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608000.9593Metagenomic Insights into the Taxonomic and Functional Features of Traditional Fermented Milk Products from Russia. Fermented milk products (FMPs) contain probiotics that are live bacteria considered to be beneficial to human health due to the production of various bioactive molecules. In this study, nine artisanal FMPs (kefir, ayran, khurunga, shubat, two cottage cheeses, bryndza, khuruud and suluguni-like cheese) from different regions of Russia were characterized using metagenomics. A metagenomic sequencing of ayran, khurunga, shubat, khuruud and suluguni-like cheese was performed for the first time. The taxonomic profiling of metagenomic reads revealed that Lactococcus species, such as Lc. lactis and Lc. cremoris prevailed in khuruud, bryndza, one sample of cottage cheese and khurunga. The latter one together with suluguni-like cheese microbiome was dominated by bacteria, affiliated to Lactobacillus helveticus (32-35%). In addition, a high proportion of sequences belonging to the genera Lactobacillus, Lactococcus and Streptococcus but not classified at the species level were found in the suluguni-like cheese. Lactobacillus delbrueckii, as well as Streptococcus thermophilus constituted the majority in another cottage cheese, kefir and ayran metagenomes. The microbiome of shubat, produced from camel's milk, was significantly distinctive, and Lentilactobacillus kefiri, Lactobacillus kefiranofaciens and Bifidobacterium mongoliense represented the dominant components (42, 7.4 and 5.6%, respectively). In total, 78 metagenome-assembled genomes with a completeness ≥ 50.2% and a contamination ≤ 8.5% were recovered: 61 genomes were assigned to the Enterococcaceae, Lactobacillaceae and Streptococcaceae families (the Lactobacillales order within Firmicutes), 4 to Bifidobacteriaceae (the Actinobacteriota phylum) and 2 to Acetobacteraceae (the Proteobacteria phylum). A metagenomic analysis revealed numerous genes, from 161 to 1301 in different products, encoding glycoside hydrolases and glycosyltransferases predicted to participate in lactose, alpha-glucans and peptidoglycan hydrolysis as well as exopolysaccharides synthesis. A large number of secondary metabolite biosynthetic gene clusters, such as lanthipeptides, unclassified bacteriocins, nonribosomal peptides and polyketide synthases were also detected. Finally, the genes involved in the synthesis of bioactive compounds like β-lactones, terpenes and furans, nontypical for fermented milk products, were also found. The metagenomes of kefir, ayran and shubat was shown to contain either no or a very low count of antibiotic resistance genes. Altogether, our results show that traditional indigenous fermented products are a promising source of novel probiotic bacteria with beneficial properties for medical and food industries.202338276185
523710.9582Phenotypic and genomic analysis of Enterococcus avium MC09 pathogenicity isolated from Scylla spp. (mud crab) in a Thai market. Enterococcus avium is a Gram-positive pathogenic bacterium classified under the Enterococcaceae family. E. avium has been isolated from diverse environmental sources, raising concerns about its potential role in the spread of antibiotic resistance. E. avium MC09, isolated from a mud crab in a Thai market, was analyzed for its antibiotic resistance and pathogenic potential in this study. The isolation of E. avium from mud crab is significant as it highlights the potential role of seafood as a reservoir for antibiotic-resistant bacteria, which may pose risks to public health throughout the food chain. Antibiotic susceptibility testing using the Kirby-Bauer disk diffusion method revealed that E. avium MC09 is resistant to clindamycin, erythromycin, streptomycin, and tetracycline, and exhibits alpha hemolysis on blood agar, indicating its potential virulence. Genomic DNA was extracted and sequenced using the Oxford Nanopore Technologies (ONT) platform, revealing the presence of resistance genes for macrolides (ermB) and tetracyclines (tetL and tetM). Furthermore, several virulence-associated genes were detected, such as srtC, ecbA, efaA, dltA, cpsA/uppS, cpsB/cdsA, cylR2, icps4I, cpsY, epsE, vctC, mgtB, ndk, lisR, and lgt suggesting a pathogenic potential. Additionally, the study identified several insertion sequences (ISs), including (IS1216, IS1216E, IS1216V, IS6770, ISEfa7, ISEfa8, and ISS1W which are commonly found in pathogenic Enterococcus strains. The presence of these IS elements further emphasizes the strain's potential for virulence and genetic adaptability. This study provides comprehensive insights into both the phenotypic and genotypic characteristics of E. avium MC09, highlighting its antimicrobial resistance and pathogenic mechanisms, and underlines the importance of monitoring antibiotic resistance in seafood-associated bacteria.202540015576
360820.9577Natural antibiotic resistance of bacteria isolated from larvae of the oil fly, Helaeomyia petrolei. Helaeomyia petrolei (oil fly) larvae inhabit the asphalt seeps of Rancho La Brea in Los Angeles, Calif. The culturable microbial gut contents of larvae collected from the viscous oil were recently examined, and the majority (9 of 14) of the strains were identified as Providencia spp. Subsequently, 12 of the bacterial strains isolated were tested for their resistance or sensitivity to 23 commonly used antibiotics. All nine strains classified as Providencia rettgeri exhibited dramatic resistance to tetracycline, vancomycin, bacitracin, erythromycin, novobiocin, polymyxin, colistin, and nitrofurantoin. Eight of nine Providencia strains showed resistance to spectinomycin, six of nine showed resistance to chloramphenicol, and five of nine showed resistance to neomycin. All 12 isolates were sensitive to nalidixic acid, streptomycin, norfloxacin, aztreonam, cipericillin, pipericillin, and cefotaxime, and all but OF008 (Morganella morganii) were sensitive to ampicillin and cefoxitin. The oil fly bacteria were not resistant to multiple antibiotics due to an elevated mutation rate. For each bacterium, the number of resistant mutants per 10(8) cells was determined separately on rifampin, nalidixic acid, and spectinomycin. In each case, the average frequencies of resistant colonies were at least 50-fold lower than those established for known mutator strain ECOR 48. In addition, the oil fly bacteria do not appear to excrete antimicrobial agents. When tested, none of the oil fly bacteria produced detectable zones of inhibition on Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, or Candida albicans cultures. Furthermore, the resistance properties of oil fly bacteria extended to organic solvents as well as antibiotics. When pre-exposed to 20 microg of tetracycline per ml, seven of nine oil fly bacteria tolerated overlays of 100% cyclohexane, six of nine tolerated 10% xylene, benzene, or toluene (10:90 in cyclohexane), and three of nine (OF007, OF010, and OF011) tolerated overlays of 50% xylene-50% cyclohexane. The observed correlation between antibiotic resistance and organic solvent tolerance is likely explained by an active efflux pump that is maintained in oil fly bacteria by the constant selective pressure of La Brea's solvent-rich environment. We suggest that the oil fly bacteria and their genes for solvent tolerance may provide a microbial reservoir of antibiotic resistance genes.200011055901
364730.9570Biofilm Forming Antibiotic Resistant Gram-Positive Pathogens Isolated From Surfaces on the International Space Station. The International Space Station (ISS) is a closed habitat in a uniquely extreme and hostile environment. Due to these special conditions, the human microflora can undergo unusual changes and may represent health risks for the crew. To address this problem, we investigated the antimicrobial activity of AGXX®, a novel surface coating consisting of micro-galvanic elements of silver and ruthenium along with examining the activity of a conventional silver coating. The antimicrobial materials were exposed on the ISS for 6, 12, and 19 months each at a place frequently visited by the crew. Bacteria that survived on the antimicrobial coatings [AGXX® and silver (Ag)] or the uncoated stainless steel carrier (V2A, control material) were recovered, phylogenetically affiliated and characterized in terms of antibiotic resistance (phenotype and genotype), plasmid content, biofilm formation capacity and antibiotic resistance transferability. On all three materials, surviving bacteria were dominated by Gram-positive bacteria and among those by Staphylococcus, Bacillus and Enterococcus spp. The novel antimicrobial surface coating proved to be highly effective. The conventional Ag coating showed only little antimicrobial activity. Microbial diversity increased with increasing exposure time on all three materials. The number of recovered bacteria decreased significantly from V2A to V2A-Ag to AGXX®. After 6 months exposure on the ISS no bacteria were recovered from AGXX®, after 12 months nine and after 19 months three isolates were obtained. Most Gram-positive pathogenic isolates were multidrug resistant (resistant to more than three antibiotics). Sulfamethoxazole, erythromycin and ampicillin resistance were most prevalent. An Enterococcus faecalis strain recovered from V2A steel after 12 months exposure exhibited the highest number of resistances (n = 9). The most prevalent resistance genes were ermC (erythromycin resistance) and tetK (tetracycline resistance). Average transfer frequency of erythromycin, tetracycline and gentamicin resistance from selected ISS isolates was 10(-5) transconjugants/recipient. Most importantly, no serious human pathogens such as methicillin resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococci (VRE) were found on any surface. Thus, the infection risk for the crew is low, especially when antimicrobial surfaces such as AGXX® are applied to surfaces prone to microbial contamination.201930941112
538940.9570Identification and characterization of potential probiotic lactic acid bacteria isolated from pig feces at various production stages. Lactic acid bacteria (LAB) were isolated, identified, and characterized from pig feces at various growth stages and feed rations in order to be used as probiotic feed additives. Lactic acid bacteria numbers ranged from 7.10 ± 1.50 to 9.40 log CFUs/g for growing and lactating pigs, respectively. Isolates (n = 230) were identified by (GTG)5-polymerase chain reaction and partial sequence analysis of 16S rRNA. Major LAB populations were Limosilactobacillus reuteri (49.2%), Pediococcus pentosaceus (20%), Lactobacillus amylovorus (11.4%), and L. johnsonii (8.7%). In-vitro assays were performed, including surface characterization and tolerance to acid and bile salts. Several lactobacilli exhibited hydrophobic and aggregative characteristics and were able to withstand gastrointestinal tract conditions. In addition, lactobacilli showed starch- and phytate-degrading ability, as well as antagonistic activity against Gram-negative pathogens and the production of bacteriocin-like inhibitory substances. When resistance or susceptibility to antibiotics was evaluated, high phenotypic resistance to ampicillin, gentamicin, kanamycin, streptomycin, and tetracycline and susceptibility towards clindamycin and chloramphenicol was observed in the assayed LAB. Genotypic characterization showed that 5 out of 15 resistance genes were identified in lactobacilli; their presence did not correlate with phenotypic traits. Genes erm(B), strA, strB, and aadE conferring resistance to erythromycin and streptomycin were reported among all lactobacilli, whereas tet(M) gene was harbored by L. reuteri and L. amylovorus strains. Based on these results, 6 probiotic LAB strains (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T, and L. johnsonii S92R) can be selected to explore their potential as direct feed additives to promote swine health and replace antibiotics.202337020571
360250.9569Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria. Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H(+) antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.200211916697
476160.9568Antimicrobial resistance and biofilm formation of penile prosthesis isolates: insights from in-vitro analysis. BACKGROUND: Inflatable penile prostheses (IPPs) have been shown to harbor biofilms in the presence and absence of infection despite exposure to various antimicrobials. Microbes persisting on IPPs following antibiotic exposure have not been adequately studied to assess biofilm formation capacity and antibiotic resistance. AIM: In this study, we aimed to assess these properties of microbes obtained from explanted infected and non-infected IPPS using an in vitro model. METHODS: 35 bacterial isolates were grown and tested against various single-agent or multiple agent antibiotic regimens including: bacitracin, cefaclor, cefazolin, gentamicin, levofloxacin, trimethoprim-sulfamethoxazole, tobramycin, vancomycin, piperacillin/tazobactam, gentamicin + piperacillin/tazobactam, gentamicin + cefazolin, and gentamicin + vancomycin. Zones of inhibition were averaged for each sample site and species. Statistics were analyzed with Holm's corrected, one-sample t-tests against a null hypothesis of 0. Isolates were also allowed to form biofilms in a 96-well polyvinyl plate and absorbance was tested at 570 nm using a microplate reader. OUTCOMES: Resistance was determined via clinical guidelines or previously established literature, and the mean and standard deviation of biofilm absorbance values were calculated and normalized to the optical density600 of the bacterial inoculum. RESULTS: Every species tested was able to form robust biofilms with the exception of Staphylococcus warneri. As expected, most bacteria were resistant to common perioperative antimicrobial prophylaxis. Gentamicin dual therapy demonstrated somewhat greater efficacy. STRENGTHS AND LIMITATIONS: This study examines a broad range of antimicrobials against clinically obtained bacterial isolates. However, not all species and antibiotics tested had standardized breakpoints, requiring the use of surrogate values from the literature. The microbes included in this study and their resistance genes are expectedly biased towards those that survived antibiotic exposure, and thus reflect the types of microbes which might "survive" in vivo exposure following revisional surgery. CLINICAL TRANSLATION: Despite exposure to antimicrobials, bacteria isolated during penile prosthesis revision for both infected and non-infected cases exhibit biofilm forming capacity and extensive antibiotic resistance patterns in vitro. These microbes merit further investigation to understand when simple colonization vs re-infection might occur. CONCLUSIONS: Although increasing evidence supports the concept that all IPPs harbor biofilms, even in the absence of infection, a deeper understanding of the characteristics of bacteria that survive revisional surgery is warranted. This study demonstrated extensive biofilm forming capabilities, and resistance patterns among bacteria isolated from both non-infected and infected IPP revision surgeries. Further investigation is warranted to determine why some devices become infected while others remain colonized but non-infected.202540062463
637770.9568Comparative metagenomics and characterization of antimicrobial resistance genes in pasteurized and homemade fermented Arabian laban. The aim of this study was to investigate bacterial diversity and function in a fermented milk drink called laban, which is traditionally served in the Middle East, Africa, and Indian subcontinent. Pasteurized laban (LBP) and unpasteurized, homemade, raw laban (LBR) underwent 16S rRNA gene amplicon and shotgun sequencing to analyze their bacterial community, presence of antimicrobial resistance genes (ARGs), and metabolic pathways. This study highlighted relatively greater diversity in LBR bacterial populations compared to LBP, despite containing similar major taxa that consisted primarily of Firmicutes followed by Proteobacteria, Bacteroidetes, and Actinobacteria. The dominant species, Streptococcus thermophilus, was relatively more abundant in LBP (80.7%) compared to LBR (47.9%). LBR had increased diversity and higher relative abundance of several known probiotic bacteria, such as Streptococcus salivarius and Lactococcus lactis, whereas Lactobacillus acidophilus was detected at a higher abundance in LBP. Pathogens like Acinetobacter baumannii, Streptococcus pneumoniae, Streptococcus pyogenes, and Escherichia coli had lower abundance in LBP compared to LBR. Thirty-three ARGs were detected in LBR compared to nine in LBP and are responsible for resistance to 11 classes of antibiotics. A significant proportion of the metagenomes from both types of laban were assigned to housekeeping functions, such as amino acid metabolism, translation, membrane transport, and carbohydrate metabolism. LBR demonstrated increased diversity in probiotics and metabolic functions compared to LBP. However, the relatively high diversity of pathogenic and opportunistic bacteria and ARGs in LBR raises safety concerns and highlights the need for a more hygienic environment for the processing of homemade fermented dairy foods.202033233218
587980.9567Isolation and phenotypic and genomic characterization of Tetragenococcus spp. from two Spanish traditional blue-veined cheeses made of raw milk. High throughput sequencing has recently revealed the presence of Tetragenococcus-related DNA sequences in dairy environments such as brine and cheeses. In the present work, a selective medium was developed to isolate Tetragenococcus spp. from two ripened, traditional, Spanish, blue-veined cheese varieties made from raw milk. The strains recovered belonged to either Tetragenococcus koreensis or Tetragenococcus halophilus species. Twenty of these isolates (15 of T. koreensis and 5 of T. halophilus) were then subjected to a battery of phenotypic and genetic tests, and six strains (4 T. koreensis and 2 T. halophilus) to genome sequencing. Wide genetic and phenotypic diversity was noted. All strains grew poorly in milk, producing small quantities of lactic and acetic acids. Most strains used lactose as a carbon source and ferment milk citrate. In agreement, genome analysis detected in the genome of the six strains analyzed gene clusters harboring several lactose/galactose-related genes and genes encoding citrate metabolic enzymes (permease, citrate lyase, and oxaloacetate decarboxylase). Most of the tested strains were resistant to erythromycin and clindamycin, and a few to other antimicrobial agents, but neither known mutations nor acquired genes conferring resistance to antibiotics were identified in their genomes. Neither were genes coding for pathogenicity or virulence factors detected. Decarboxylase-encoding genes involved in biogenic amine production were not identified, in keeping with the strains' negative biogenic amine-producer phenotype. Genome comparison revealed vast arrays of genes (similar in number to those described in other lactic acid bacteria) coding for components of proteolytic and lipolytic systems. Tetragenococcus strains showing desirable traits plus the absence of detrimental features might be exploitable in the form of secondary, adjunct or ripening cultures to ensure the typical bouquet of traditional blue-veined cheeses is obtained, or to diversify the final flavor in other varieties.202235427955
604690.9566Safety Evaluations of Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI. Over the past decade, a variety of lactic acid bacteria have been commercially available to and steadily used by consumers. However, recent studies have shown that some lactic acid bacteria produce toxic substances and display properties of virulence. To establish safety guidelines for lactic acid bacteria, the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) has suggested that lactic acid bacteria be characterized and proven safe for consumers’ health via multiple experiments (e.g., antibiotic resistance, metabolic activity, toxin production, hemolytic activity, infectivity in immune-compromised animal species, human side effects, and adverse-outcome analyses). Among the lactic acid bacteria, Bifidobacterium and Lactobacillus species are probiotic strains that are most commonly commercially produced and actively studied. Bifidobacterium bifidum BGN4 and Bifidobacterium longum BORI have been used in global functional food markets (e.g., China, Germany, Jordan, Korea, Lithuania, New Zealand, Poland, Singapore, Thailand, Turkey, and Vietnam) as nutraceutical ingredients for decades, without any adverse events. However, given that the safety of some newly screened probiotic species has recently been debated, it is crucial that the consumer safety of each commercially utilized strain be confirmed. Accordingly, this paper details a safety assessment of B. bifidum BGN4 and B. longum BORI via the assessment of ammonia production, hemolysis of blood cells, biogenic amine production, antimicrobial susceptibility pattern, antibiotic resistance gene transferability, PCR data on antibiotic resistance genes, mucin degradation, genome stability, and possession of virulence factors. These probiotic strains showed neither hemolytic activity nor mucin degradation activity, and they did not produce ammonia or biogenic amines (i.e., cadaverine, histamine or tyramine). B. bifidum BGN4 and B. longum BORI produced a small amount of putrescine, commonly found in living cells, at levels similar to or lower than that found in other foods (e.g., spinach, ketchup, green pea, sauerkraut, and sausage). B. bifidum BGN4 showed higher resistance to gentamicin than the European Food Safety Authority (EFSA) cut-off. However, this paper shows the gentamicin resistance of B. bifidum BGN4 was not transferred via conjugation with L. acidophilus ATCC 4356, the latter of which is highly susceptible to gentamicin. The entire genomic sequence of B. bifidum BGN4 has been published in GenBank (accession no.: CP001361.1), documenting the lack of retention of plasmids capable of transferring an antibiotic-resistant gene. Moreover, there was little genetic mutation between the first and 25th generations of B. bifidum BGN4. Tetracycline-resistant genes are prevalent among B. longum strains; B. longum BORI has a tet(W) gene on its chromosome DNA and has also shown resistance to tetracycline. However, this research shows that its tetracycline resistance was not transferred via conjugation with L. fermentum AGBG1, the latter of which is highly sensitive to tetracycline. These findings support the continuous use of B. bifidum BGN4 and B. longum BORI as probiotics, both of which have been reported as safe by several clinical studies, and have been used in food supplements for many years.201829747442
6049100.9566Probiotic Properties and Antioxidant Activity In Vitro of Lactic Acid Bacteria. The properties of probiotics such as lactic acid bacteria (LAB) have been widely studied over the last decades. In the present study, four different LAB species, namely Lactobacillus gasseri ATCC 33323, Lacticaseibacillus rhamnosus GG ATCC 53103, Levilactobacillus brevis ATCC 8287, and Lactiplantibacillus plantarum ATCC 14917, were investigated in order to determine their ability to survive in the human gut. They were evaluated based on their tolerance to acids, resistance to simulated gastrointestinal conditions, antibiotic resistance, and the identification of genes encoding bacteriocin production. All four tested strains demonstrated high resistance to simulated gastric juice after 3 h, and the viable counts revealed declines in cell concentrations of less than 1 log cycle. L. plantarum showed the highest level of survival in the human gut, with counts of 7.09 log CFU/mL. For the species L. rhamnosus and L. brevis, the values were 6.97 and 6.52, respectively. L. gasseri, after 12 h, showed a 3.96 log cycle drop in viable counts. None of the evaluated strains inhibited resistance to ampicillin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, or chloramphenicol. With regard to bacteriocin genes, the Pediocin PA gene was identified in Lactiplantibacillus plantarum ATCC 14917, Lacticaseibacillus rhamnosus GG ATCC 53103, and Lactobacillus gasseri ATCC 33323. The PlnEF gene was detected in Lactiplantibacillus plantarum ATCC 14917 and Lacticaseibacillus rhamnosus GG ATCC 53103. The Brevicin 174A and PlnA genes were not detected in any bacteria. Moreover, the potential antioxidant activity of LAB's metabolites was evaluated. At the same time, the possible antioxidant activity of metabolites of LAB was first tested using the free radical DDPH(•) (a, a-Diphenyl-β-Picrylhydrazyl) and then evaluated with regard to their radical scavenging activity and inhibition against peroxyl radical induced DNA scission. All strains showed antioxidant activity; however, the best antioxidant activity was achieved by L. brevis (94.47%) and L. gasseri (91.29%) at 210 min. This study provides a comprehensive approach to the action of these LAB and their use in the food industry.202337317238
5904110.9565Antimicrobial susceptibilities of Lactobacillus, Pediococcus and Lactococcus human isolates and cultures intended for probiotic or nutritional use. OBJECTIVES: To determine MICs of 16 antimicrobials representing all major classes for 473 taxonomically well-characterized isolates of lactic acid bacteria (LAB) encompassing the genera Lactobacillus, Pediococcus and Lactococcus. To propose tentative epidemiological cut-off (ECOFF) values for recognizing intrinsic and acquired antimicrobial resistances in numerically dominant species. METHODS: On the basis of depositors' information, LAB were grouped in categories of probiotic, nutritional, probiotic or nutritional research, human and animal isolates and tested for their antibiotic susceptibilities by broth microdilution using LAB susceptibility test medium (LSM). Tentative ECOFFs were defined according to the recommendations of the European Committee on Antimicrobial Susceptibility Testing. Isolates showing acquired antimicrobial resistance(s) were selected for PCR-based detection of resistance gene(s) and in vitro conjugative transfer experiments. RESULTS: Tentative ECOFF values of 13 antibiotics were determined for up to 12 LAB species. Generally, LAB were susceptible to penicillin, ampicillin, ampicillin/sulbactam, quinupristin/dalfopristin, chloramphenicol and linezolid. LAB exhibited broad or partly species-dependent MIC profiles of trimethoprim, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and fusidic acid. Three probiotic Lactobacillus strains were highly resistant to streptomycin. Although erythromycin, clindamycin and oxytetracycline possessed high antimicrobial activities, 17 Lactobacillus isolates were resistant to one or more of these antibiotics. Eight of them, including six probiotic and nutritional cultures, possessed erm(B) and/or tet(W), tet(M) or unidentified members of the tet(M) group. In vitro intra- and interspecies filter-mating experiments failed to show transfer of resistance determinants. CONCLUSIONS: Finding of acquired resistance genes in isolates intended for probiotic or nutritional use highlights the importance of antimicrobial susceptibility testing in documenting the safety of commercial LAB.200717369278
6075120.9564Molecular screening of beneficial and safety determinants from bacteriocinogenic lactic acid bacteria isolated from Brazilian artisanal calabresa. Despite of the beneficial relevance of several lactic acid bacteria (LAB) in the food industry, micro-organisms belonging to this group can determine spoilage in food products and carry a number of virulence and antibiotic resistance-related genes. This study aimed on the characterization of beneficial and safety aspects of five bacteriocinogenic LAB strains (Lactobacillus curvatus 12-named L. curvatus UFV-NPAC1), L. curvatus 36, Weissela viridescens 23, W. viridescens 31 and Lactococcus garvieae 36) isolated from an artisanal Brazilian calabresa, a traditional meat sausage. Regarding their beneficial aspects, all tested isolates were positive for mub, while EF226-cbp, EF1249-fbp and EF2380-maz were detected in at least one tested strain; none of the isolates presented map, EFTu or prgB. However, evaluated strains presented a variable pattern of virulence-related genes, but none of the strains presented gelE, cylA, efsA, cpd, int-Tn or sprE. Moreover, other virulence-related genes evaluated in this study were detected at different frequencies. L. curvatus 12 was generated positive results for ace, ccf, int, ermC, tetL, aac(6')-Ie-aph(2″)-Ia, aph(2″)-Ib, aph(2″)-Ic, bcrB, vanB and vanC2; L. curvatus 36: hyl, asa1, esp, int, ermC, tetK, aph(3')-IIIa, aph(2'')-Ic and vanC2; L. garvieae 32: asa1, ant(4')-Ia, aph(2'')-Ib, catA, vanA and vanC1; W. viridescens 23: esp, cob, ermB, aph(3')-IIIa, aph(2'')-Ic, vanA, vanB and vanC2; W. viridescens 31: hyl, esp, ermC, aph(3')-IIIa, aph(2'')-Ib, aph(2'')-Ic, catA, vanA and vanB. Despite presenting some beneficial aspects, the presence of virulence and antibiotic resistance genes jeopardize their utilization as starter or biopreservatives cultures in food products. Considering the inhibitory potential of these strains, an alternative would be the use of their bacteriocins as semi-purified or pure technological preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: The food industry has a particular interest in using bacteriocinogenic lactic acid bacteria (LAB) as starter, probiotics and/or biopreservatives in different food products. Characterization of additional beneficial features is important to identify new, multifunctional potential probiotic strains. However, these strains can only be applied in food products only after being properly characterized according their potential negative aspects, such as virulence and antibiotic resistance genes. A wide characterization of beneficial and safety aspects of bacteriocinogenic LAB is determinant to guide the proper utilization of these strains, or their purified bacteriocins, by the food industry.201931250457
5395130.9562Assessment of Antibiotic Susceptibility within Lactic Acid Bacteria and Coagulase-Negative Staphylococci Isolated from Hunan Smoked Pork, a Naturally Fermented Meat Product in China. The aim of this study was to evaluate the antibiotic susceptibility of lactic acid bacteria (LAB) and coagulase-negative staphylococci (CNS) strains isolated from naturally fermented smoked pork produced in Hunan, China. A total of 48 strains were isolated by selective medium and identified at the species level by 16S rRNA gene sequencing as follows: Staphylococcus carnosus (23), Lactobacillus plantarum (12), Lactobacillus brevis (10), Lactobacillus sakei (1), Weissella confusa (1), and Weissella cibaria (1). All strains were typed by RAPD-PCR, and their susceptibility to 15 antibiotics was determined and expressed as the minimum inhibitory concentration (MIC) using agar dilution method. High resistance to penicillin G, streptomycin, gentamycin, vancomycin, chloramphenicol, norfloxacin, ciprofloxacin, kanamycin, and neomycin was found among the isolates. All the strains were sensitive to ampicillin, while the susceptibility to tetracycline, oxytetracycline, erythromycin, lincomycin, and roxithromycin varied. The presence of relevant resistance genes was investigated by PCR and sequencing, with the following genes detected: str(A), str(B), tet(O), tet(M), ere(A), and catA. Eleven strains, including 3 S. carnosus, 6 L. plantarum, and 2 L. brevis, harbored more than 3 antibiotic resistance genes. Overall, multiple antibiotic resistance patterns were widely observed in LAB and S. carnosus strains isolated from Hunan smoked pork. Risk assessment should be carried out with regard to the safe use of LAB and CNS in food production. PRACTICAL APPLICATION: We evaluated the antibiotic resistance of lactic acid bacteria and coagulase-negative staphylococci strains isolated from Chinese naturally fermented smoked pork. Our results may provide important data on establishing breakpoint standards for LAB and CNS and evaluating the safety risk of these strains for commercial use.201829786847
3652140.9562Distribution of Transferable Antibiotic Resistance Genes in Laboratory-Reared Edible Mealworms (Tenebrio molitor L.). In the present study, the distribution of antibiotic resistance genes in laboratory-reared fresh mealworm larvae (Tenebrio molitor L.), their feeding substrates (carrots and wheatmeal), and frass was assessed. Microbial counts on selective media added with antibiotics highlighted the presence of lactic acid bacteria resistant to ampicillin and vancomycin and, more specifically, enterococci resistant to the latter antibiotic. Moreover, staphylococci resistant to gentamicin, erythromycin, tetracycline, and vancomycin were detected. Enterobacteriaceae resistant to ampicillin and gentamicin were also found, together with Pseudomonadaceae resistant to gentamicin. Some of the genes coding for resistance to macrolide-lincosamide-streptogramin B (MLS(B)) [erm(A), erm(C)], vancomycin [vanA, vanB], tetracycline [tet(O)], and β-lactams [mecA and blaZ] were absent in all of the samples. For the feeding substrates, organic wheatmeal was positive for tet(S) and tet(K), whereas no AR genes were detected in organic carrots. The genes tet(M), tet(K), and tet(S) were detected in both mealworms and frass, whereas gene aac-aph, coding for resistance to amynoglicosides was exclusively detected in frass. No residues for any of the 64 antibiotics belonging to 10 different drug classes were found in either the organic wheatmeal or carrots. Based on the overall results, the contribution of feed to the occurrence of antibiotic resistance (AR) genes and/or antibiotic-resistant microorganisms in mealworm larvae was hypothesized together with vertical transmission via insect egg smearing.201830510544
5542150.9561Analysis of bacteriological pollution and the detection of antibiotic resistance genes of prevailing bacteria emanating from pig farm seepage. Management and disposal of pig farm seepage constitute a serious environmental challenge, and seepage discharge from agricultural waste-water is considered to be one of the greatest contributors of organic substances, bacterial pathogens, and antibiotic resistance genes into the environment. The objectives of this study were to assess the level of bacteriological pollution and to identify the resident antibiotic-resistant genes of culturable bacteria from a studied pig farm seepage. Enumeration of the viable bacterial cell of plated bacteria suspensions (10(-1) to 10(-8)  cfu/mL) was performed; also, identification of pure bacterial colonies was done using an API 20E bacterial identification kit. CLSI guidelines for antimicrobial susceptibility testing were adopted to determine the antibiotic susceptibility/resistance of the cultured bacterial isolates. Identification of resident-resistant genes was done using molecular biology procedures. The results on viable cells in seepage samples ranged from 4.30 × 10(2) to 1.29 × 10(9 ) cfu/mL. Pseudomonas luteola, Enterococcus vulneris, Salmonella choleraesuis spp arizonae, Escherichia coli, Enterobacter cloacae, Proteus mirabillis etc. were isolated from the pig farm soil samples. Almost all of the cultured isolates were resistant to Penicillin G, Vancomycin, Oxytetracycline, Spectinomycin, and Lincomycin. The most frequent resistant genes detected in the isolates were Van A, Van B, InuA, aph (3")-llla, bla(TEM,) Otr A, and Otr B. It was inferred from the study that Pig farm seepage has the ability to cause bacterial pollution that may negatively impact the natural environment, by introducing bacteria pathogens that harbor antibiotic-resistant genes.201930414264
2816160.9560Water supply and feed as sources of antimicrobial-resistant Enterococcus spp. in aquacultures of rainbow trout (Oncorhyncus mykiss), Portugal. The role of European fish farms in the spread of antimicrobial-resistance in the environment and food chain, as well as possible sources of their contamination by clinically relevant antimicrobial-resistance bacteria is scarcely known. This study aimed to assess the contribution of Portuguese rural trout farms on dispersion of Enterococcus with antimicrobial-resistance and putative virulence genes in the environment and food chain, as well as to identify farms contamination sources. We also assessed the presence of Enterococcus with low-levels of antimicrobial-resistance using epidemiological cut-offs (ECOFFs). Enterococcus spp. (n=391) from water/sediment recovered upstream, within and downstream trout tanks, feed, trout (2 aquacultures; no antibiotic use) and marketed trout (8 supermarkets) showed variable resistance to tetracycline, erythromycin, ciprofloxacin, chloramphenicol, quinupristin-dalfopristin, nitrofurantoin or aminoglycosides. Antimicrobial-resistance rates were similar among upstream, within and downstream trout tank samples (P>0.05), positioning water-supplying aquacultures as a source of multidrug-resistant (MDR) strains. Nevertheless, predominance of MDR E. faecium in feed, trout tanks and trout comparing to upstream samples, suggests feed as an additional aquaculture contamination source. The observation of E. faecium and E. faecalis susceptible to ampicillin and gentamicin by clinical breakpoints but with low-levels of resistance to those antimicrobials by ECOFFs breakpoints is of concern, as they might evolve throughout secondary genetic events to resistance levels with human clinical impact. Multiple MDR clones carrying copper tolerance (tcrB/cueO), putative virulence or other genes often associated with clinical strains (e.g. E. faecium with IS16/ptsD/sgrA) were observed, some in distinct samples (e.g. upstream and within trout tanks). They included major human and animal Enterococcus lineages, suggesting human and non-aquatic animal origins. The results highlight the need to define the maximum acceptance level of antimicrobial-resistance genes/bacteria to assess water quality and to monitor antimicrobial-resistance strains on feed, essential requirements to maintain a sustainable aquaculture production.201829996407
6165170.9559Evaluation of kasugamycin for fire blight management, effect on nontarget bacteria, and assessment of kasugamycin resistance potential in Erwinia amylovora. The emergence and spread of streptomycin-resistant strains of Erwinia amylovora in Michigan has necessitated the evaluation of new compounds effective for fire blight control. The aminoglycoside antibiotic kasugamycin (Ks) targets the bacterial ribosome and is particularly active against E. amylovora. The efficacy of Ks formulated as Kasumin 2L for control of fire blight was evaluated in six experiments conducted over four field seasons in our experimental orchards in East Lansing, MI. Blossom blight control was statistically equivalent to the industry standard streptomycin in all experiments. E. amylovora populations remained constant on apple flower stigmas pretreated with Kasumin and were ≈100-fold lower than on stigmas treated with water. Kasumin applied to apple trees in the field also resulted in a 100-fold reduced total culturable bacterial population compared with trees treated with water. We performed a prospective analysis of the potential for kasugamycin resistance (Ks(R)) development in E. amylovora which focused on spontaneous resistance development and acquisition of a transferrable Ks(R) gene. In replicated lab experiments, the development of spontaneous resistance in E. amylovora to Ks at 250 or 500 ppm was not observed when cells were directly plated on medium containing high concentrations of the antibiotic. However, exposure to increasing concentrations of Ks in media (initial concentration 25 μg ml(-1)) resulted in the selection of Ks resistance (at 150 μg ml(-1)) in the E. amylovora strains Ea110, Ea273, and Ea1189. Analysis of mutants indicated that they harbored mutations in the kasugamycin target ksgA gene and that all mutants were impacted in relative fitness observable through a reduced growth rate in vitro and decreased virulence in immature pear fruit. The possible occurrence of a reservoir of Ks(R) genes in orchard environments was also examined. Culturable gram-negative bacteria were surveyed from six experimental apple orchards that had received at least one Kasumin application. In total, 401 Ks(R) isolates (42 different species) were recovered from apple flowers and leaves and orchard soil samples. Although we have not established the presence of a transferrable Ks(R) gene in orchard bacteria, the frequency, number of species, and presence of Ks(R) enterobacterial species in orchard samples suggests the possible role of nontarget bacteria in the future transfer of a Ks(R) gene to E. amylovora. Our data confirm the importance of kasugamycin as an alternate antibiotic for fire blight management and lay the groundwork for the development and incorporation of resistance management strategies.201120923369
3066180.9559Staphylococci and fecal bacteria as bioaerosol components in animal housing facilities in the Zoological Garden in Chorzów. Zoos are places open for a large number of visitors, adults and children, who can admire exotic as well as indigenous animal species. The premises for animals may contain pathogenic microbes, including those exhibiting antibiotic resistance. It poses a threat to people remaining within the zoo premises, both for animal keepers who meet animals on a daily basis and visitors who infrequently have contact with animals. There are almost no studies concerning the presence on the concentration of airborne bacteria, especially staphylococci and fecal bacteria in animal shelters in the zoo. There is no data about antibiotic resistance of staphylococci in these places. The results will enable to determine the scale of the threat that indicator bacteria from the bioaerosol pose to human health within zoo premises. This study conducted in rooms for 5 animals group (giraffes, camels, elephants, kangaroos, and Colobinae (species of monkey)) in the Silesian Zoological Garden in Chorzów (Poland). The bioaerosol samples were collected using a six-stage Andersen cascade impactor to assess the concentrations and size distribution of airborne bacteria. Staphylococci were isolated from bioaerosol and tested for antibiotic resistance. In our study, the highest contamination of staphylococci and fecal bacteria was recorded in rooms for camels and elephants, and the lowest in rooms for Colobinae. At least 2/3 of bacteria in bioaerosol constituted respirable fraction that migrates into the lower respiratory tract of the people. In investigated animal rooms, the greatest bacteria contribution was recorded for bioaerosol fraction sized 1.1-3.3μm. Bacterial concentrations were particularly strong in spring and autumn, what is related to shedding fur by animals. Among the isolated staphylococci which most often occurred were Staphylococcus succinus, S. sciuri, and S. vitulinus. The highest antibiotic resistance was noted in the case of Staphylococcus epidermidis, while the lowest for S. xylosus. In addition to standard cleaning of animal rooms, periodic disinfection should be considered. Cleaning should be carried out wet, which should reduce dust, and thus the concentrations of bacteria in the air of animal enclosures.202134061267
6032190.9559Toxigenic potential and heat survival of spore-forming bacteria isolated from bread and ingredients. Fifty-four spore-forming bacterial strains isolated from bread ingredients and bread, mainly belonging to the genus Bacillus (including Bacillus cereus), together with 11 reference strains were investigated to evaluate their cytotoxic potential and heat survival in order to ascertain if they could represent a risk for consumer health. Therefore, we performed a screening test of cytotoxic activity on HT-29 cells using bacterial culture filtrates after growing bacterial cells in Brain Heart Infusion medium and in the bread-based medium Bread Extract Broth (BEB). Moreover, immunoassays and PCR analyses, specifically targeting already known toxins and related genes of B. cereus, as well as a heat spore inactivation assay were carried out. Despite of strain variability, the results clearly demonstrated a high cytotoxic activity of B. cereus strains, even if for most of them it was significantly lower in BEB medium. Cytotoxic activity was also detected in 30% of strains belonging to species different from B. cereus, although, with a few exceptions (e.g. Bacillus simplex N58.2), it was low or very low. PCR analyses detected the presence of genes involved in the production of NHE, HBL or CytK toxins in B. cereus strains, while genes responsible for cereulide production were not detected. Production of NHE and HBL toxins was also confirmed by specific immunoassays only for B. cereus strains even if PCR analyses revealed the presence of related toxin genes also in some strains of other species. Viable spore count was ascertained after a heat treatment simulating the bread cooking process. Results indicated that B. amyloliquefaciens strains almost completely survived the heat treatment showing less than 2 log-cycle reductions similarly to two strains of B. cereus group III and single strains belonging to Bacillus subtilis, Bacillus mojavensis and Paenibacillus spp. Importantly, spores from strains of the B. cereus group IV exhibited a thermal resistance markedly lower than B. cereus group III with high values of log-cycle reductions. In conclusion, our results indicate that spore-forming bacteria contaminating bread ingredients and bread could represent a source of concern for consumer health related to the presence of strains, such as strains of B. cereus group III and single strains of other species, showing the ability to produce toxic substances associated to a thermal resistance enough to survive the bread cooking conditions.201525555227