Fourier-transform infrared (FTIR) spectroscopy can detect biomolecular changes in bacterial cells in response to drugs or other stimuli. Fully developing this area requires an understanding of IR spectral changes associated with the growth of unperturbed cells. Such an understanding is still lacking, however. To address this issue, attenuated total reflectance (ATR) FTIR spectroscopy has been used to probe changes in the composition of Staphylococcus aureus ATCC 6538 cells during exponential growth, with a 30 min time resolution. We find prominent spectral changes in proteins, nucleic acids, and carbohydrates evolving from the early (30–120 min) to the late (240–360 min) log phase of growth. Principal component analysis (PCA) shows that spectra obtained for cells during the early and late log phases of growth can be discriminated against with 100% accuracy. Protein-related spectral features are most significant in spectra collected at 30- and 90-min post-inoculation and provide a robust basis for temporal differentiation. Spectral changes that occur during the first 30 min after inoculation are shown to reverse over the next 30–120 min, indicating dynamic adaptations during cellular growth. Overall, we demonstrate a band assignment strategy based on time resolution, underscoring the utility of FTIR spectroscopy in dynamic studies of bacterial cells.

Aims: Anti-virulence compounds can control pathogens with reduced selection for antimicrobial resistance. There is little understanding of how these compounds impact biofilm structure and development through time. We hypothesised that 4-ethoxybenzoic acid (4EB), an anti-virulence compound, disrupts normal growth for the four phases of Staphylococcus aureus ATCC 6538 biofilm development (attachment, multiplication, exodus, and maturation). Methods and Results: Flow-cell grown biofilms were fed Luria Bertani (LB) broth (control) or LB broth with 0.8 mg/mL 4EB (treatment). Treatment inhibited the progress of multiplication phase and caused a 6-hour delay in the onset of exodus phase. Transcriptional analysis showed patterns of nuc, saeS, and saeR expression consistent with the delayed exodus phenotype. Imaging by confocal laser scanning microscopy followed by digital image analysis determined that 4EB interfered with biofilm structure formation, including reductions in height (57%/44%) and biovolume (73%/63%) during the multiplication and maturation phases, respectively, with statistically insignificant effects during exodus phase (1.9%/15%). These measurements indicated that the occurrence of exodus phase was not impacted by 4EB. Gene expression analysis using flow cell effluent found significant downregulation of genes including atl (−3.1 fold change) during multiplication phase and agrA and saeR (−5.8 and −5.2 fold change, respectively) during maturation. Principal component analysis with 24 measured parameters confirmed that 4EB treatment primarily affected multiplication and maturation phases.

Fourier transform infrared (FTIR) spectroscopy can detect biomolecular changes in bacterial cells in response to drugs or other stimuli. To fully develop that area requires an understanding of IR spectral changes associated with the growth of unperturbed cells. Such an understanding is still lacking, however. To address this issue, attenuated total reflectance (ATR) FTIR spectroscopy has been used to probe changes in the composition of Staphylococcus aureus ATCC 6538 cells during exponential growth, with 30-minute time resolution. We find prominent spectral changes associated with proteins, nucleic acids and carbohydrates on evolving from the early (30-120 min) to the late (240-360 min) log phase of growth. Principal component analysis (PCA) shows that spectra obtained for cells during the early and late log phases of growth can be discriminated against with 100% accuracy. Protein-related spectral features are most significant in spectra collected at 30- and 90-minutes post-inoculation and provide a robust basis for temporal differentiation. Spectral changes that occur during the first 30 minutes after inoculation are shown to reverse over the next 30-120 minutes, indicating dynamic adaptations during cellular growth. Overall, we demonstrate a band assignment strategy based on time resolution, underscoring the utility of FTIR spectroscopy in dynamic studies of bacterial cells.

Aims: Rapid urbanization of the Chattahoochee River has decreased the water quality with higher levels of anthropogenic bacteria from nonpoint source pollution. Introduced bacteria are variable across urban watersheds but only monitored by the abundance of fecal indicator bacteria. Staphylococcus aureus is a halotolerant, opportunistic pathogen associated with aquatic pollution, but impact on the microbial ecology is not well understood in freshwater systems. Describing the halotolerant subset of the aquatic microbiome, can expand upon the health risk of bacterial pollution. Methods and results: Surface water samples along the Chattahoochee River were collected and halotolerant bacteria were cultured using selective agar, typical for growing Staphylococcus species. Bacteria colonies were isolated then characterized by morphology, biochemical tests, and antibiotic resistance screening. Antibiotic resistance profiles showed isolates with a high percentage of resistance to penicillin (86.2%) and novobiocin (17.2%). Biofilm formation was common with 41.4% of isolates formed moderate-to-strong biofilms. 16S rRNA gene sequencing identified isolates to not be Staphylococcus spp. but within two predominant groups, Bacillus spp. and Priestia spp. Conclusions: The Priestia genus is poorly known in freshwater systems; though the combination of antibiotic resistance, biofilm formation, and spore-forming traits indicate that it has key survival characteristics. Halotolerant bacteria harbor opportunistic human pathogens and a public health risk because the bacteria have a high prevalence of antibiotic resistance and biofilm capability, which contribute to environmental persistence and reservoirs for antibiotic resistance genes. This environmental resistome is a notable and developing characteristic of the urban aquatic microbiome.