Background 

Multi drug-resistant Salmonella species represents a threat to the perceived and actual safety of beef in the United States.  Antibiotic resistance genes can be transferred between bacteria through several DNA transfer mechanisms, or they can spontaneously arise within the bacterium.  In either mechanism, the acquisition of new antimicrobial resistance genes by pathogens can pose a risk to consumers. 

When bacteria are stressed by their environment (or other conditions), they utilize a “SOS” response mechanism that, among other things, causes them take up DNA from the environment, and creates a situation where the bacterial chromosome is more plastic, meaning it incorporates new DNA into it more easily.  This enhanced plasticity of the genome under stress means that a bacterium can (in some cases) alter its genome in a desperate gamble to enhance its survivability.  These changes can quickly alter the resistance profile of a pathogen by inserting a resistance gene (or genes) into the genome.  Additionally, DNA repair mechanisms are reduced, meaning spontaneous mutations are not repaired, resulting in many point mutations that can confer resistance to some antibiotics.   

Chemostats are continuous flow culture devices that simulate the constant digesta flow through the gastrointestinal tract of cattle under highly controlled, replicable conditions.  Using these culture systems we can alter a single factor and determine the effect of stresses through environmental changes on bacteria.  This allows us to determine subtle differences in the competitive fitness of bacteria in a competitive environment that selects for ability to grow constantly, a good model of bacterial existence in the gut. 

The stated objectives for this work were: Do changing environmental stresses affect the ability of Salmonella to transfer MDR genes between species in continuous flow cultures?  Environmental changes were: (1) Acetate to Propionate Ratio; (2) pH; (3) Temperature; (4) Anionic Salts; (5) Mitomycin C Treatment; and (6) Growth Rate Increase.

Methodology 

All experiments utilized BioFlow III continues flow culture chemostats that are equipped with temperature jackets to maintain temperature at 39°C.  All experiments were performed using Viande-Levure (VL) Broth that contained (g/liter): tryptose (Becton Dickinson, Sparks, MD) 10, glucose 2.5, NaCl 5, yeast extract (Sigma–Aldrich, St. Louis, MO) 5, beef extract (Becton Dickinson) 2.4, Bacto agar (Becton Dickinson) 0.6, and cysteine hydrochloride (Sigma–Aldrich) 0.4.  Control chemostat was maintained stably for more than 8 months.    We grew a S. Kentucky isolate originally from beef cattle that was sensitive to all antibiotics except for Tetracycline in 4 chemostats in co-culture with a S. Typhimurium isolate from dairy cattle that was MDR (but not resistant to Tetracycline) and could grow on plates supplemented with novobiocin and nalidixic acid.  Bacterial populations reached a steady-state equilibrium at a growth rate of 0.05 h-1 similar to that found in the ruminant gut.  Novobiocin and Nalidixic acid were added at 10 and 15 μg/ml (a level of that the Typhimurium strain was resistant to, but that the Kentucky strain was not) to 2 of the chemostats to create a 2 x 2 factorial design (see below).  We then added one of several stressors to the chemostat as separate experiments.

Samples were removed from each chemostat in each run at -1, 0, 1, 2, 4, 8, 24, 48, 72, 96 and 120 hours after the initiation of each change.  We determined total populations of Salmonella and populations of each serotype by differential plating on BGA media containing Tet or Nov/Nal, respectively.  Isolates (6 from the highest dilution plate) recipient strain (non-MDR) will be examined for changes in their antibiotic resistance profile.   Antimicrobial susceptibility was determined using the Sensititre automated antimicrobial susceptibility system according to the manufacturer’s directions (Trek Diagnostic Systems, Westlake, OH).  Broth microdilution was used according to methods described by the National Committee for Clinical Laboratory Standards.  The National Antibiotic Resistance Monitoring System (NARMS) isolate panel was used to determine minimum inhibitory concentrations for the following antimicrobials: NARMS panel (cefoxitin, amikacin, chloramphenicol, tetracycline, ceftriaxone, amoxicillin/clavulanic acid, ciprofloxacin, gentamicin, nalidixic acid, ceftiofur, sulfisoxazole, trimethoprim/sulphamethoxazole, kanamycin, ampicillin and streptomycin).  Resistance breakpoints were determined using the NCCLS interpretive standards or the NARMS 2000 Annual Report.  Escherichia coli ATCC 25922, E. coli ATCC 35218, and Enterococcus faecalis ATCC 29212 were used as quality control organisms.     

In the second series of experiments, we utilized a tet-resistant S. Reading isolate in co-culture with a S. Enteritidis MDR in the presence of the most effective stressor(s) from the previous study.   

Findings 

The populations of S. Kentucky remained fairly constant around 10⁷ CFU/ml while S. Typhimurium populations remained constant around 10⁶ CFU/ml. We expected the populations of Kentucky to drop rapidly but rebound as they became resistant to Novobiocin and Nalidixic acid.  Indeed, we saw approximately a 10- to 100-fold drop (to 10⁴ to 10⁵ CFU/ml in S. Kentucky populations following introduction of Nov/Nal into the chemostats, however Nov/Nal resistant Kentucky isolates were never found.  S. Typhimurium populations however, did not change (increase) significantly following the addition of Nov/Nal which would be expected given the steady-state relationship between the two serotypes of Salmonella, and the populations returned to their previous levels relative to one another. The introduction of various metabolic and environmental stresses was expected to increase the rate at which the Salmonella Kentucky isolates became resistant to other antibiotics due to transfer from the S. Typhimurium MDR strain grown in co-culture. However, in this study that did not occur.     

It is theorized that the lack of transfer of antibiotic resistance seen in this study is an artifact of the fact that bacteria mate better given a solid surface than in liquid broth. A logical explanation for this is simply that the solid surface studies hold bacteria in close physical proximity for the period of time needed to transfer a complete plasmid via conjugation.  

We expected there to be a significant amount of transfer in the continuous flow culture system.  We are currently repeating it with two other Salmonella species, but to this date, again there have been no resistant isolates detected.  In order to shed further light on the issue of stress (that has not been satisfactorily examined in this study) we will examine the effects of these stressors on the transmission of antibiotic resistance genes in more traditional filter-mating experiments.  

Implications   

We found in our studies using a continuous-flow culture system that mimics the flow through the gastrointestinal tract of cattle in a controllable model system.  On a solid agar plate, Salmonella Typhimurium transferred gentamycin (an antibiotic) resistance genes to Salmonella Kentucky, but in continuous flow culture, the transfer of antibiotic resistance genes between a happened at a much lower, nearly undetectable, rate.  Furthermore, the severe stresses placed on the Salmonella species in continuous culture did not increase this frequency significantly.  This difference is likely due simply to motion in the continuous flow system not allowing the bacteria to physically mate long enough to facilitate genetic transfer.  Further research is needed and being performed to determine if these stresses can affect the transfer of antibiotic resistance genes in solid-surface conditions.