140_High-throughput determination of bacterial growth kinetics using a BMG LABTECH microplate reader

Mark A Webber, Laura JV Piddock, Division of Immunity and Infection, University of Birmingham, 05/2006
  • Growth rates of biocide resistant mutants of salmonella and their parent strains were determined and compared
  • Growth curves were monitored in absorbance mode automatically over 24 h 
  • Biocide exposure selects for strains with increased tolerance to biocides and there is no obvious fitness cost

Introduction

Salmonella enterica serovar Typhimurium are amongst the leading causes of gastrointestinal disease. Multiple antibiotic resistance (MAR) in salmonella has been increasing and over 30% of isolates of S. Typhimurium. Efflux is one mechanism that can confer MAR, efflux pumps are membrane proteins that actively export a wide range of toxic substrates including antibiotics, dyes, and biocides to the cells’ external environment, thereby preventing accumulation of toxic agents and mediating resistance to these agents as a result.

The wide spectrum of substrates recognized by efflux systems has prompted concern that exposure of a bacterium to one substrate could select for over-expression of an efflux system and consequent resistance to all other substrates. It is possible that exposure of a bacterium to a biocide could result in selection of an efflux mutant which has reduced susceptibility to antibiotics, which it has not previously encountered. In this study, mutants of salmonella resistant to biocides were selected. The growth rates of biocide resistant mutants were determined and compared to parent strains and the effect of the addition of biocides during growth was determined.

Materials & Methods

Selection of biocide resistant mutants
Previously, a panel of Salmonella Typhimurium was used to select biocide resistant mutants. SL1344 was used as a reference control strain, L108 is a derivative of SL1344 lacking the TolC porin and L358 is a multiple antibiotic resistant DT104 isolate. L378 is a cipro-floxacin resistant isolate from poultry and L357 is a representative DT104 isolate. The biocides AQAS (a quaternary ammonium compound), Superkill (a mix of aldehydes), Farm Fluid S (a mixture of tar oils and phenolics), and Virkon S (peroxygen generating compound) were used to select for tolerant mutants by incorporation in agar and overnight incubation after inoculation with each strain.

Analysis of growth kinetics of biocide tolerant mutants
The rate of growth was determined over 24 h at 37°C using a BMG LABTECH microplate reader. 100 μL of sterile LB broth was dispensed into clear sterile 96-well microplates and inoculated with overnight culture of each strain to give a final inoculum of 4%. Readings were taken every ten minutes of absorbance of each well (scanned at 600 nm) in the microplates over the 24 h time period. Each strain was analysed in triplicate wells on at least three separate occasions to give nine data sets for analysis. Additionally, each strain was challenged with either 0.5 X, or the MIC (minimum inhibitory concentration) of the selective biocide for the parent after two hours growth in order to determine whether the inhibitory ability of each biocide was reduced in mutants, respective to parent strains.

Results & Discussion

None of the biocide selected mutants were compromised in their ability to grow. Mutants derived from L358 (S2, S22, S23) after exposure to Superkill grew significantly better than L358 in the absence of biocide (Figure 1).
 

Fig. 1: Growth of Superkill selected mutants S2, S22, S23 and parent L358 at 37°C after inoculation with 4% vol/vol of overnight culture. Superkill was added to cultures at 2 h.

The majority of biocide selected mutants were more resistant to the addition of biocides to the media than their respective parent strains, including those strains for which the MIC of the selective biocide had remained unchanged when compared to the parent (table 1). AQAS selected mutant A27 and A26 were able to grow significantly (p >0.01) better upon both the addition of 0.5 X and the MIC of L378 and L108, respectively, to the media (figures 2 and 3).

 

Fig. 2: Growth of AQAS selected mutant A27 and parent L378 at 37°C after inoculation with 4% vol/vol of overnight culture. AQAS was added to cultures at 2 h.

Fig. 3: Growth of AQAS selected mutant A26 and parent L108 at 37°C after inoculation with 4% vol/vol of overnight culture. AQAS was added to cultures at 2 h.

Virkon selected mutants V6 and V7 both grew significantly (p > 0.01) better than L354 when challenged with 0.5 X or the MIC of Virkon for L354 (figure 4).

Fig. 4: Growth of Virkon selected mutants V6, V7 and parent L354 at 37°C after inoculation with 4% vol/vol of overnight culture. Virkon was added to cultures at 2 h.No significant differences were observed between Farm Fluid S selected mutants F1 and F2 and their parent L357 in biocide free broth or when exposed to 0.5 X the MIC of Farm Fluid S. However both mutants grew significantly (p > 0.05) better when challenged with the MIC of Farm Fluid S (0.1% and 0.2% final concentration) as shown in figure 5.

Fig. 5: Growth of Farm Fluid selected mutants F1, F2 and parent L357 at 37°C after inoculation with 4% vol/vol of overnight culture. Farm Fluid S was added to cultures at 2 h.Table 1: Parent strains are in bold and mutants are listed below their respective parents. Mutants with increased resistance to biocide compared to their parents are indicated in red.

 

    MIC (%)
Strain and Genotype Selective agent

Virkon

Superkill AQAS FFS
L357 (DT104 ‘A’)   0.4 0.025 0.1 0.2
F1 FFS (1X MIC)       0.2
F2 FFS (1X MIC)       0.2
L378 (CipR VLA52)   0.4 0.025 1.6 0.2
A27       >3.2  
L108 (tolC::aph from SL1344)   0.4 0.006 <0.003 0.025
A26 AQAS (2X MIC)     0.12  
L358 (MDR DT104)   0.4 0.025 1.6 0.2
S2 S’kill (1X MIC)   0.025    
S22 S’kill (2X MIC)   0.025    
S23 S’kill (2X MIC)   0.025    
SL1344   0.4 0.025 0.1 0.2
V6 V’kon (2X MIC) 0.8      
V7 V’kon (2X MIC) 0.8      

 

Conclusion

This data clearly indicates that biocide exposure selects for strains with increased tolerance to biocides at sub-MIC concentrations and at the MIC and that there is no obvious fitness cost in these strains when compared to their parents in biocide free broth. 

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