The sub-MIC selective window decreases along the digestive tract: determination of the minimal selective concentration of oxytetracycline in sterilised intestinal contents.

Introduction: The administration of antibiotics can expose the digestive microbiota of humans and animals to sub-inhibitory concentrations, potentially favouring the selection of resistant bacteria. The minimal selective concentration (MSC) is a key indicator to understand this process. The MSC is defined as the lowest concentration of an antibiotic that promotes the growth of a resistant strain over a susceptible isogenic strain. It represents the lower limit of the sub-minimal inhibitory concentration (MIC) selective window, where resistant mutants can be selected. Previous studies focused on determining the MSC under standard culture conditions, whereas our research aimed to determine the MSC in a model that approximates in vivo conditions. Methods: We investigated the MSC of oxytetracycline (OTC) in Mueller-Hinton broth (MHB) and sterilised intestinal contents (SIC) from the jejunum, caecum and rectum (faeces) of pigs, using two isogenic strains of Escherichia coli (one susceptible and one resistant to OTC). Additionally, the MIC of OTC against the susceptible strain was determined to assess the upper limit of the sub-MIC selective window. Results: Our study took a novel approach, and the results indicated that MIC and MSC values were lower in MHB than in SIC. In the latter, these values varied depending on the intestinal segment, with distal compartments exhibiting higher MIC and MSC values. Moreover, the sub-MIC selective window of OTC in SIC narrowed from the jejunum to the rectum, with a significantly closer MSC to MIC in faecal SIC. Discussion: The results suggest that OTC binds to digestive contents, reducing the fraction of free OTC. However, binding alone does not fully explain our results, and interactions between bacteria and intestinal contents may play a role. Furthermore, our findings provide initial estimates of low concentrations facilitating resistance selection in the gut. Finally, this research enhances the understanding of antimicrobial resistance selection, emphasising the intricate interplay between antibiotics and intestinal content composition in assessing the risk of resistance development in the gut.

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

Carnobacterium maltaromaticum as bioprotective culture against spoilage bacteria in ground meat and cooked ham.

This study assessed the bioprotective effect of Carnobacterium maltaromaticum (CM) against Pseudomonas fluorescens (PF) and Brochothrix thermosphacta (BT) in ground beef and sliced cooked ham stored in high- and low-oxygen-modified atmospheres (66/4/30% O2/N2/CO2 and 70/30% N2/CO2, respectively). Both meat products were inoculated with CM, PF, and BT individually or in combination and stored for 7 days (3 days at 4 °C + 4 days at 8 °C) for ground beef and 28 days (10 days at 4 °C + 18 days at 8 °C) for sliced cooked ham. Each food matrix was assigned to 6 treatments: NC (no bacterial inoculation, representing the indigenous bacteria of meat), CM, BT, PF, CM + BT, and CM + PF. Bacterial growth, pH, instrumental color, and headspace gas composition were assessed during storage. CM counts remained stable from inoculation and throughout the shelf-life. CM reduced the population of inoculated and indigenous spoilage bacteria, including BT, PF, and enterobacteria, and showed a negligible impact on the physicochemical quality parameters of the products. Furthermore, upon simulating the shelf-life of ground beef and cooked ham, a remarkable extension could be observed with CM. Therefore, CM could be exploited as a biopreservative in meat products to enhance quality and shelf-life.

Carnobacterium maltaromaticum as bioprotective culture in vitro and in cooked ham.

The bioprotective effects of Carnobacterium maltaromaticum (CM) strains were assessed in vitro and in sliced cooked ham. CM strains were tested in vitro against Listeria monocytogenes (LM), Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST). In vitro effect was evaluated using co-culture (with and without EDTA) and cell-free supernatant (CFS). CFS was tested by agar well diffusion and minimum inhibitory concentration. In cooked ham, the inhibitory effect of CM on L. innocua (LI) and on the physicochemical parameters were evaluated for 7 days at 4 °C. In co-cultures at -1 °C and 4 °C, all CM isolates inhibited LM. A slight inhibition was observed against the Gram-negative bacteria with the addition of EDTA. CFS did not show inhibitory effect under the studied conditions. In cooked ham, CM inhibited LI growth and did not affect the physicochemical parameters of the product during storage. CM strains show potential to be used as bioprotective cultures in cold-stored cooked ham and improve its safety.

Effect of sex and sub-zero storage temperature on the microbial and oxidative stability of beef packed in a high-oxygen atmosphere after different vacuum ageing times.

This study aimed to evaluate the effect of sex and sub-zero storage temperature on the microbial and oxidative stability of Belgian Blue beef packed in a high-oxygen atmosphere after different ageing times. Longissimus thoracis et lumborum from Belgian Blue young bulls and cull cows were aged at -1 or 4 °C for 80 days in vacuum. Every 20 days, samples were repackaged in a high-oxygen atmosphere (70/30% O2/CO2) and stored for 7 days (2 days at 4 °C + 5 days at 8 °C). Ageing at -1 °C had a protective effect against the growth of lactic acid bacteria and Enterobacteriaceae and myoglobin oxidation. Brochothrix thermosphacta was the limiting parameter for ageing longer than 20 days at -1 °C, permitting a subsequent 7-day shelf-life in a high-oxygen atmosphere. Meat from young bulls was more sensitive to oxidation than meat from cull cows. Extending Belgian Blue meat ageing for >20 days had a negative impact on retail shelf-life.

Estrogenic evaluation and organochlorine identification in blubber of North Sea harbour porpoise (Phocoena phocoena) stranded on the North Sea coast.

Thirteen individual organochlorine compounds at 3 concentrations (80, 400, and 2000 ng/mL culture medium), as well as mixtures, were assayed for the estrogen receptor (ER) activation or inhibition, using a luciferase reporter gene assay (RGA). None of the PCB 138, 153, or 180 or their mixture induced a response in the RGA. o,p'-DDT was the most potent xenoestrogen from the DDT group, inducing a response already at 80 ng/mL. From the HCH and HCB group, only ß-HCH (at 400 and 2000 ng/mL) and δ-HCH (at 2000 ng/mL) displayed estrogenic activities. These 13 organochlorines were determined by GC-MS in 12 samples of North Sea harbor porpoise blubber. The PCBs were the main contaminants. Within each group, PCB 153 (6.0 × 10(2)~4.2 × 10(4) µg/kg), p,p'-DDE (5.1 × 10(2)~8.6 × 10(3) µg/kg), and HCB (7.6 × 10(1)~1.5 × 10(3) µg/kg) were the compounds found in highest concentrations. The hormonal activity of the porpoise blubber samples was also assayed in RGA, where two samples showed estrogenic activity, seven samples showed antiestrogenic activity, and one sample showed both estrogenic and antiestrogenic activity. Our results suggest that the 13 POPs measured by GC-MS in the samples cannot explain alone the estrogenicity of the extracts.