Rapid Microbiological Testing: Monitoring the Development of Bacterial Stress

The ability to respond to adverse environments effectively along with the ability to reproduce are sine qua non conditions for all sustainable cellular forms of life. Given the availability of an appropriate sensing modality, the ubiquity and immediacy of the stress response could form the basis for a new approach for rapid biological testing. We have found that measuring the dielectric permittivity of a cellular suspension, an easily measurable electronic property, is an effective way to monitor the response of bacterial cells to adverse conditions continuously. The dielectric permittivity of susceptible and resistant strains of Escherichia coli and Staphylococcus aureus, treated with gentamicin and vancomycin, were measured directly using differential impedance sensing methods and expressed as the Normalized Impedance Response (NIR). These same strains were also heat-shocked and chemically stressed with Triton X-100 or H2O2. The NIR profiles obtained for antibiotic-treated susceptible organisms showed a strong and continuous decrease in value. In addition, the intensity of the NIR value decrease for susceptible cells varied in proportion to the amount of antibiotic added. Qualitatively similar profiles were found for the chemically treated and heat-shocked bacteria. In contrast, antibiotic-resistant cells showed no change in the NIR values in the presence of the drug to which it is resistant. The data presented here show that changes in the dielectric permittivity of a cell suspension are directly correlated with the development of a stress response as well as bacterial recovery from stressful conditions. The availability of a practical sensing modality capable of monitoring changes in the dielectric properties of stressed cells could have wide applications in areas ranging from the detection of bacterial infections in clinical specimens to antibiotic susceptibility testing and drug discovery

A Glutamine Transport Gene, glnQ, Is Required for Fibronectin Adherence and Virulence of Group B Streptococci

Group B streptococci (GBS) are a leading cause of neonatal sepsis and meningitis. GBS adhere to fibronectin when it is attached to a solid phase. We isolated a Tn917 transposon mutant, COH1-GT1, which shows decreased adherence to fibronectin. COH1-GT1 also shows decreased adherence to and invasion of respiratory epithelial cells in vitro and decreased virulence in vivo. COH1-GT1 contains a Tn917 insertion in a homolog of glnQ, a gene from Escherichia coli which is required for glutamine transport and codes for a cytoplasmic ATP-binding cassette protein. To confirm that the decreased fibronectin adherence of COH1-GT1 was due to the mutation in glnQ, we constructed COH1-GT2, a strain with a nonpolar site-directed mutation in glnQ. COH1-GT2 showed decreased binding to fibronectin. We also demonstrated that complementation of glnQ in trans restored fibronectin adherence to COH1-GT1. COH1-GT1 shows decreased uptake of radiolabeled glutamine and is resistant to the toxic glutamine analog γ-l-glutamylhydrazide, demonstrating that the glnQ gene is required for glutamine transport in GBS. glnQ lacks a signal sequence and is a cytoplasmic protein in E. coli and thus is unlikely to act as a fibronectin adhesin. glnQ is transcribed in an operon with a putative glutamine permease gene, glnP, which has a novel predicted structure containing three distinct domains linked in a single gene. The first two domains are putative glutamine binding domains with homology to the E. coli periplasmic glutamine binding gene glnH. The third is a putative permease domain with homology to the E. coli glutamine permease gene glnP. RT-PCR analysis demonstrated that glnP and glnQ are contained within a single transcript. Transcription of scpB, encoding the only known fibronectin-binding adhesin of GBS, is unaffected. We speculate that glnQ may regulate expression of fibronectin adhesins by affecting cytoplasmic glutamine levels and that regulation may be posttranscriptional

Screening of symbiotic frankiae for host specificity by restriction fragment length polymorphism analysis.

Restriction fragment length polymorphism analysis of numerous Frankia strains, using a nifDH probe, separated the strains into three distinct groups based on hybridization patterns. The groups identified in this study were well correlated with host specificity groups identified in earlier cross-inoculation studies