Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability

Cefquinome and ceftiofur are β-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC–MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g−1 to 1000 ng g−1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g−1 to 2000 ng g−1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since β-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL−1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.

PSV-41 Antibacterial activity of ferulic acid and sodium chlorate against Escherichia coli F18 and K88 during incubations with porcine fecal bacteria

The influence of ferulic acid (FA) and sodium chlorate (SC) was evaluated in two trials on the growth of Escherichia coli F18 and K88 (F18 and K88) incubated with porcine fecal bacteria. Treatments were 2 levels of FA (0 and 5 mg/mL) and 2 levels of SC (0 and 10 mM/mL). In trial one, ½-strength Mueller Hinton broth mixed with porcine feces (0.5% w/v) was inoculated with a novobiocin and naladixic acid resistant F18-strain. This fecal suspension was transferred to tubes (3/treatment) and anaerobically incubated at 39 oC for enumeration at 0, 6 and 24 h using MacConkey agar supplemented with novobiocin and naladixic acid with aerobic incubation at 37 oC. An interaction (FA x SC) at 6 and 24 h was observed (P < 0.01). At 6 h of incubation, SC alone or combined with FA had the lowest counts (P < 0.05); FA alone was lower than control but higher than SC or SC+FA (P < 0.05). At 24 h, FA alone or combined with SC had the lowest counts (P < 0.05); SC was lower than control but higher than FA or SC+FA (P < 0.05). In trial 2 were used the same procedures of trial 1, except that K88 was used. There was an interaction at 6 h (P < 0.01) where the lowest counts were in FA+SC (P < 0.05). SC alone or FA alone were lower than control but higher than SC+FA (P < 0.05). There was no interaction at 24 h (P = 0.16), where FA reduced the K88 counts (P < 0.01), however it was not affected by SC (P = 0.12). In conclusion, SC reduced E. coli counts; however, at 24 h of incubation greater reductions were observed when FA alone or combined with SC was added into the incubation fluid with porcine feces.

PSV-40 Antibacterial activity of sodium chlorate and essential oils against Escherichia coli (F18 and K88) during incubations with porcine fecal bacteria

In two trials was evaluated the influence of sodium chlorate (SC) and essential oils (EO) on the growth of Escherichia coli F18 and K88 (F18 and K88) incubated with porcine fecal bacteria. The treatments were 2 levels of SC (0 and 10 mM/mL) and 2 levels of Activo® (0 and 1.5%; vol/vol). Activo® (EW Nutrition, Des Moines, IA) is a blend of oregano oil and cinnamon oil (EO) with water and citric acid. In trial one, ½-strength Mueller Hinton broth mixed with porcine feces (0.5% w/vol) was inoculated with a novobiocin and naladixic acid resistant F18-strain. This fecal suspension was transferred to tubes (3/treatment) and anaerobically incubated at 39 oC for enumeration at 0, 6 and 24 h using MacConkey agar supplemented with novobiocin and naladixic acid with aerobic incubation at 37 oC. An interaction (EO x SC) at 6 and 24 was observed (P < 0.01). At 6 and 24 h, EO alone or combined with SC had the lowest counts of F18 (P < 0.05); SC alone had lower counts of F18 than control (P < 0.05). In trial 2 were used the same procedures of trial 1, except that K88 was inoculated in the porcine fecal suspension. There was an interaction at 6 h (P < 0.01) where EO had the lowest counts of K88. The control showed the highest counts of K88 (P < 0.05). There was no interaction at 24 h (P = 0.14). The counts of K88 were reduced by EO (P < 0.01), however the counts were not affected by SC (P = 0.14). It was concluded that SC reduced the counts of E. coli F18, but it had minimal effect on E. coli K88 in the challenged porcine feces; essential oils were effective to reduce the pathogenic bacteria in the porcine feces.

Draft Genome Sequence of the Bacteriophage vB_Eco_slurp01

Bacteriophage vB_Eco_slurp01 was isolated from porcine feces using Escherichia coli MG1655 as a host. With a genome size of 348 kb, vB_Eco_slurp01 is one of the largest bacteriophages isolated to date.

In Vitro Effects of Thymol-β-d-Glucopyranoside on Salmonella enterica Serovar Typhimurium and Escherichia coli K88†

ABSTRACT Although thymol is bactericidal against many pathogens in vitro, its in vivo effectiveness against pathogens in the lower gastrointestinal tract is limited because of its rapid absorption in the proximal gut. Thymol-β-d-glucopyranoside (β-thymol), a conjugated form of thymol, can deliver thymol to the lower gastrointestinal tract and has shown antibacterial effects. In the present study, we examined the in vitro effects of β-thymol on Salmonella enterica serovar Typhimurium (ST) and Escherichia coli K88 (K88). We inoculated one-half strength Mueller-Hinton broth with 5.8 ± 0.09 log CFU/ml novobiocin- and naladixic acid–resistant (NN) ST (NVSL 95-1776) and 5.1 ± 0.09 log CFU ml−1 NN-resistant K88, with or without porcine feces (0.1% [wt/vol]) (fecal incubations). The resultant bacterial suspensions were distributed under N2 to triplicate sets of tubes to achieve initial concentrations of 0, 3, 6, and 12 mM for ST treatments and 0, 3, 12, and 30 mM for K88 treatments. Samples were incubated at 39°C and then plated onto NN-containing brilliant green agar and NN-containing MacConkey agar; ST and K88 CFU concentrations were determined via 10-fold dilutions, and viable cell counts were performed at 0, 6, and 24 h. No differences in ST CFU counts were observed in β-thymol–treated tubes without the added porcine feces (i.e., pure culture) at 6 or 24 h. However, in tubes that contained fecal incubations, ST CFU counts were reduced (P < 0.05) from controls at 6 h in tubes treated with 6 and 12 mM β-thymol, whereas in tubes treated with 3, 6, and 12 mM β-thymol the CFU counts were reduced (P < 0.05) at 24 h. No differences were observed in K88 CFU counts in pure culture or in fecal incubations at 6 h, but K88 CFU counts were reduced (P < 0.05) in both pure and fecal incubations at 24 h. The results from this study demonstrate that β-thymol, in the presence of fecal suspensions, has anti-Salmonella and anti–E. coli effects, suggesting a role of β-glycoside–hydrolyzing microbes for the release of bactericidal thymol from β-thymol.