Therefore, environmental, animal husbandry, and food product surveillance, as well as, clinical infection control, must be strengthened to avoid the extra spread of MDR pathogens, especially MDR STEC strains.

Objective: Species identification of Staphylococcus is the prerequisite for precise assessment of
 microbial dynamics including their transmission and pathogenic significance in
 the dairy herd environment. The present study aimed to develop simplex PCR
 assays for rapid and specific identification of nine different Staphylococcus species. 
 
 Methods: Specific
 primers targeting sodA gene for S. aureus, S. chromogenes, S. hominis,
 S. haemolyticus, S. hyicus; gap gene for S. sciuri, S. auricularis, S. simulans;
 and rdr gene for S. epidermidis were designed. The PCR assays were evaluated against
 28 ATCC reference strains and 209 composite milk samples. Partial 16S rRNA sequencing was performed to
 reconfirm the results. 
 
 Results: The
 PCR assays allowed species level identification of 348 staphylococcal field
 isolates recovered from 209 milk samples. The identification pattern was S. aureus (n=101), S. chromogenes (n=89), S.
 epidermidis (n=57), S. sciuri
 (n=43), S. haemolyticus (n=34), S. hyicus (n=13), S. hominis (n=5), S.
 auricularis (n=3) and S. simulans (n=3). The PCR based species
 identification was in 100% concordance with the partial 16S rRNA gene sequencing approach.
 
 Conclusion: The simplex PCR assays can be used as a precise tool
 for routine identification of Staphylococcus
 species from bovine milk as Staphylococcus
 species including coagulase-negative staphylococci is recognized a major cause
 of bovine mastitis in different parts of the world including India. J Microbiol Infect Dis 2018; 8(3):120-127

Background: Rapidly growing mycobacteria (RGM) are ubiquitous in the environment, can be isolated from soil and wa­ter, and demonstrate visible growth on culture media within seven days. Mycobacterium smegmatis is an acid-alcohol fast bacterium, which belong to RGM group. The diagnosis of M. smegmatis infections may be quite difficult by conventional methods; therefore, biochemistry associated to nucleic acid-based approaches provided fast and accurate identification. Although this specie may be associated to animals and humans infections, there is few cases description. Nontuberculous mycobacterial bovine mastitis is uncommon, and bovine mastitis by M. smegmatis has been reported but non-confirmed case once in the past. This paper reports M. smegmatis recovered from a cattle with relapsing pyogranulomatous mastitis.Case: Milk samples from an adult Holstein cow showing relapsing pyogranulomatous mastitis history and by pronounced glandular hardening were cultivated and analyzed accordingly to standard milk cultivation protocols. The animal had been subject to several intramammary and parenteral antibiotic therapies protocols without adequate response. After 48 h incubation, a slow and sparse growth of slightly pigmented, shiny and smooth colonies was observed on the blood agar plate. The bacterium isolated was named as strain 55/08. The morphological and biochemical profile were tested, and the ability of the isolate to grow at Lowenstein-Jensen slants was confirmed. The isolated have showed positive reaction to catalase, glucose, sucrose, mannitol and nitrate. The pigment formation was observed for 14 days incubation, and the colonies produce pigment after prolonged time. Gram and Ziehl-Neelsen staining revealed poorly pigmented, irregular, slender Gram-positive and acid fast rods. The staining and biochemical profile showed closed isolated relationship to M. smegmatis. A discriminatory identification based in the 16S rRNA gene sequence analysis was performed. The total DNA from the strain 55/08 was extracted and the partial 16S rRNA sequence was amplified, using prokaryotic universal primer pairs and the extract DNA as template, by PCR assay following the purification and sequencing of the amplicons. A total of 1,443 nucleotides form consensus sequence were alignment to M. smegmatis and other mycobacteria 16S rRNA avail­able sequences. The sequence analysis confirmed the M. smegmatis identification as etiological agent of bovine relapsing pyogranulomatous mastitis. M. smegmatis strain 55/08 partial 16S rRNA gene sequence was submitted to GenBank. The phylogenetic relationship of the strain 55/08 with other mycobacteria was performed in order to confirm the identification of the isolate as M. smegmatis. Discussion: Nontuberculous mycobacteria are uncommon causes of bovine mastitis. Some old reports have described M. smegmatis as etiological agent of mastitis, but without definitive diagnostic. M. smegmatis mammary quarter introduction may be related to the repeated intramammary treatment protocols, because this mycobacteria is related to environmental infections. The relapsing pyogranulomatous mastitis infection could be associated to other bacteria species. However, the phenotypic and molecular characterization which was performed demonstrated the accurate identification of the isolated as M. smegmatis. Milk contaminated by M. smegmatis may be a potential infection source for human and other animal species. This report reinforces the need to optimize quality programs and laboratorial diagnosis to further the accurate microorganism identification in milk samples.Keywords: RGM mycobacteria, relapsing mastitis, M. smegmatis, molecular identification.

A Comparison of Techniques used to Distinguish Strains of Prevotella intermedia from Prevotella nigrescens

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

Species of the genus Dendrodoris are defined as “radula-less” dorids (Nudibranchia: Doridina: Porostomata: Dendrodorididae). These sea slugs are colorful nudibranchs distributed across the world’s oceans. Because of color variations and anatomical similarities, species discrimination has been disputed in several Dendrodoris species. We determined the partial mitochondrial COI gene sequences and 18S rRNA gene sequences of five Dendrodoris species (D. arborescens, D. denisoni, D. guttata, D. nigra, and D. rubra) and Doriopsilla miniata in Japan to clarify the species identity of each dendrodorid species. We examined a total of 50 specimens from the intertidal zone at 10 sites in four regions along the Pacific coast of Japan. Partial COI gene sequences significantly differed among the six species. Although six partial 18S rRNA gene sequences were obtained for the six species, sequences differed little among species and did not clarify details of the Dendrodoris phylogeny. We also described the morphological features of veliger larvae of three Dendrodoris species. (D. arborescens, D. guttata, and D. rubra) The veligers of D. arborescens had a brown-pigmented, cup-shaped shell, and its foot was not retracted into the shell. In contrast, the veligers of D. guttata and D. rubra had a transparent, coiled shell with an operculum. Mean shell lengths of D. arborescens, D. rubra, and D. guttata were 171.5 ± 8.0, 172.8 ± 4.9, and 222.3 ± 15.2 μm, respectively. The present molecular analysis and larval characteristics indicated that each of the six species is a distinct species.

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees

From our recent survey of non-pigmented rapidly growing mycobacteria in the Parisian water system, three groups of isolates (taxons 1-3) corresponding to possible novel species were selected for taxonomic study. The three taxa each formed creamy white, rough colonies, had an optimal growth temperature of 30 °C, hydrolyzed Tween 80, were catalase-positive at 22 °C and expressed arylsulfatase activity. All three were susceptible to amikacin, ciprofloxacin and tigecycline. The three taxa produced specific sets of mycolic acids, including one family that has never previously been described, as determined by thin layer chromatography and nuclear magnetic resonance. The partial rpoB sequences (723 bp) showed 4-6 % divergence from each other and more than 5 % differences from the most similar species. Partial 16S rRNA gene sequences showed 99 % identity within each species. The most similar sequences for 16S rRNA genes (98-99 % identity over 1444-1461 bp) were found in the Mycobacterium fortuitum group, Mycobacterium septicum and Mycobacterium farcinogenes. The three taxa formed a new clade (bootstrap value, 99 %) on trees reconstructed from concatenated partial 16S rRNA, hsp65 and rpoB sequences. The above results led us to propose three novel species for the three groups of isolates, namely Mycobacterium lutetiense sp. nov. [type strain 071T=ParisRGMnew_1T (CIP 110656T=DSM 46713T)], Mycobacterium montmartrense sp. nov. [type strain 196T=ParisRGMnew_2T (CIP 110655T=DSM 46714T)] and Mycobacteriu marcueilense sp. nov. [type strain of 269T=ParisRGMnew_3T (CIP 110654T=DSM 46715T)].

Comparative transcriptome analysis reveals the immune response of turbot (Scophthalmus maximus) induced by inactivated bivalent vaccine

The cultivated and uncultivated bacterial communities of an activated sludge plant were studied. Two samples were taken and a total of 516 bacterial isolates were classified into groups using their whole-cell protein patterns. The distribution of bacteria into protein-pattern groups differed significantly between the two samples, suggesting variation in culturable bacterial flora. Partial 16S rRNA gene sequences were determined for representatives of the commonest protein-pattern groups. Most of the sequences obtained were previously unknown, but relatively closely related to known sequences of organisms belonging to the alpha, beta or gamma subclasses of the proteobacteria, the first two subclasses being predominant. This classification of bacteria isolated on a diluted nutrient-rich medium differed from recent culture-dependent studies using nutrient-rich media. The uncultivated bacterial community was studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of the cloned sequences was identical to those determined for culturable organisms; or to those in the GenBank database. They were, however, related to the alpha or beta subclasses of the proteobacteria, or to the gram-positive bacteria with a high G + C DNA content.