Assessment of faecal contamination in selected concrete and earthen ponds stocked with Clarias gariepinus

Background This study was carried out to monitor the levels of faecal pollution markers in catfish (Clarias gariepinus) and their growing waters in selected earthen and concrete ponds. Water and catfish samples were collected weekly in the months of February, March, April, May, June and July, 2019. The concentrations of enteric bacteria in the water and catfish samples were determined using membrane filtration and pour plate methods, respectively. The rate of bioaccumulation of faecal indicator bacteria was obtained by dividing the log concentration of each organism in catfish by the corresponding log concentration in the growing waters. Result The concentration of faecal coliforms in catfish samples from concrete and earthen ponds ranged from 1.41 to 2.28 log10 CFU/100 ml and 1.3 to 2.47 log10 CFU/100 ml respectively and in growing waters from the concrete and earthen ponds; 1.43 to 2.41 log10 CFU/100 ml and 1.50 to 2.80 log10 CFU/100 ml respectively. Faecal coliforms exhibited positive relationships with alkalinity in water samples from the earthen (r = 0.61) and concrete ponds (r = 0.62). Salmonella and faecal coliforms had the highest and least bioaccumulation in catfish raised in earthen pond, respectively, whereas Salmonella and enterococci had the highest and least bioaccumulation in catfish raised in concrete pond, respectively. Faecal coliforms and E. coli had the highest and least counts in water samples from the earthen pond during the dry and wet months, Salmonella and E. coli had the highest and least counts in water samples from the concrete pond during the dry and wet months. Conclusion There were high levels of bacterial faecal pollution markers in water and C. gariepinus from the earthen and concrete ponds. Physicochemical characteristics of the water and seasonality played major roles in the rate of bioaccumulation of the faecal pollution markers in C. gariepinus raised in the earthen and concrete ponds.

Contamination of hay and haylage with enteric bacteria and selected antibiotic resistance genes following fertilization with dairy manure or biosolids.

The present study evaluated if enteric bacteria or antibiotic resistance genes carried in fecal amendments contaminate the hay at harvest, representing a potential route of exposure to ruminants that consume the hay. In field experiments, dairy manure was applied to a hay field for three successive growing seasons, and biosolids applied to a hay field for one growing season. Various enteric bacteria in the amendments were enumerated by viable plate count, and selected gene targets were quantified by qPCR. Key findings include the following: At harvest, hay receiving dairy manure or biosolids did not carry more viable enteric bacteria than did hay from unamended control plots. Fermentation of hay did not result in a detectable increase in viable enteric bacteria. The application of dairy manure or biosolids did result in a few gene targets being more abundant on hay at the first harvest. Fermentation of hay did result in an increase in the abundance of gene targets, but this occurred both with hay from amended and control plots. Overall, application of fecal amendments will result in an increase in the abundance of some gene targets associated with antibiotic resistance on first cut hay.

Enteric α-synuclein impairs intestinal epithelial barrier through caspase-1-inflammasome signaling in Parkinson’s disease before brain pathology

AbstractsBowel inflammation, impaired intestinal epithelial barrier (IEB), and gut dysbiosis could represent early events in Parkinson’s disease (PD). This study examined, in a descriptive manner, the correlation among enteric α-synuclein, bowel inflammation, impairments of IEB and alterations of enteric bacteria in a transgenic (Tg) model of PD before brain pathology. Human A53T α-synuclein Tg mice were sacrificed at 3, 6, and 9 months of age to evaluate concomitance of enteric inflammation, IEB impairments, and enteric bacterial metabolite alterations during the early phases of α-synucleinopathy. The molecular mechanisms underlying the interplay between α-synuclein, activation of immune/inflammatory responses and IEB alterations were investigated with in vitro experiments in cell cultures. Tg mice displayed an increase in colonic levels of IL-1β, TNF, caspase-1 activity and enteric glia activation since 3 months of age. Colonic TLR-2 and zonulin-1 expression were altered in Tg mice as compared with controls. Lipopolysaccharide levels were increased in Tg animals at 3 months, while fecal butyrate and propionate levels were decreased. Co-treatment with lipopolysaccharide and α-synuclein promoted IL-1β release in the supernatant of THP-1 cells. When applied to Caco-2 cells, the THP-1-derived supernatant decreased zonulin-1 and occludin expression. Such an effect was abrogated when THP-1 cells were incubated with YVAD (caspase-1 inhibitor) or when Caco-2 were incubated with anakinra, while butyrate incubation did not prevent such decrease. Taken together, early enteric α-synuclein accumulation contributes to compromise IEB through the direct activation of canonical caspase-1-dependent inflammasome signaling. These changes could contribute both to bowel symptoms as well as central pathology.

Efflux-Mediated bile Resistance in Gram-Positive Pathogens

Gram-positive pathogens are causing many serious infections that affect humans and result in mild to severe diseases worldwide. In order to survive and initiate infection, enteric pathogens must resist the physiochemical defence factors in the human intestinal tract. One of these defence factors is bile, a potent antibacterial like compound in the intestine. Efflux pumps are the important mechanism by which bacteria resist antibacterial agents such as bile. Efflux of antimicrobial substances outside the bacterial cell is considered as a key factor for intestinal colonization and virulence of enteric pathogens. This paper will review the research conducted on efflux–mediated bile resistance in Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis and Clostridium perfringens. These bacteria colonize in the human & animal gastrointestinal tract and they have a multiple mechanism to resist the innate defences in the gut and antibacterial activity of bile. However, bile resistance in these bacteria is not fully understood. The evidence from this review suggests that Gram-positive pathogens have the ability to active transport of bile. Further research is needed to know how these pathogens sense bile and how bile regulates its virulence factor. In general, therefore, it seems that understanding the specific mechanism of bile resistance in enteric bacteria including gram-positive pathogens may involve in the development of novel strategies to control and treatment of gastrointestinal infections.

C4-dicarboxylates as growth substrates and signaling molecules for commensal and pathogenic enteric bacteria in mammalian intestine

The C4-dicarboxylates (C4-DC) L-aspartate and L-malate have been identified as playing an important role in the colonization of mammalian intestine by enteric bacteria, such as Escherichia coli and Salmonella Typhimurium, and succinate as a signaling molecule for host–enteric bacteria interaction. Thus, endogenous and exogenous fumarate respiration and related functions are required for efficient initial growth of the bacteria. L-aspartate represents a major substrate for fumarate respiration in the intestine and a high-quality substrate for nitrogen assimilation. During nitrogen assimilation, DcuA catalyzes an L-aspartate/fumarate antiport and serves as a nitrogen shuttle for the net uptake of ammonium only, whereas DcuB acts as a redox shuttle that catalyzes the L-malate/succinate antiport during fumarate respiration. The C4-DC two-component system DcuS-DcuR is active in the intestine and responds to intestinal C4-DC levels. Moreover, in macrophages and in mice, succinate is a signal that promotes virulence and survival of S . Tm and pathogenic E. coli . On the other hand, intestinal succinate is an important signaling molecule for the host and activates response and protective programs. Therefore, C4-DCs play a major role in supporting colonization of enteric bacteria and as signaling molecules for the adaptation of host physiology.

Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

Background: The gastrointestinal (GI) tract is the reservoir for multidrug-resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals. Results: Here, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including MDR clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota. Conclusions: This study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step towards the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract.

Increased Rate of Enteric Bacteria As Cause of Periprosthetic Joint Infections In Patients With Liver Cirrhosis

Introduction: Periprosthetic joint infections (PJI) are a major complication in joint-arthroplasty. While Rifampicin is one of the few antimicrobial agents, that penetrate the bacterial biofilm and therefore is often used as an additional agent to treat PJI, rifampicin-resistant pathogens are also known to be cross-resistant to other approved rifamycins (rifambutin, rifaximin and rifapentine). Moreover, rifaximin, a broad-spectrum antibiotic with poor intestinal absorption, is used to prevent episodes of hepatic encephalopathy. As transient resistances to rifampin may emerge in patients after taking rifaximin the aim of this study was to examine the microbial pattern of periprosthetic joint infections in cirrhotic patients and to test the hypothesis that intake of rifaximin increases the rate of resistance to rifampicin in periprosthetic joint infections. Methods: A cohort of cirrhotic patients and PJI (n=25) was analysed on the characteristics of bacterial isolates from sonication and tissue analysis. In a second step a subgroup analysis on the development of rifampicin resistant bacterial specimens, depending on the intake of rifaximin (8 rifaximin intake patients vs. 13 non rifaximin intake patients) was performed. Results: Gram-negative bacteria were found in nearly 30% of the specimens. By comparison of the single bacterial isolates, rifampicin resistance was detected in 69.2% (9/13) of the rifaximin-intake samples. In contrast, the non-rifaximin-intake isolates only were resistant to rifampicin in 22.2% (4/18) of the cases (p=0.01). The odds ratio for developing a rifampicin-resistance through rifaximin intake was calculated as OR=13.5.Conclusion: Periprosthetic joint infections have a high incidence of being caused by gram-negative bacteria in cirrhotic patients. Due to this change in microbial pattern and the innate resistance to rifampicin of most of gram-negative bacteria, the therapy with rifampicin should be carefully considered. The association between the use of rifaximin and developed resistance to rifampicin has a major impact on the treatment of PJI.

Evaluation of Antioxidant and Antibacterial Activities of White Mulberry (Morus alba L.) Fruit Extracts

The fruit of mulberry trees (Morus sp.), mulberries, are traditionally utilised as a nutritional food and provide health benefits as well as skin nourishment in Thailand. White mulberries (Morus alba L.) from Chiang Mai and Mae Hong Son provinces were evaluated for their antioxidant and antibacterial activities. The antioxidant activities as well as the total phenolic, flavonoid and anthocyanin content of the aqueous and ethanolic extracts were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) assays. The aqueous extracts of mulberries exhibited the highest antioxidant activity, which was associated with a higher phenolic and anthocyanin content. In testing the potent antibacterial activity against Escherichia coli, Salmonella Typhi, Shigella dysenteriae, Staphylococcus aureus and Vibrio cholerae, the mulberry extracts proved to be quite efficient, especially following water extraction. Time-kill and antibacterial adhesion assays further indicated that aqueous mulberry extracts could inhibit bacterial growth and prevent adhesions of pathogenic enteric bacteria on intestinal epithelial cells. It thus appears that mulberries can potentially be consumed as a good source of antioxidants, containing antimicrobial properties against some pathogenic bacteria which cause gastrointestinal tract infections.

Characterizing and Overcoming Resistance to Aminomethylspectinomycins in Gram-negative Bacteria

Spectinomycin (SPC) is a broad-spectrum aminocyclitol antibiotic. Its use in agriculture has led to widespread resistance in enteric bacteria, necessitating the development of more effective analogs. Aminomethyl spectinomycins (amSPC) are modified spectinomycins with increased potency against many bacterial species. These species include Legionella pneumophila, which harbors a chromosomally encoded aminoglycoside modifying enzyme (AME). In this study, we follow up on this observation and examine the extent to which the amSPCs are substrates for AMEs through adenylation (ANTs) and phosphorylation (APH). APH(9)-Ia and ANT(3")(9) were expressed in E. coli BL21(DE3) and purified using the Ni-affinity chromatography. The ability of AMEs to modify and inactivate amSPCs has been examined by two unique biochemical assays, including an agar-based enzyme assay. Binding of APH (9)-Ia and ANT (3")(9) to spectinomycin and amSPCs has been studied using Thermal Denaturation assay and MicroScale Thermophoresis (MST). The microbiological role of these enzymes has been examined by minimum inhibitory concentration (MIC) shifts using an arabinose inducible expression of APH (9)-Ia and ANT (3")(9) in E.coli K12 and JW ΔtolC strains. Our agar-based enzyme assay shows the inactivation of spectinomycin by APH(9)-Ia. Phosphorylated spectinomycin and adenylated spectinomycin products upon incubation with APH(9)-Ia and ANT(3",9), respectively, have been identified using MALDI-MS. APH(9)-Ia induction studies in E. coli tolC knock-out strains reveal a MIC increase against spectinomycin in the presence of 2% arabinose compared to no shift with amSPCs. ANT (3")(9) showed an increase in MIC against spectinomycin as well as amSPCs. In conclusion, amSPCs are not inactivated by APH (9)-Ia in vivo but are inactivated by ANT (3")(9). Most Gram-negative bacteria isolated in clinics possess one or more AMEs. By overcoming modification by AMEs, amSPCs can be a valuable tool in overcoming resistance in Gram-negative bacterial infections. We also conducted a high throughput screen of a polar small molecule library against two multi-drug resistant clinical isolates of Escherichia coli that encode aminoglycoside modifying enzyme for small molecule potentiators of amSPCs to yield 12 possible potentiating molecules that have been confirmed by dose-response analysis. Future work as a continuation of this project will involve further analysis of any existing synergy between the potentiating molecules and amSPCs and target validation of these potentiators.