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Journal of Applied Microbiology Jul 2021The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis...
Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.
AIM
The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches.
METHODS AND RESULTS
Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 10 to 2·2 × 10 cells 100 mL .
CONCLUSIONS
Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices.
SIGNIFICANCE AND IMPACT OF THE STUDY
The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.
Topics: Agriculture; Animals; Arcobacter; Campylobacteraceae; DNA Primers; DNA, Bacterial; Feces; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; RNA, Ribosomal, 16S; Reproducibility of Results; Sensitivity and Specificity; Species Specificity; Water Microbiology
PubMed: 33174331
DOI: 10.1111/jam.14926 -
Foods (Basel, Switzerland) Feb 2022There is growing interest in Baltic herring () and other undervalued, small-sized fish species for human consumption. Gutting or filleting of small-sized fish is...
There is growing interest in Baltic herring () and other undervalued, small-sized fish species for human consumption. Gutting or filleting of small-sized fish is impractical; hence, the aim of this study was to explore the suitability of the whole (ungutted) herring for food use. The microbiological quality of commercially fished whole and gutted herring was analysed with culture-dependent methods combined with identification of bacterial isolates with MALDI-TOF Mass Spectrometry and culture-independent 16S rRNA gene amplicon sequencing. Whole and gutted herring had between 2.8 and 5.3 log CFU g aerobic mesophilic and psychrotrophic bacteria and between 2.2 and 5.6 log CFU g H₂S-producing bacteria. Enterobacteria counts remained low in all the analysed herring batches. The herring microbiota largely comprised the phyla Proteobacteria, Firmicutes, and Actinobacteria (71.7% to 95.0%). , , and were the most frequently isolated genera among the viable population; however, with the culture-independent approach, followed by were the most abundant genera. In some samples, a high relative abundance of the phylum Epsilonbacteraeota, represented by the genus , was detected. This study reports the bacterial diversity present in Baltic herring and shows that the microbiological quality was acceptable in all the analysed fish batches.
PubMed: 35205969
DOI: 10.3390/foods11040492 -
Journal of Food Protection Jul 2021Campylobacter spp. and Arcobacter butzleri are foodborne pathogens associated with the consumption of contaminated raw chicken meat. At the industry level, the...
ABSTRACT
Campylobacter spp. and Arcobacter butzleri are foodborne pathogens associated with the consumption of contaminated raw chicken meat. At the industry level, the combination of new and common antimicrobials could be used as a strategy to control the presence of pathogens in chicken carcasses. The objective of this study was to determine the bacteriostatic and bactericidal effects of a mixture of chlorine (Cl) and sodium gallate (SG) on a mixture of two Campylobacter species (Campylobacter jejuni and Campylobacter coli) and A. butzleri. Using a central composite experimental design, it was established that the optimum inhibitory SG-Cl concentration for Campylobacter spp. was 44 to 45 ppm. After 15 h of incubation, Campylobacter species growth was reduced by 37.5% and the effect of Cl was potentiated by SG at concentrations above 45 ppm. In the case of A. butzleri, optimum levels of 28 and 41 ppm were observed for SG and Cl, respectively; no synergism was reported, as this bacterium was more sensitive to lower Cl concentrations than Campylobacter. After a 20-min pretreatment with peracetic acid (50 ppm), the optimum condition to achieve a >1.0-Log CFU/mL reduction of Campylobacter spp. was exposure to 177 ppm of Cl and 44 ppm of SG for 56 min. As A. butzleri showed lower resistance to the bacteriostatic effect of the Cl-SG combination, it was assumed that optimum bactericidal conditions for Campylobacter spp. were effective to control the former; this was confirmed with subsequent validation of the model. The SG-Cl combination has bactericidal properties against Campylobacter and A. butzleri, and it may be a useful strategy to improve sanitary practices applied in the poultry industry.
Topics: Animals; Arcobacter; Campylobacter; Chickens; Chlorine; Food Microbiology; Meat; Sodium
PubMed: 33428726
DOI: 10.4315/JFP-20-181 -
Frontiers in Cellular and Infection... 2022Intestinal autochthonous bacteria play important roles in the maintenance of the physiological homeostasis of animals, especially contributing to the host immune system....
Intestinal autochthonous bacteria play important roles in the maintenance of the physiological homeostasis of animals, especially contributing to the host immune system. In the present study, the variation of autochthonous bacterial community in the intestinal tract of 2-7 months-old tiger pufferfish and bacterial communities in the seawater of recirculating aquaculture system (RAS) and the following offshore sea cage aquaculture system (OSCS) were analyzed during the aquaculture period from May to October 2021. Proteobacteria was found to be the most dominant phyla in both intestinal and seawater bacterial communities, which accounted for 68.82% and 65.65% of the total bacterial abundance, respectively. was the most core bacterial taxon in the intestinal bacterial community, with the most dominant abundance (42.89%) at the genus level and dominant positions in co-occurrence relationships with other bacterial taxa (node-betweenness value of 150). Enterococcaceae was specifically enriched in the intestinal bacterial community of pufferfishes from RAS, while Vibrionaceae was enriched in the intestinal bacterial community from OSCS. The F-values of beta diversity analysis between intestinal and seawater bacterial communities generally increased from May (6.69) to October (32.32), indicating the increasing differences between the intestinal and seawater bacterial communities along with the aquaculture process. Four bacterial taxa of sp., , sp. and had significant correlations with immune response parameters, and they were suggested to be the indicators for immune status and pathological process of pufferfish. The knowledge about the specific core bacteria, potentially pathogenic bacteria and the change of bacterial community in the intestinal tract of cultured pufferfish is of great scientific significance and will contribute to the understanding of intestinal bacterial homeostasis and biosecurity practice in pufferfish aquaculture.
Topics: Animals; Takifugu; Bacteria; Proteobacteria
PubMed: 36583108
DOI: 10.3389/fcimb.2022.1062512 -
Pathogens (Basel, Switzerland) Mar 2020Routine diagnostic methods for the aetiologic agents of diarrhoea in most developing countries are usually not sensitive enough, leading to under-diagnosis. Thus, this...
Routine diagnostic methods for the aetiologic agents of diarrhoea in most developing countries are usually not sensitive enough, leading to under-diagnosis. Thus, this study investigated possible mixed diarrhoeal aetiology by using cultures and real-time polymerase chain reactions (PCR) in children younger than four years old in the Northwest Province, South Africa. In total, 505 stool samples were collected from symptomatic and asymptomatic children who were attending three clinics and the Brits hospital in Madibeng District, between September 2016 and December 2017. Rotavirus, norovirus, , , and diarrhoeagenic (DEC) were targeted. spp. (24.6%), (15.8%) and DEC (19.6%) were detected using PCR; only spp. (29.7%) and DEC (26.9%) were detected through the culture. (36%), (28%), (12%), and (15.8%) were the only spp. of and r identified. The gene (31.4%) of enteropathogenic /enterohaemorrhagic (EPEC/EHEC) was the most prevalent DEC virulence gene (VG) identified. and were detected at 23.4% and 20%, respectively. Mixed viral aetiology (7.3%) and the co-infection of and (49%) were recorded. A mixed bacterial-viral aetiology was observed in 0.6% of the specimens. Sensitive diagnostic procedures like PCR should be considered to provide the best treatment to children experiencing diarrhoea.
PubMed: 32155961
DOI: 10.3390/pathogens9030198 -
The ISME Journal Aug 2023Dinitrogen (N) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent...
Dinitrogen (N) fixation is the major source of reactive nitrogen in the ocean and has been considered to occur specifically in low-latitude oligotrophic oceans. Recent studies have shown that N fixation also occurs in the polar regions and thus is a global process, although the physiological and ecological characteristics of polar diazotrophs are not yet known. Here, we successfully reconstructed diazotroph genomes, including that of cyanobacterium UCYN-A (Candidatus 'Atelocyanobacterium thalassa'), from metagenome data corresponding to 111 samples isolated from the Arctic Ocean. These diazotrophs were highly abundant in the Arctic Ocean (max., 1.28% of the total microbial community), suggesting that they have important roles in the Arctic ecosystem and biogeochemical cycles. Further, we show that diazotrophs within genera Arcobacter, Psychromonas, and Oceanobacter are prevalent in the <0.2 µm fraction in the Arctic Ocean, indicating that current methods cannot capture their N fixation. Diazotrophs in the Arctic Ocean were either Arctic-endemic or cosmopolitan species from their global distribution patterns. Arctic-endemic diazotrophs, including Arctic UCYN-A, were similar to low-latitude-endemic and cosmopolitan diazotrophs in genome-wide function, however, they had unique gene sets (e.g., diverse aromatics degradation genes), suggesting adaptations to Arctic-specific conditions. Cosmopolitan diazotrophs were generally non-cyanobacteria and commonly had the gene that encodes the cold-inducible RNA chaperone, which presumably makes their survival possible even in deep, cold waters of global ocean and polar surface waters. This study shows global distribution pattern of diazotrophs with their genomes and provides clues to answering the question of how diazotrophs can inhabit polar waters.
Topics: Seawater; Nitrogen Fixation; Ecosystem; Oceans and Seas; Cyanobacteria
PubMed: 37217593
DOI: 10.1038/s41396-023-01424-x -
Frontiers in Microbiology 2020(formerly ) is a globally emerging foodborne and zoonotic pathogen. However, little is known about the species' genomic features and diversity, antibiotic resistance...
(formerly ) is a globally emerging foodborne and zoonotic pathogen. However, little is known about the species' genomic features and diversity, antibiotic resistance and virulence. In this study, 27 strains from water poultry in Thuringia, Germany, were investigated using whole-genome sequencing. Four of these strains were sequenced using long- and short-read sequencing methods to obtain circularized genomes. The German strains belong to the cluster I. Cluster I genomes exhibited a high degree of genetic diversity in which variable sites comprised 9.1% of the core genome. The German strains formed three subgroups that contained 2, 6, and 9 strains, respectively. The genomic analysis of cluster I revealed variable presence of mobile elements and that 65% of the strains lack CRISPR systems. The four circularized genomes carried a ∼2 Mbp chromosome and a single megaplasmid (size 98.1-154.5 Kbp). The chromosome was densely packed with coding sequences (∼92%) and showed inversions and shifts in the gene blocks between different strains. Antimicrobial resistance was assessed using a gradient strip diffusion method and showed that all 27 strains were resistant to cefotaxime and susceptible to erythromycin, gentamicin, and ampicillin. Sixteen strains were also resistant to ciprofloxacin, whereas 23 were resistant to streptomycin. The genetic prediction of antibiotic resistance identified numerous efflux pumps similar to those found in . All strains harbored two beta-lactamase genes which may explain the cefotaxime resistance. A correlation between the A point mutation (Thr-85-Ile) and ciprofloxacin resistance was partially discovered in 15 out of 16 strains. virulence profiling showed a wide range of virulence factors including a full chemotaxis system and most of the flagellar genes. In contrast to , no urease cluster was found. This study provides new insights into the genomic variability of strains of cluster I. The different genetic makeup of these strains may contribute to the virulence of strains and the severity of the infections in humans.
PubMed: 32754133
DOI: 10.3389/fmicb.2020.01549 -
Antibiotics (Basel, Switzerland) Jan 2022The purpose of this study was to test the in vitro effects of ampicillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and tetracycline on planktonic cells of...
The purpose of this study was to test the in vitro effects of ampicillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and tetracycline on planktonic cells of -like microorganisms and on their biofilm formation ability. The minimum inhibitory concentrations (MICs) were determined by the microdilution method. Further, biofilm formation ability in the presence of various concentrations of antibiotics was evaluated by a modified Christensen method. Most of the 60 strains exhibited high susceptibility to gentamicin (98.3%), ciprofloxacin (95.0%), and erythromycin (100.0%). High level of resistance was observed to clindamycin and tetracycline with MIC and MIC in range of 4-32 mg/L and 32-128 mg/L, respectively. Combined resistance to both clindamycin and tetracycline was found in 38.3% of tested strains. In general, higher biofilm formation was observed especially at lower concentrations of antibiotics (0.13-2 mg/L). However, a significant decrease in biofilm formation ability of LMG 25694 was exhibited with ampicillin and clindamycin at concentrations above 32 or 8 mg/L, respectively. Biofilm formation represents a potential danger of infection and also a risk to human health, in particular due to antimicrobial-resistant strains and the ability to form a biofilm structure at a concentration that is approximately the MIC determined for planktonic cells.
PubMed: 35052964
DOI: 10.3390/antibiotics11010087 -
Animal Microbiome May 2021Oral diseases are common in dogs, with microbiota playing a prominent role in the disease process. Oral cavity habitats harbor unique microbiota populations that have...
BACKGROUND
Oral diseases are common in dogs, with microbiota playing a prominent role in the disease process. Oral cavity habitats harbor unique microbiota populations that have relevance to health and disease. Despite their importance, the canine oral cavity microbial habitats have been poorly studied. The objectives of this study were to (1) characterize the oral microbiota of different habitats of dogs and (2) correlate oral health scores with bacterial taxa and identify what sites may be good options for understanding the role of microbiota in oral diseases. We used next-generation sequencing to characterize the salivary (SAL), subgingival (SUB), and supragingival (SUP) microbial habitats of 26 healthy adult female Beagle dogs (4.0 ± 1.2 year old) and identify taxa associated with periodontal disease indices.
RESULTS
Bacterial species richness was highest for SAL, moderate for SUB, and lowest for SUP samples (p < 0.001). Unweighted and weighted principal coordinates plots showed clustering by habitat, with SAL and SUP samples being the most different from one another. Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, Actinobacteria, and Spirochaetes were the predominant phyla in all habitats. Paludibacter, Filifactor, Peptostreptococcus, Fusibacter, Anaerovorax, Fusobacterium, Leptotrichia, Desulfomicrobium, and TG5 were enriched in SUB samples, while Actinomyces, Corynebacterium, Leucobacter, Euzebya, Capnocytophaga, Bergeyella, Lautropia, Lampropedia, Desulfobulbus, Enhydrobacter, and Moraxella were enriched in SUP samples. Prevotella, SHD-231, Helcococcus, Treponema, and Acholeplasma were enriched in SAL samples. p-75-a5, Arcobacter, and Pasteurella were diminished in SUB samples. Porphyromonas, Peptococcus, Parvimonas, and Campylobacter were diminished in SUP samples, while Tannerella, Proteocalla, Schwartzia, and Neisseria were diminished in SAL samples. Actinomyces, Corynebacterium, Capnocytophaga, Leptotrichia, and Neisseria were associated with higher oral health scores (worsened health) in plaque samples.
CONCLUSIONS
Our results demonstrate the differences that exist among canine salivary, subgingival plaque and supragingival plaque habitats. Salivary samples do not require sedation and are easy to collect, but do not accurately represent the plaque populations that are most important to oral disease. Plaque Actinomyces, Corynebacterium, Capnocytophaga, Leptotrichia, and Neisseria were associated with higher (worse) oral health scores. Future studies analyzing samples from progressive disease stages are needed to validate these results and understand the role of bacteria in periodontal disease development.
PubMed: 34001282
DOI: 10.1186/s42523-021-00100-9 -
MicrobiologyOpen Oct 2020Arcobacter spp. are commonly present on meat products. However, the source of contamination on chicken meat is under dispute. Since different studies reported...
Arcobacter spp. are commonly present on meat products. However, the source of contamination on chicken meat is under dispute. Since different studies reported contradictory results on the occurrence of Arcobacter spp. inside the intestinal tract of chicken, our study examined four intestinal compartments at four significant production steps during broiler slaughter and processing in the slaughterhouse. Altogether, 157 intestinal tracts from 19 flocks were examined qualitatively and semiquantitatively applying a selective enrichment. Further verification was performed by mPCR and rpoB sequencing. Arcobacter spp. were only detected sporadically in intestinal contents after bleeding (2/32) and in none after scalding (0/32). After defeathering, Arcobacter spp. were detected in 62% (18/29) of the intestinal contents with 28% (8/29) of the duodenal, 21% (6/29) of the jejunal, 3% (1/29) of the cecal, and 55% (16/29) of the colonic samples tested positive with loads up to 24,000 MPN/g in the colonic content. Further 88% (7/8) of colonic tissue samples were tested positive. After evisceration, the prevalences (58/64) and loads of Arcobacter spp. display comparable levels in the intestinal contents like after defeathering. In conclusion, our data point out that Arcobacter spp. are most likely detected in the colonic intestinal compartment of the chicken after defeathering and evisceration. Therefore, not only cross-contamination originating from the environment inside the slaughterhouse may cause carcass contamination with Arcobacter spp. on broiler chicken carcasses. The detection of Arcobacter spp. in duodenal and jejunal contents as well as in the colonic tissue indicates that there possibly exists an Arcobacter reservoir inside the chicken.
Topics: Abattoirs; Animals; Arcobacter; Bacterial Proteins; Chickens; Food Contamination; Food Handling; Food Microbiology; Intestines; Meat; Polymerase Chain Reaction
PubMed: 32830916
DOI: 10.1002/mbo3.1106