Transcriptomic modifications of the thyroid gland upon exposure to phytosanitary-grade fipronil: Evidence for the activation of compensatory pathways.

Fipronil is a phenylpyrazole insecticide used for the control of a variety of pest for domestic, veterinary and agricultural uses. Fipronil exposure is associated to thyroid disruption in the rat. It increases thyroid hormone (TH) hepatic clearance. The effect on thyroxine (T4) clearance is about four fold higher than the effect on T4 plasma concentrations suggesting that the thyroid gland might develop compensatory mechanisms. The aim of this study was to document the potential effects of fipronil treatment on the thyroid transcriptome together with its effects on TSH and TH blood levels under well characterized internal exposure to fipronil and its main metabolite fipronil sulfone. Fipronil (3 mg/kg/d by gavage for 14 days) clearance increased while its half-life decreased (about 10 fold) throughout treatment. Fipronil treatment in adult female rats significantly decreased total T4 and free triiodothyronine (T3) concentrations. Key genes related to thyroid hormone synthesis and/or cellular dynamic were modulated by fipronil exposure. RT-PCR confirmed that thyroglobulin gene expression was upregulated. A trend toward higher Na/I symporter expression was also noted, while sulfotransferase 1a1 gene expression was down-regulated. The expression of genes potentially involved in thyroid cell dynamic were upregulated (e.g. prostaglandin synthase 1, amphiregulin and Rhoa). Our results indicate that both pathways of TH synthesis and thyroid cell dynamics are transcriptional targets of fipronil and/or its main sulfone metabolite. The underlying mechanisms remain to be elucidated.

Impact of Low and High Doses of Marbofloxacin on the Selection of Resistant Enterobacteriaceae in the Commensal Gut Flora of Young Cattle: Discussion of Data from 2 Study Populations.

In the context of requested decrease of antimicrobial use in veterinary medicine, our objective was to assess the impact of two doses of marbofloxacin administered on young bulls (YBs) and veal calves (VCs) treated for bovine respiratory disease, on the total population of Enterobacteriaceae in gut flora and on the emergence of resistant Enterobacteriaceae. In two independent experiments, 48 YBs from 6 commercial farms and 33 VCs previously colostrum deprived and exposed to cefquinome were randomly assigned to one of the three groups LOW, HIGH, and Control. In LOW and HIGH groups, animals received a single injection of, respectively, 2 and 10 mg/kg marbofloxacin. Feces were sampled before treatment, and at several times after treatment. Total and resistant Enterobacteriaceae enumerating were performed by plating dilutions of fecal samples on MacConkey agar plates that were supplemented or not with quinolone. In YBs, marbofloxacin treatment was associated with a transient decrease in total Enterobacteriaceae count between day (D)1 and D3 after treatment. Total Enterobacteriaceae count returned to baseline between D5 and D7 in all groups. None of the 48 YBs harbored marbofloxacin-resistant Enterobacteriaceae before treatment. After treatment, 1 out of 20 YBs from the Control group and 1 out of 14 YBs from the HIGH group exhibited marbofloxacin-resistant Enterobacteriaceae. In VCs, the rate of fluoroquinolone-resistant Enterobacteriaceae significantly increased after low and high doses of marbofloxacin treatment. However, the effect was similar for the two doses, which was probably related to the high level of resistant Enterobacteriaceae exhibited before treatment. Our results suggest that a single treatment with 2 or 10 mg/kg marbofloxacin exerts a moderate selective pressure on commensal Enterobacteriaceae in YBs and in VCs. A fivefold decrease of marbofloxacin regimen did not affect the selection of resistances among commensal bacteria.

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.

Prevalence and characterization of the seven major serotypes of Shiga toxin-producing Escherichia coli (STEC) in veal calves slaughtered in France.

Shiga toxin (Stx)-producing Escherichia coli (STEC) belonging to the "top 7″ serotypes (i.e. O157:H7, O26:H11, O45:H2, O103:H2, O111:H8, O121:H19 and O145:H28) are considered as the main pathogenic enterohemorrhagic E. coli (EHEC). As ruminants, including calves, are a reservoir of pathogenic STEC, we investigated the prevalence, major virulence genes and genetic relatedness of top7 STEC in veal calves slaughtered in France, through the analysis of 500 fecal samples collected over one year. Thirty top7 STEC isolates were recovered from 28 calves. The two serotypes O103:H2 and O26:H11 accounted for 73% of STEC strains, followed by O145:H28 and O157:H7. STEC super-shedding levels were identified for two calves carrying STEC O103:H2 and O157:H7, respectively. Thirty-nine atypical enteropathogenic E. coli (aEPEC) were also recovered from calves. Overall, a prevalence of 5.6% top7 STEC-positive calves was found, thus higher than that previously determined for the French slaughtered adult cattle (1.8%), confirming the impact of animals age on STEC carriage. Most top7 STEC strains carried the stx1a subtype suggesting a low pathogenicity for humans. Seasonal variation in STEC carriage was also observed, with two peaks of higher prevalence during spring and fall. Genetic similarity of top7 STEC isolates was found for calves originating from the same fattening facilities, reflecting STEC circulation between animals kept in groups. This study indicates that veal calves grown for meat production are at higher risk of shedding top7 STEC compared to adult cattle. They thus represent ideal targets for the implementation of farm interventions aimed at reducing STEC burden in cattle and the food chain.

High Fecal Prevalence of mcr-Positive Escherichia coli in Veal Calves at Slaughter in France.

The aim of this study was to determine the percentage of healthy veal calves carrying mcr-positive E. coli strains at the time of slaughter in France. Fecal samples were selectively screened for mcr-positive E. coli isolates using media supplemented with colistin. Screening for mcr genes was also carried out in E. coli isolates resistant to critically important antimicrobials used in human medicine recovered from the same fecal samples. Overall, 28 (16.5%) out of the 170 veal calves tested carried mcr-positive E. coli. As some calves carried several non-redundant mcr-positive strains, 41 mcr-positive E. coli were recovered. Thirty-one and seven strains were positive for mcr-1 and mcr-3 genes, respectively, while no strain was positive for the mcr-2 gene. Co-carriage of mcr-1 and mcr-3 was identified in three strains. All mcr-positive E. coli isolates, except one, were multidrug-resistant, with 56.1% being ciprofloxacin-resistant and 31.7% harboring blaCTX-M genes. All mcr-3-positive E. coli carried blaCTX-M genes, mainly blaCTX-M-55. This study highlights the high prevalence of mcr-positive E. coli strains in feces of veal calves at the time of slaughter. It also points out the multidrug (including ciprofloxacin) resistance of such strains and the co-occurrence of mcr-3 genes with blaCTX-M-55 genes.

Prediction of Minocycline Activity in the Gut From a Pig Preclinical Model Using a Pharmacokinetic -Pharmacodynamic Approach.

The increase of multidrug-resistant (MDR) bacteria has renewed interest in old antibiotics, such as minocycline, that can be active against various MDR Gram-negative pathogens. The elimination of minocycline by both kidneys and liver makes it suitable for impaired renal function patients. However, the drawback is the possible elimination of a high amount of drug in the intestines, with potential impact on the digestive microbiota during treatment. This study aimed to predict the potential activity of minocycline against Enterobacterales in the gut after parenteral administration, by combining in vivo and in vitro studies. Total minocycline concentrations were determined by UPLC-UV in the plasma and intestinal content of piglets following intravenous administration. In parallel, the in vitro activity of minocycline was assessed against two Escherichia coli strains in sterilized intestinal contents, and compared to activity in a standard broth. We found that minocycline concentrations were 6-39 times higher in intestinal contents than plasma. Furthermore, minocycline was 5- to 245-fold less active in large intestine content than in a standard broth. Using this PK-PD approach, we propose a preclinical pig model describing the link between systemic and gut exposure to minocycline, and exploring its activity against intestinal Enterobacterales by taking into account the impact of intestinal contents.

Selection of ESBL-producing Escherichia coli in the gut of calves experimentally fed with milk containing antibiotic residues.

In the bovine sector, the spread of Enterobacterales producing extended-spectrum and AmpC ß-lactamases (ESBL/AmpC) mostly concerns veal calves, and the use of waste milk containing antibiotic residues has been recurrently incriminated. In this study, calves were experimentally fed with milk containing either 2,000 µg/L or 20,000 µg/L of the critically important antibiotic cefquinome. The total counts of enterobacterales and ESBL-producing E. coli were monitored using non-selective and selective media. Our data highlighted the important combination of two main factors (cefquinome exposure and initial ESBL colonization level) in the ESBL selection and amplification process in the gut of calves. Results also proved the dose-independent effect of cefquinome administration on the selection and amplification of ESBL-producing E. coli. Finally, the blaCTX-M-1/IncI1 ST3 plasmid was systematically recovered after cefquinome exposure, highlighting its epidemic success. Altogether, this work is one of the rare experimental studies providing quantitative information on the impact of waste milk containing antimicrobials on the ESBL load in calves' microbiota, and the first one using cefquinome. These data emphasise the need for global guidelines on the use of waste milk on dairy farms in order to decrease the antimicrobial resistance burden in this sector.

Early Inoculation of Microbial Suspension in Suckling Piglets Affects the Transmission of Maternal Microbiota and the Associated Antibiotic Resistance Genes.

Antibiotic resistance of microbes thriving in the animal gut is a growing concern for public health as it may serve as a hidden reservoir for antibiotic resistance genes (ARGs). We compared 16 control piglets to 24 piglets fed for 3 weeks with S1 or S2 fecal suspensions from two sows that were not exposed to antibiotics for at least 6 months: the first suspension decreased the erythromycin resistance gene ermB and the aminoglycoside phosphotransferase gene conferring resistance to kanamycine (aphA3), while the second decreased the tetracycline resistance gene tetL, with an unexpected increase in ARGs. Using 16S RNA sequencing, we identified microbial species that are likely to carry ARGs, such as the lincosamide nucleotidyltransferase lnuB, the cephalosporinase cepA, and the tetracycline resistance genes tetG and tetM, as well as microbes that never co-exist with the tetracycline resistance gene tetQ, the erythromycin resistance gene ermG and aphA3. Since 73% of the microbes detected in the sows were not detected in the piglets at weaning, a neutral model was applied to estimate whether a microbial species is more important than chance would predict. This model confirmed that force-feeding modifies the dynamics of gut colonization. In conclusion, early inoculation of gut microbes is an interesting possibility to stimulate gut microbiota towards a desirable state in pig production, but more work is needed to be able to predict which communities should be used.

Variability of the Ability of Complex Microbial Communities to Exclude Microbes Carrying Antibiotic Resistance Genes in Rabbits.

Reducing antibiotic use is a necessary step toward less antibiotic resistance in livestock, but many antibiotic resistance genes can persist for years, even in an antibiotic-free environment. In this study, we investigated the potential of three fecal complex microbial communities from antibiotic-naive does to drive the microbiota of kits from antibiotic-exposed dams and outcompete bacteria-carrying antibiotic-resistant genes. The fecal complex microbial communities were either orally delivered or simply added as fresh fecal pellets in four to five nests that were kept clean from maternal feces. Additionally, four nests were cleaned for the maternal feces and five nests were handled according to the common farm practice (i.e., cleaning once a week) as controls. At weaning, we measured the relative abundance of 26 antibiotic resistance genes, the proportion of Enterobacteriaceae resistant to tetracycline and sulfonamide antibiotics, and the taxonomic composition of the microbiota by sequencing the 16S rRNA genes of one kit per nest. Changing the surrounding microbes of the kits can hinder the transmission of antibiotic resistance genes from one generation to the next, but the three communities widely differed in their ability to orient gut microbes and in their impact on antibiotic resistance genes. The most efficient delivery of the microbial community reduced the proportion of resistant Enterobacteria from 93 to 9%, decreased the relative abundance of eight antibiotic resistance genes, and changed the gut microbes of the kits at weaning. The least efficient did not reduce any ARG or modify the bacterial community. In addition, adding fecal pellets was more efficient than the oral inoculation of the anaerobic suspension derived from these fecal pellets. However, we were unable to predict the outcome of the exclusion from the data of the donor does (species composition and abundance of antibiotic resistance genes). In conclusion, we revealed major differences between microbial communities regarding their ability to exclude antibiotic resistance genes, but more work is needed to understand the components leading to the successful exclusion of antibiotic resistance genes from the gut. As a consequence, studies about the impact of competitive exclusion should use several microbial communities in order to draw general conclusions.

Prevalence of Beta-Lactam and Quinolone/Fluoroquinolone Resistance in Enterobacteriaceae From Dogs in France and Spain-Characterization of ESBL/pAmpC Isolates, Genes, and Conjugative Plasmids.

Quantitative data on fecal shedding of antimicrobial-resistant bacteria are crucial to assess the risk of transmission from dogs to humans. Our first objective was to investigate the prevalence of quinolone/fluoroquinolone-resistant and beta-lactam-resistant Enterobacteriaceae in dogs in France and Spain. Due to the particular concern about possible transmission of extended-spectrum cephalosporin (ESC)-resistant isolates from dogs to their owners, we characterized the ESBL/pAmpC producers collected from dogs. Rectal swabs from 188 dogs, without signs of diarrhea and that had not received antimicrobials for 4 weeks before the study, were quantified for total and resistant Enterobacteriaceae on selective media alone or containing relevant antibiotic concentrations. Information that might explain antibiotic resistance was collected for each dog. Extended-spectrum cephalosporin-resistant isolates were subjected to bacterial species identification (API20E), genetic lineage characterization (MLST), ESBL/pAmpC genes identification (sequencing), and plasmid characterization (pMLST). Regarding beta-lactam resistance, amoxicillin- (AMX) and cefotaxime- (CTX) resistant Enterobacteriaceae were detected in 70 and 18% of the dogs, respectively, whereas for quinolone/fluoroquinolone-resistance, Nalidixic acid- (NAL) and ciprofloxacin- (CIP) resistant Enterobacteriaceae were detected in 36 and 18% of the dogs, respectively. Medical rather than preventive consultation was a risk marker for the presence of NAL and CIP resistance. CTX resistance was mainly due to a combination of specific ESBL/pAmpC genes and particular conjugative plasmids already identified in human patients: bla CTX-M-1/IncI1/ST3 (n = 4), bla CMY-2/IncI1/ST12 (n = 2), and bla CTX-M-15/IncI1/ST31 (n = 1). bla SHV-12 (n = 3) was detected in various plasmid lineages (InI1/ST3, IncI1/ST26, and IncFII). ESBL/pAmpC plasmids were located in different genetic lineages of E. coli, with the exception of two strains in France (ST6998) and two in Spain (ST602). Our study highlights dogs as a potential source of Q/FQ-resistant and ESBL/pAmpC-producing bacteria that might further disseminate to humans, and notably a serious risk of future acquisition of CTX-M-1 and CMY-2 plasmids by the owners of dogs.

Mixing of Shiga toxin-producing and enteropathogenic Escherichia coli in a wastewater treatment plant receiving city and slaughterhouse wastewater.

Wastewater of human and animal may contain Shiga toxin-producing (STEC) and enteropathogenic (EPEC) Escherichia coli. We evaluated the prevalence of such strains in a wastewater treatment plant (WWTP) receiving both city and slaughterhouse wastewater. PCR screenings were performed on 12,248 E. coli isolates. The prevalence of STEC in city wastewater, slaughterhouse wastewater and treated effluent was 0.22%, 0.07% and 0.22%, respectively. The prevalence of EPEC at the same sampling sites was 0.63%, 0.90% and 0.55%. No significant difference was observed between the sampling points. Treatment had no impact on these prevalences. Enterohemorrhagic E. coli (EHEC) O157:H7 and O111:H8 were isolated from the treated effluent rejected into the river. The characteristics of STEC and EPEC differed according to their origin. City wastewater contained STEC with various stx subtypes associated with serious human disease, whereas slaughterhouse wastewater contained exclusively STEC with stx2e subtype. All the EPEC strains were classified as atypical and were screened for the ε, γ1 and ß1 subtypes, known to be associated with the EHEC mainly involved in human infections in France. In city wastewater, eae subtypes remained largely unidentified; whereas eae-ß1 was the most frequent subtype in slaughterhouse wastewater. Moreover, the EPEC isolated from slaughterhouse wastewater were positive for other EHEC-associated virulence markers, including top five serotypes, the ehxA gene, putative adherence genes and OI-122 associated genes. The possibility that city wastewater could contain a pool of stx genes associated with human disease and that slaughterhouse wastewater could contain a pool of EPEC sharing similar virulence genes with EHEC, was highlighted. Mixing of such strains in WWTP could lead to the emergence of EHEC by horizontal gene transfer.

Dominant plasmids carrying extended-spectrum ß-lactamases blaCTX-M genes in genetically diverse Escherichia coli from slaughterhouse and urban wastewaters.

Wastewater treatment plants (WWTP) receiving effluents from food-producing animals and humans may contribute to the spread of extended-spectrum ß-lactamases (ESBL)-carrying plasmids. This study was designed to investigate extended-spectrum cephalosporin resistant Escherichia coli strains, CTX-M distributions and the genetic lineage of blaCTX-M -carrying plasmids from urban and slaughterhouse wastewaters. The level of extended-spectrum cephalosporin-resistant E. coli in slaughterhouse wastewater entering the WWTP was negligible compared with that of urban wastewater. The blaCTX-M-1 gene was predominant in slaughterhouse wastewater whereas diverse blaCTX-M genes were encountered in urban wastewater and WWTP outlet. Characterization of the main CTX-M-producing E. coli isolates by antibiotic resistance phenotyping, genotyping and typing of plasmids carrying blaCTX-M genes revealed that blaCTX-M-1 and blaCTX-M-15 genes were harboured by the predominant blaCTX-M-1 IncI1/ST3 and blaCTX-M-15 F31:A4:B1 plasmids, which were recovered from unrelated E. coli genotypes in both slaughterhouse and urban wastewaters. This study highlighted the spread of predominant blaCTX-M-1 and blaCTX-M-15 plasmid lineages in diverse E. coli genotypes from humans and food-producing animals, their mixing in WWTP and final release into the aquatic environment. This could have a serious negative impact on public health and requires further evaluation.

Persistence and prevalence of pathogenic and extended-spectrum beta-lactamase-producing Escherichia coli in municipal wastewater treatment plant receiving slaughterhouse wastewater.

We compared the prevalence of pathogenic and extended-spectrum beta-lactamase (ESBL) - producing Escherichia coli in effluents of a municipal wastewater treatment plant (WWTP) receiving wastewater from a slaughterhouse. A total of 1248 isolates were screened for the presence of virulence genes associated with enterohemorrhagic E. coli (EHEC) (stx1, stx2, and eae) and extraintestinal pathogenic E. coli (ExPEC) (sfa/focDE, kpsMT K1, hlyA, papEF, afa/draBC, clbN, f17A and cnf). The prevalence of atypical enteropathogenic E. coli (EPEC) was 0.7%, 0.2% and 0.5% in city wastewater, slaughterhouse wastewater and in the treated effluent, respectively. One stx1a and stx2b-positive E. coli isolate was detected in city wastewater. The prevalence of ExPEC was significantly higher in city wastewater (8.4%), compared to slaughterhouse wastewater (1.2%). Treatment in the WWTP did not significantly impact the prevalence of ExPEC in the outlet effluent (5.0%) compared to city wastewater. Moreover, the most potentially pathogenic ExPEC were isolated from city wastewater and from the treated effluent. ESBL-producing E. coli was also mainly detected in city wastewater (1.7%), compared to slaughterhouse wastewater (0.2%), and treated effluent (0.2%). One ESBL-producing E. coli, isolated from city wastewater, was eae-ß1 positive. These results showed that pathogenic and/or ESBL-producing E. coli were mainly detected in human wastewater, and at a lesser extend in animal wastewater. Treatment failed to eliminate these strains which were discharged into the river, and then these strains could be transmitted to animals and humans via the environment.

Oral absorption of ampicillin: role of paracellular route vs. PepT1 transporter.

The beta-lactam antibiotic ampicillin has a relatively poor oral bioavailability in animals and man (30-40%), and its widespread agricultural use in livestock may be contributing to the emergence of antibiotic resistance in the environment. The aim of this study was to define the absorption mechanism by which ampicillin crosses the small intestinal epithelium. The improved rat everted gut sac system was used, with an emphasis on the role of the PepT1 transporter. The absorption kinetics, effects of pH and the use of competitive substrates failed to provide any substantive evidence that the transporter played a major role in ampicillin absorption. Ethylenediaminetetraacetic acid enhanced the absorption, and tissue levels remained low, suggesting that paracellular transport was predominant. pH and competition studies with glycylsarcosine, the widely used PepT1 substrate, also failed to show any transporter activity. Despite evidence from studies with Caco-2 cells that beta-lactam antibiotics are transported by the PepT1 transporter in rat small intestine, the results rather suggest that paracellular diffusion is the major mechanism of absorption, at least for beta-lactam antibiotics with poor bioavailability, such as ampicillin. We suggest that the use of Caco-2 cells underestimates the role of the paracellular route in the absorption of hydrophilic drugs in vivo, and may exaggerate the role of influx transporters.

Influence of inoculum size on the selection of resistant mutants of Escherichia coli in relation to mutant prevention concentrations of marbofloxacin.

We demonstrate using an in vitro pharmacodynamic model that the likelihood of selection of Escherichia coli mutants resistant to a fluoroquinolone was increased when the initial size of the bacterial population, exposed to fluoroquinolone concentrations within the mutant selection window, was increased.