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Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature.

Singh AK, Drolia R, Bai X, Bhunia AK - PLoS ONE (2015)

Bottom Line: Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns.Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered.The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

View Article: PubMed Central - PubMed

Affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125-250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25-5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25-50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

No MeSH data available.


Related in: MedlinePlus

Minimum inhibitory concentration (MIC) of streptomycin against top 20 Salmonella enterica serovars.(A) Effect of streptomycin at 100 μg/mL (Strep100) on top 20 human-origin S. enterica serovars arranged in the order of outbreak incidence as per the National Salmonella Surveillance Annual Report, 2009. (B) Analysis of MIC of the streptomycin-sensitive (<100 μg/mL) S. enterica serovars. Absorbance of blank (LB broth) was 0.109 ± 0.052. (C) MIC of streptomycin-resistant S. enterica serovars Typhimurium (13ENT1277, 13ENT1140, 13ENT1288, and 13ENT0899). Broken straight line indicate threshold absorbance value (0.2), and an absorbance > 0.2 indicated growth of cultures in the wells of micro-titer plate.
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pone.0135035.g002: Minimum inhibitory concentration (MIC) of streptomycin against top 20 Salmonella enterica serovars.(A) Effect of streptomycin at 100 μg/mL (Strep100) on top 20 human-origin S. enterica serovars arranged in the order of outbreak incidence as per the National Salmonella Surveillance Annual Report, 2009. (B) Analysis of MIC of the streptomycin-sensitive (<100 μg/mL) S. enterica serovars. Absorbance of blank (LB broth) was 0.109 ± 0.052. (C) MIC of streptomycin-resistant S. enterica serovars Typhimurium (13ENT1277, 13ENT1140, 13ENT1288, and 13ENT0899). Broken straight line indicate threshold absorbance value (0.2), and an absorbance > 0.2 indicated growth of cultures in the wells of micro-titer plate.

Mentions: Initially, the top 20 human-origin S. enterica serovars were tested against the streptomycin at a concentration of 100 μg/mL, and all but four serovars; S. Enteritidis PT21, Muenchen 12ENT1182, Mississippi E345, and Schwarzengrund 13ENT82 were found resistant (Fig 2A). The minimum inhibitory concentration (MIC) was analyzed by using micro-titer plate broth dilution method after taking spectrophotometric absorbance measurements at 595 nm [37]. The MIC of Enteritidis PT21 and Mississippi E345, was 12.5 and 25 μg/mL, respectively; while MIC for both Muenchen 12ENT1182, and Schwarzengrund 13ENT82 was 50 μg/mL (Fig 2B). None of the S. Typhimurium serovars from our collection was sensitive to this concentration. Select four strains of S. Typhimurium strains (13ENT1277, 13ENT1140, 13ENT1288, 13ENT0899) were further examined for their response to a streptomycin concentration of higher than 100 μg/ml and the MIC for strains 13ENT1277, 13ENT1140, and 13ENT1288 was 250 μg/mL, and for the strain 13ENT0899, it was 125 μg/mL (Fig 2C). These MIC values of streptomycin coincides with the earlier MIC values (48–256 μg/mL) reported for 51 S. Typhimurium isolates of nosocomial origin with multiple-drug resistance traits [34]. Based on these results, in all future experiments, we used sub-inhibitory concentrations of streptomycin at 1.25 μg/mL to 5 μg/mL for streptomycin-sensitive serovars; and 25 μg/mL and 50 μg/mL for streptomycin-resistant serovars to visualize their effect on the optical scattering properties of the colonies. A concentration of 100 μg/mL of streptomycin was not selected for streptomycin-resistant serovar, since it affected the S. Typhimurium growth taking more than 24 h to reach the desired colony size (~1 mm) for BARDOT analysis.


Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature.

Singh AK, Drolia R, Bai X, Bhunia AK - PLoS ONE (2015)

Minimum inhibitory concentration (MIC) of streptomycin against top 20 Salmonella enterica serovars.(A) Effect of streptomycin at 100 μg/mL (Strep100) on top 20 human-origin S. enterica serovars arranged in the order of outbreak incidence as per the National Salmonella Surveillance Annual Report, 2009. (B) Analysis of MIC of the streptomycin-sensitive (<100 μg/mL) S. enterica serovars. Absorbance of blank (LB broth) was 0.109 ± 0.052. (C) MIC of streptomycin-resistant S. enterica serovars Typhimurium (13ENT1277, 13ENT1140, 13ENT1288, and 13ENT0899). Broken straight line indicate threshold absorbance value (0.2), and an absorbance > 0.2 indicated growth of cultures in the wells of micro-titer plate.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4529181&req=5

pone.0135035.g002: Minimum inhibitory concentration (MIC) of streptomycin against top 20 Salmonella enterica serovars.(A) Effect of streptomycin at 100 μg/mL (Strep100) on top 20 human-origin S. enterica serovars arranged in the order of outbreak incidence as per the National Salmonella Surveillance Annual Report, 2009. (B) Analysis of MIC of the streptomycin-sensitive (<100 μg/mL) S. enterica serovars. Absorbance of blank (LB broth) was 0.109 ± 0.052. (C) MIC of streptomycin-resistant S. enterica serovars Typhimurium (13ENT1277, 13ENT1140, 13ENT1288, and 13ENT0899). Broken straight line indicate threshold absorbance value (0.2), and an absorbance > 0.2 indicated growth of cultures in the wells of micro-titer plate.
Mentions: Initially, the top 20 human-origin S. enterica serovars were tested against the streptomycin at a concentration of 100 μg/mL, and all but four serovars; S. Enteritidis PT21, Muenchen 12ENT1182, Mississippi E345, and Schwarzengrund 13ENT82 were found resistant (Fig 2A). The minimum inhibitory concentration (MIC) was analyzed by using micro-titer plate broth dilution method after taking spectrophotometric absorbance measurements at 595 nm [37]. The MIC of Enteritidis PT21 and Mississippi E345, was 12.5 and 25 μg/mL, respectively; while MIC for both Muenchen 12ENT1182, and Schwarzengrund 13ENT82 was 50 μg/mL (Fig 2B). None of the S. Typhimurium serovars from our collection was sensitive to this concentration. Select four strains of S. Typhimurium strains (13ENT1277, 13ENT1140, 13ENT1288, 13ENT0899) were further examined for their response to a streptomycin concentration of higher than 100 μg/ml and the MIC for strains 13ENT1277, 13ENT1140, and 13ENT1288 was 250 μg/mL, and for the strain 13ENT0899, it was 125 μg/mL (Fig 2C). These MIC values of streptomycin coincides with the earlier MIC values (48–256 μg/mL) reported for 51 S. Typhimurium isolates of nosocomial origin with multiple-drug resistance traits [34]. Based on these results, in all future experiments, we used sub-inhibitory concentrations of streptomycin at 1.25 μg/mL to 5 μg/mL for streptomycin-sensitive serovars; and 25 μg/mL and 50 μg/mL for streptomycin-resistant serovars to visualize their effect on the optical scattering properties of the colonies. A concentration of 100 μg/mL of streptomycin was not selected for streptomycin-resistant serovar, since it affected the S. Typhimurium growth taking more than 24 h to reach the desired colony size (~1 mm) for BARDOT analysis.

Bottom Line: Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns.Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered.The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

View Article: PubMed Central - PubMed

Affiliation: Molecular Food Microbiology Laboratory, Department of Food Science, Purdue University, West Lafayette, Indiana, United States of America.

ABSTRACT
We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125-250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25-5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25-50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.

No MeSH data available.


Related in: MedlinePlus