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Antifungal agents are not always efficient in resolving vulvovaginal candidiasis (VVC), a common genital infection caused by overgrowth of Candida spp., including Candida albicans, or preventing recurrent infections. Although lactobacilli (which are dominant microorganisms constituting healthy human vaginal microbiota) are important barriers against VVC, the Lactobacillus metabolite concentration needed to suppress VVC is unknown. Therefore, we quantitatively evaluated Lactobacillus metabolite concentrations to determine their effect on Candida spp., including 27 vaginal strains of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Lacticaseibacillus rhamnosus, and Limosilactobacillus vaginalis, with inhibitory abilities against biofilms of Candida clinical isolates. Lactobacillus culture supernatants suppressed viable bacteria by approximately 24%-92% relative to preformed Candida biofilms, but their suppression differed between strains, not species. Lactate production was necessary to suppress preformed biofilms and hyphal elongation of C. albicans, whereas hydrogen peroxide was not always essential. Both lactic acid and hydrogen peroxide were required to suppress Candida planktonic cell growth. Lactobacillus strains that significantly inhibited biofilm formation in culture supernatant also inhibited Candida adhesion to epithelial cells in an actual live bacterial adhesion competition test. Healthy human microflora and their metabolites may play important roles in the development of new antifungal agent against VVC caused by C. albicans.

Tetracyclines (TCs) are widely used for livestock, and the high prevalence of TC-resistant Escherichia coli in livestock has become a serious concern worldwide. In Japan, the National Action Plan on Antimicrobial Resistance in 2016 aimed to reduce the TC resistance rate in E. coli derived from livestock. Flavophospholipol (FPL), used as a feed additive, has an inhibitory effect on the spread of plasmid-mediated antimicrobial resistance. The number of TC-resistant E. coli was determined in pigs administered TCs and/or FPL to clarify the effect of FPL on reducing the number of TC-resistant E. coli in pigs. TC-resistant E. coli and their plasmids were then analyzed. The pigs were divided into four groups: control, doxycycline (DOXY; a TC), FPL, and a DOXY-FPL combination. Their feces were collected from the nursing period to the day before being transported to the slaughterhouse, followed by estimation of TC-resistant E. coli (colony-forming units [CFU]/g). The number of TC-resistant E. coli increased with the use of DOXY, suggesting that DOXY administration provides a selective pressure for TC-resistant E. coli. Supplementation with FPL as a feed additive significantly suppressed the increase in the number of TC-resistant E. coli, especially during the DOXY administration period. Transfer and growth inhibition analyses were performed for TC-resistant isolates. FPL inhibited the conjugational transfer and growth of a few TC-resistant E. coli isolates. These results suggest that FPL is effective against the spread of TC-resistant E. coli.

Background Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that is used as an anti-diarrheal medicine in Japan. However, the impact of this probiotic on the gut microbiome has not been fully elucidated, especially, when used with antimicrobials. Material and methods In an in vivo study, CBM 588 monotherapy, clindamycin monotherapy, CBM 588 and clindamycin (combination therapy), or normal saline (control) was orally administered to mice for 4 days, and fecal samples were collected for 18 days to enumerate C. butyricum. We also extracted DNA from these fecal samples for metagenomics analysis by amplification of the V3-V4 region of the bacterial 16S rRNA gene and MiSeq Illumina sequencing. In addition, the concentrations of some short chain fatty acids were assessed in the fecal samples. A histological analysis was also conducted. Results On day 4 (the last treatment day), there was no difference in the total counts of C. butyricum between the CBM 588 monotherapy and combination therapy groups (5.21 ± 0.78 vs. 5.13 ± 0.45 log10 cfu/g, p = 0.86). Clindamycin treatment resulted in dramatic increases in the phylum Firmicutes, especially Enterobacteriaceae, Clostridiaceae, Lactobacillus, and Enterococcus, compared with the other groups during the treatment period. CBM 588 treatment modified the bacterial community composition at lower phylogenetic levels. Some bacterial taxa, such as Bifidobacterium, Coprococcus, and Bacteroides, were significantly increased in the combination therapy group when compared with the other groups. In the metabolic analysis, CBM 588 enhanced lactic acid production. It also enhanced the efficiency of lactic acid use for the production of butyric acid. Only the clindamycin monotherapy group showed abnormal colon tissue, with superficial epithelial necrosis and the presence of inflammatory cells. Conclusion CBM 588 treatment modulated the gut microbiota composition under dysbiosis due to the use of an antimicrobial with strong activity against anaerobes and significantly reduced epithelial damage.

Flavophospholipol (FPL) is an antimicrobial feed additive that has been approved for use in livestock animals and has the potential to decrease horizontal dissemination of antimicrobial resistance genes. Since previous studies showed that FPL has an inhibitory effect on plasmid transfer, in vitro experiments have proven the efficacy of FPL in reducing the conjugative transfer of plasmids encoding the extended-spectrum β-lactamase (ESBL) and vanA genes. These are among the most important antimicrobial resistance loci known. ESBL-producing Escherichia coli and vancomycin-resistant Enterococcus faecalis (VRE) were exposed to several concentrations of FPL, and transfer frequency and plasmid curing activity were determined. FPL inhibited the conjugative transfer of plasmids harboring ESBL and vanA genes in a concentration-dependent manner in all strains. Further transfer experiments revealed that FPL could decrease or increase transfer frequency depending on plasmid type when transfer frequency was at low levels. The plasmid curing activity of FPL was also observed in ESBL-producing E. coli in a concentration-dependent manner, suggesting that they partially contribute to the inhibition of conjugative transfer. These results suggest that the use of FPL as a feed additive might decrease the dissemination of ESBL and vanA genes among livestock animals.

Clostridium butyricum MIYAIRI 588 (CBM 588) is a nonpathogenic, anaerobic, gram-positive bacillus characterized by the production of short-chain fatty acids, including butyrate. The safety and tolerance of CBM 588 was investigated as a feed additive for broiler chickens, weaned piglets, and turkeys. CBM 588 administered to broilers at doses up to 5 × 10 7 CFU/g feed for 42 days produced no detrimental effects on zootechnical performance, natural mortality, hematology, or biochemical parameters. Piglets receiving CBM 588 at doses up to 5 × 10 7 CFU/g feed for 42 days showed no significant differences from controls in zootechnical performance, mortality, or morbidity. Finally, CBM 588 administered to turkeys at doses up to 2.5 × 10 7 CFU/g feed for 84 days produced no detrimental effects on zootechnical performance, hematology, or biochemical parameters. Some improvements in zootechnical performance were seen with CBM 588, including improved average daily gain (ADG) and feed conversion for broilers from days 1 to 21 as well as final body weight and overall ADG for turkeys. Overall, CBM 588 administered in feed at dose up to 5 × 10 7 CFU/g (broilers and piglets) or 2.5 × 10 7 CFU/g (turkeys) was shown to be safe and well-tolerated in all tested animals and may provide some nutritional benefit when added to standard commercial feed.

Supplementary_Material for In vivo safety evaluation of the Clostridium butyricum MIYAIRI 588 strain in broilers, piglets, and turkeys by Kentaro Oka, Elinor McCartney, Tadashi Ariyoshi, Hayami Kudo, Borja Vilá, Lineke de Jong, Krzysztof Kozłowski, Jan Jankowski, Sarah Morgan, Claire Kruger, and Motomichi Takahashi in Toxicology Research and Application

Background Shiga toxin-producing Escherichia coli (STEC) causes serious gastrointestinal illness. Although O157 is predominant, non-O157 infections have been increasingly reported worldwide. We used whole-genome sequencing (WGS) to investigate molecular characteristics and phylogeny of STEC isolates. Methods A total of 22 STEC isolates from symptomatic patients (n = 13) and asymptomatic carriers (n = 9) in a Japanese region during 2016–2017 were used. Serogroups were O157, O26 and O103 (n = 5, 12, and 5, respectively). WGS was performed using an Illumina Miseq. Genomic analysis was performed using web-based tools by the Center for Genomic Epidemiology. Single nucleotide polymorphism detection and construction of phylogenetic tree were performed using Mauve software. Results Of 76 virulence genes, 32 (42%) were detected (Figure 1). Eighteen (82%) and 7 (32%) isolates contained stx1 and stx2, respectively. Twelve (91%) contained eae. stx2 was more frequent in isolates from patients (P < 0.05), whereas cba was more frequent in isolates from carriers (P < 0.05). stx2, etpD were more frequent in O157 isolates (P < 0.05, respectively), whereas stx1, efa1, cif, tccP, cba, lpfA were more frequent in non-O157 isolates (P < 0.05, respectively). Nine acquired resistance gene (aph(3′)-Ia, blaTEM-1b, dfrA5, dfrA8, strA, strB, sul2, tetA, tetB) were detected, while at least one was found in 6 (27%) isolates. Isolates from patients (5/13, 38%) were likely to have more resistance genes than isolates from carriers (1/9, 11%) (P = 0.33). Genotyping and multilocus sequence typing revealed all O26 isolates belonged to O26:H11 ST21, O103 belonged to O103:H2 ST17 and novel O103:H8 ST2836, while O157 belonged to O157:H7 ST11 and ST2966 (Figure 2). Phylogenetic tree showed O103:H8 ST2836 isolates clustered with O26, separated from O103:H2 ST17 (Figure 3). In a cluster of O26:H11 ST21 isolates, isolates from carriers formed a subcluster. O157 isolates clustered in a separate lineage. O157:H7 ST2966 isolates evolved from ST11. Conclusion Of the non-O157 isolates, O26:H11 ST21, which contained as many virulence genes as O157, was prevalent among both patients and carriers in our region, highlighting the importance of monitoring genomic characteristics of non-O157 STEC. Disclosures All authors: No reported disclosures.

Shiga toxin-producing Escherichia coli (STEC) can cause severe gastrointestinal disease and colonization among food handlers. In Japan, STEC infection is a notifiable disease, and food handlers are required to undergo routine stool examination for STEC. However, the molecular epidemiology of STEC is not entirely known. We investigated the genomic characteristics of STEC from patients and asymptomatic food handlers in Miyagi Prefecture, Japan. Whole-genome sequencing (WGS) was performed on 65 STEC isolates obtained from 38 patients and 27 food handlers by public health surveillance in Miyagi Prefecture between April 2016 and March 2017. Isolates of O157:H7 ST11 and O26:H11 ST21 were predominant (n = 19, 29%, respectively). Non-O157 isolates accounted for 69% (n = 45) of all isolates. Among 48 isolates with serotypes found in the patients (serotype O157:H7 and 5 non-O157 serotypes, O26:H11, O103:H2, O103:H8, O121:H19 and O145:H28), adhesion genes eae, tir, and espB, and type III secretion system genes espA, espJ, nleA, nleB, and nleC were detected in 41 to 47 isolates (85–98%), whereas isolates with other serotypes found only in food handlers were negative for all of these genes. Non-O157 isolates were especially prevalent among patients younger than 5 years old. Shiga-toxin gene stx1a, adhesion gene efa1, secretion system genes espF and cif, and fimbrial gene lpfA were significantly more frequent among non-O157 isolates from patients than among O157 isolates from patients. The most prevalent resistance genes among our STEC isolates were aminoglycoside resistance genes, followed by sulfamethoxazole/trimethoprim resistance genes. WGS revealed that 20 isolates were divided into 9 indistinguishable core genomes (

Clostridium butyricum MIYAIRI 588 (CBM 588) is a nonpathogenic, anaerobic, gram-positive bacillus characterized by the production of short-chain fatty acids, including butyrate. The safety and tolerance of CBM 588 was investigated as a feed additive for broiler chickens, weaned piglets, and turkeys. CBM 588 administered to broilers at doses up to 5 × 107 CFU/g feed for 42 days produced no detrimental effects on zootechnical performance, natural mortality, hematology, or biochemical parameters. Piglets receiving CBM 588 at doses up to 5 × 107 CFU/g feed for 42 days showed no significant differences from controls in zootechnical performance, mortality, or morbidity. Finally, CBM 588 administered to turkeys at doses up to 2.5 × 107 CFU/g feed for 84 days produced no detrimental effects on zootechnical performance, hematology, or biochemical parameters. Some improvements in zootechnical performance were seen with CBM 588, including improved average daily gain (ADG) and feed conversion for broilers from days 1 to 21 as well as final body weight and overall ADG for turkeys. Overall, CBM 588 administered in feed at dose up to 5 × 107 CFU/g (broilers and piglets) or 2.5 × 107 CFU/g (turkeys) was shown to be safe and well-tolerated in all tested animals and may provide some nutritional benefit when added to standard commercial feed info:eu-repo/semantics/publishedVersion