Mass spectrometry profiling of single bacterial cells reveals metabolic regulation during antibiotics induced bacterial filamentation

Bacterial antimicrobial resistance(AMR)is a severe threat to global health and development.Under the stimulation of antibiotics,bacterial cells can undergo filamentation and generate daughter cells with stronger AMR.The current research on bacterial AMR mechanism is mainly conducted with a population of cells.However,bacterial cells exhibit heteroresistance,making the study at population level not reliable.Herein,we developed single bacterial cell metabolic profiling by mass spectrometry(MS)to study bacterial AMR at single-cell level.By utilizing a microprobe controlled by a microoperation platform,single filamentous extended spectrum beta-lactamase(ESBL)producing Escherichia coli(ESBL-E.coli)cells generated by ceftriaxone sodium stimulation can be extracted and spray-ionized for MS analysis.Heterogeneous among ESBL-E.coli cells under the same antibiotic stimulus condition was observed from mass spectra as well as cell morphology.The metabolic profiles by MS of different individual cells can be clustered into subgroups well in accordance with bacterial cell length.Metabolic pathways including arginine and proline metabolism,as well as cysteine and methionine metabolism were disclosed to play an important role in the bacterial SOS-associated filamentation against antibiotics.The microprobe electrospray ionization-MS-based single bacterial cell analysis method is promising in the study of various bacterial AMR mechanism and can reveal the heterogeneity of bacterial AMR from-cell-to-cell.

Molecular mechanism of the qnrA genemediated quionlone resistance in Gram-negative bacteria

To explore the prevalence of the plasmid-mediated quinolone resistance gene qnrA in Gramnegative bacteria and to investigate its molecular genetic background and resistance profile in isolates harboring this gene, a total of 629 nalidixic acid-resistant isolates of non-repetitive Gram-negative bacteria were collected from clinical specimens between April 2004 and April 2006 and these isolates were screened for qnrA gene by PCR using specific primers combined with DNA sequencing. The extended spectrum β-1actamase （ESBL） or AmpC-producing isolates were distinguished by the phenotypic confirmatory test combined with DNA sequencing, and the antibiotics susceptibility test for qnrA-positive isolates was carried out by Kirby-Bauer and E-test method. To detect the location of the qnrA gene, plasmid conjugation and Southern hybridization were performed and the integron structure containing the qnrA gene was cloned by PCR strategy and sequenced by primer walking. It was demonstrated that the incidence of the qnrA-positive strains in nalidixic acid-resistant bacteria was 1.9% （12/629）, in which the detection rates for Klebiesiella pneumoniae. Enterobacter cloacae, Enterobacter aerogenes, Citrobacterfreundii and Salmonella choeraesuis were 2.2% （3/138）, 17. 1% （6/35）, 9. 1% （1/11）, 12.5% （1/8）, and 14.3% （1/7）, respectively. The qnrA gene was found to be embedded in the complex sull-type integron located on plasmids with varied size （80-180 kb）. Among them, 4 qnrA-positive isolates carried integron In37 and 8 isolates carried a novel integron, temporarily desig- nated as InX. All the qnrA-positive isolates were ESBL-producing and transferable for the multi-drug resistance. It is concluded that the plasmid-mediated drug-resistance mechanism exists in the quinolone resistant strains of isolates from hospitals in Guangdong area, but the incidence was rather low. Nevertheless, it is still possible that the horizontal transfer of the resistant qnrA gene might lead to the spreading of drug-resistance.

Applications and challenges for single-bacteria analysis by flow cytometry

Flow cytometry(FCM)is a powerful technique for single-bacteria analysis via simultaneous light-scattering and fluorescence measurements.By offering high-throughput,quantitative,and multiparameter analysis at the single-cell level,FCM has gained an increased popularity in microbiological research,food safety monitoring,water quality control,and clinical diagnosis.Here we will review the recent applications of flow cytometry in areas such as(1)total bacterial cell count,(2)bacterial viability analysis,(3)specific bacterial detection and identification,(4)characterization of physiological changes under environmental perturbations,and(5)biological function studies.Nevertheless,despite these widespread applications,challenges still remain for the detection of small sizes of bacteria and biochemical features that cannot be brightly stained via fluorescence.Recent improvement in FCM instrumentation will be discussed,and particularly the development of high sensitivity flow cytometry for advanced analysis of single bacterial cells will be highlighted.

Bacterial communities in different locations, seasons and segments of a dairy wastewater treatment system consisting of six segments

A dairy wastewater treatment system composed of the 1st segment（no aeration） equipped with a facility for the destruction of milk fat particles, four successive aerobic treatment segments with activated sludge and a final sludge settlement segment was developed. The activated sludge is circulated through the six segments by settling sediments（activated sludge） in the 6th segment and sending the sediments beck to the 1st and 2nd segments.Microbiota was examined using samples from the non-aerated 1st and aerated 2nd segments obtained from two farms using the same system in summer or winter. Principal component analysis showed that the change in microbiota from the 1st to 2nd segments concomitant with effective wastewater treatment is affected by the concentrations of activated sludge and organic matter（biological oxygen demand [BOD]）, and dissolved oxygen（DO） content. Microbiota from five segments（1st and four successive aerobic segments） in one location was also examined. Although the activated sludge is circulating throughout all the segments, microbiota fluctuation was observed. The observed successive changes in microbiota reflected the changes in the concentrations of organic matter and other physicochemical conditions（such as DO）, suggesting that the microbiota is flexibly changeable depending on the environmental condition in the segments. The genera Dechloromonas, Zoogloea and Leptothrix are frequently observed in this wastewater treatment system throughout the analyses of microbiota in this study.