Flagella and curli fimbriae are important for the growth of Salmonella enterica serovars in hen eggs

Salmonella enterica serovar Enteritidis is unable to multiply in the albumen of fresh eggs and must gain access to the yolk contents in order to multiply to a high level (> 10(6) c.f.u. per ml egg contents). As human Salmonella infections resulting from the consumption of infected eggs more frequently involve serovar Enteritidis phage type (PT) 4 than other serovars or PTs, a number of isolates of various S. enterica serovars were examined for their ability to multiply to a high level in eggs over a period of 8 days storage at 20 degreesC. Their behaviour was compared to that of a range of defined fimbrial and flagella mutants of S. Enteritidis. Strains that did not express flagella were unable to multiply in eggs, and those deficient for curli fimbriae, including strains of S. Enteritidis PT6, displayed high-level growth in significantly fewer eggs than those able to express curli. Most S. Enteritidis strains multiplied to a high level in between 5 and 10 % of eggs during 8 days storage. One PT4 strain, though, showed high levels of growth in more than 25 % of eggs over this period, significantly higher than the other PTs or the two other isolates of PT4 tested. This ability may be important for the association of PT4 infection with the consumption of eggs.

Effect of triclosan or a phenolic farm disinfectant on the selection of antibiotic-resistant Salmonella enterica

Objective: To determine the effect of growth of five strains of Salmonella enterica and their isogenic multiply antibiotic-resistant (MAR) derivatives with a phenolic farm disinfectant or triclosan (biocides) upon the frequency of mutation to resistance to antibiotics or cyclohexane. Methods: Strains were grown in broth with or without the biocides and then spread on to agar containing ampicillin, ciprofloxacin or tetracycline each at 4x MIC or agar overlaid with cyclohexane. Incubation was for 24 and 48 h and the frequency of mutation to resistance was calculated for strains with and without prior growth with the biocides. MICs were determined and the presence of mutations in the acrR and marR regions was determined by sequencing and the presence of mutations in gyrA by light-cycler analysis, for a selection of the mutants that arose. Results: The mean frequency of mutation to antibiotic or cyclohexane resistance was increased similar to10- to 100-fold by prior growth with the phenolic disinfectant or triclosan. The increases were statistically significant for all antibiotics and cyclohexane following exposure to the phenolic disinfectant (P less than or equal to 0.013), and for ampicillin and cyclohexane following exposure to triclosan (P less than or equal to 0.009). Mutants inhibited by >1 mg/L ciprofloxacin arose only from strains that were MAR. Reduced susceptibility to ciprofloxacin (at 4x MIC for parent strains) alone was associated with mutations in gyrA. MAR mutants did not contain mutations in the acrR or marR region. Conclusions: These data renew fears that the use of biocides may lead to an increased selective pressure towards antibiotic resistance.

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

Aims: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [ indicative of a multiple antibiotic resistance ( MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. Methods and Results: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli ( 10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive ( n = 5) and - resistant ( n = 5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli ( n = 389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations ( MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers ( Pless than or equal to 0(.)023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. Conclusions: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. Significance and Impact of the Study: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.

Commonly used farm disinfectants can select for mutant Salmonella enterica serovar Typhimurium with decreased susceptibility to biocides and antibiotics without compromising virulence

Objectives: To determine if one passage of Salmonella enterica serovar Typhimurium in the presence of farm disinfectants selected for mutants with decreased susceptibility to disinfectants and/or antibiotics. Methods: Eight Salmonella Typhimurium strains including field isolates and laboratory mutants were exposed to either a tar oil phenol (PFD) disinfectant, an oxidizing compound disinfectant (OXC), an aldehyde based disinfectant (ABD) or a dairy sterilizer disinfectant (based on quaternary ammonium biocide) in agar. The susceptibility of mutants obtained after disinfectant exposure to antibiotics and disinfectants was determined as was the accumulation of norfloxacin. The proteome of SL1344 after exposure to PFD and OXC was analysed using two-dimensional liquid chromatography mass spectrometry. Results: Strains with either acrB or tolC inactivated were more susceptible to most disinfectants than other strains. The majority (3/5) of mutants recovered after disinfectant exposure required statistically significantly longer exposure times to disinfectants than their parent strains to generate a 5 log kill. Small decreases in antibiotic susceptibility were observed but no mutants were multiply antibiotic-resistant (MAR). Notably exposure to ABD decreased susceptibility to ciprofloxacin in some strains. Mutants with increased disinfectant tolerance were able to survive and persist in chicks as well as in parent strains. Analysis of proteomes revealed significantly increased expression of the AcrAB-TolC efflux system after PFD exposure. Conclusions: Data presented demonstrate that efflux pumps are required for intrinsic resistance to some disinfectants and that exposure to disinfectants can induce expression of the AcrAB-TolC efflux system, but that single exposure was insufficient to select for MAR strains.

Direct experimental evidence that early-life farm environment influences regulation of immune responses

Background: In mammals, early-life environmental variations appear to affect microbial colonization and therefore competent immune development, and exposure to farm environments in infants has been inversely correlated with allergy development. Modelling these effects using manipulation of neonatal rodents is difficult due to their dependency on the mother, but the relatively independent piglet is increasingly identified as a valuable translational model for humans. This study was designed to correlate immune regulation in piglets with early-life environment. Methods: Piglets were nursed by their mother on a commercial farm, while isolatorreared siblings were formula fed. Fluorescence immunohistology was used to quantify T-reg and effector T-cell populations in the intestinal lamina propria and the systemic response to food proteins was quantified by capture ELISA. Results: There was more CD4+ and CD4+CD25+ effector T-cell staining in the intestinal mucosa of the isolator-reared piglets compared with their farm-reared counterparts. In contrast, these isolator-reared piglets had a significantly reduced CD4+CD25+Foxp3+ regulatory T-cell population compared to farm-reared littermates, resulting in a significantly higher T-reg-to-effector ratio in the farm animals. Consistent with these findings, isolator-reared piglets had an increased serum IgG anti-soya response to novel dietary soya protein relative to farm-reared piglets. Conclusion: Here, we provide the first direct evidence, derived from intervention, that components of the early-life environment present on farms profoundly affects both local development of regulatory components of the mucosal immune system and immune responses to food proteins at weaning. We propose that neonatal piglets provide a tractable model which allows maternal and treatment effects to be statistically separated.