An optotracer-based antibiotic susceptibility test targeting by biofilm

Lucia Dong^*

Department of Chemical Engineering, University of Nanjing Agricultural, China

Abstract

A medical problem with worldwide implications is antimicrobial resistance. To prevent inefficient medication usage and the emergence of resistance, appropriate antimicrobial susceptibility testing is essential for drug development, patient diagnosis, and therapy [1]. Although bacterial biofilms play a significant role in persistent infections and illnesses brought on by medical devices, planktonic cultures are used to assess the effectiveness of medicines [2]. Here, we offer an optotracing-based biofilm AST using Salmonella as a model to address the demand for antibiotics that target bacteria living in biofilms [3]. The extracellular matrix components may be recorded in real-time using our non-disruptive approach, which allows for the precise identification of the biofilm lifestyle [4]. Thus, it is possible to confirm biofilm development before an antibiotic challenge and get pre-treatment information [5]. We performed a wide screen of the effects of antibiotics from various classes by introducing Kirby-Bauer discs [6]. Substances that have both stimulating and inhibiting effects on the ECM. Further research on these substances was conducted using agar-based dose-response biofilm-AST tests [7]. We obtained fresh data that directly reflected the treated biofilm by measuring the ECM based on the number of Curli and viewing the size and shape of the biofilm [8]. This demonstrated the effectiveness of numerous antibiotics in dismantling biofilms that had already formed, and it revealed fascinating potential resistance mechanisms that may have developed in response to therapies [9].

Keywords

Ast Curli • Ebbabiolight 680 • Optotracing • Real-Time

Introduction

Expanding the usage of the biofilm-AST will help treat infections more successfully and prevent the emergence of resistance by revealing more information about the susceptibilities and resistances of microorganisms. Specifically as a source of persistent infections and infections brought on by medical devices, biofilm infections pose a serious concern in medicine [10]. Aggregates that join with or cling to the host's natural or man-made surfaces [11]. A organised group of cells encircled by a self-made polymer matrix represents a biofilm. The biofilm mode of development renders bacteria more resilient to the host's innate and adaptive defence systems, as well as the effects of antibiotics and disinfectants, when compared to planktonic growth. This makes treating biofilm infections difficult [12]. The currently available antibiotics were created based on how they affected bacteria that were growing planktonic ally, not in the biofilm phase [13]. In a similar vein, planktonic developing bacteria are used for antibiotic susceptibility testing in clinical microbiology laboratories all over the world [14]. This suggests that the current ASTs' recommended antibiotic dose and therapy are ineffective against biofilm infections, placing the patient at risk of unsuccessful treatment [15]. While improper antibiotic usage can have negative effects on the person who is afflicted, it also affects the entire world as a cause of antimicrobial resistance. A number of causes, including the creation of a new set of determinants and the overuse of antibiotics in both clinical and agricultural contexts, contribute to the spread of AMR. Common infectious infections will probably cause protracted sickness, disability, and death if antibiotics lose their efficacy. There is an urgent need for innovative techniques that expand our understanding of biofilm physiology, composition, and structure since current forecasts indicate that resistance will result in 300 million premature deaths by the year. In order to produce adjuvant antibiotics and antibiotics specific to biofilms, as well as to diagnose and treat biofilm infections, this information is desperately needed. That is there is a debate in the industry on the introduction of standard techniques for microbial biofilm ASTs into clinical practise. The availability of acceptable bioassays is a key factor in the realisation of such approaches. Azeredo and colleagues have impressively examined the wide range of methods accessible for biofilm investigations. However, several techniques damage the biofilm structure or utilise colours that are harmful to microorganisms. While these techniques are effective for end-stage analysis, they are unable to identify the production of biofilms in real-time. To meet this demand, we have created a number of non-disruptive methods based on optotracer, which are fluorescent tracer molecules that are non-toxic and optically active and that, when added to nutrient broth or agar, report bacterially generated polysaccharides and peptides in real-time. By enabling real-time measurement and display of the optotracing approaches have produced greater understandings of the creation and composition of Salmonella, including bacterial proliferation and biofilm development, including the synthesis of the extracellular matrix. By examining their potential to create an AST specifically for the biofilm lifestyle, we here expand on our use of optotracer for biofilm investigation. A role for the optotracer EbbaBiolight 680 in analysing anti-biofilm activity based on the ECM rather than cellular viability and decrease of total biomass is suggested by the specific detection of Salmonella's ECM components.

Discussion

We here discuss the creation of an optotracerbased biofilm-AST to meet the requirement to produce data that is more relevant than planktonic-based assays, hence enhancing the prediction of treatment effectiveness of antibiotics for biofilm infections. The semi-high throughput optotracing biofilm assay, which was just published, is the foundation for the biofilm- AST. Salt agar with tracer molecule EbbaBiolight Ebba Biotech AB was applied to each well of a sterile Costar tissue culture treatment well plate. The temperature of the plates was allowed to stabilise. The GFP-expressing S. Enteritis’s strain, which was obtained from an exponential phase culture, was placed on the plate’s mm distant from the centre of each well. Plates were incubated to permit the development of pretreatment biofilms. Automated fluorescence microscopy was used to confirm biofilm formation, and phase contrast microscopy was used to assess the biofilms' physical dimensions. We created agar-containing 6-well plates by inserting an upturned sterile pipette tip into each well to create a cylindrical chamber that was 8.5 mm in diameter. To verify that bacterial clumps have been properly disaggregated, Samples underwent optical microscope analysis. Spread plating on LB agar was used to calculate the CFU counts of the bacterial suspensions when only single cells were visible. The amount of Curli ECM, defined as RFU from bound to Curli, in biofilms treated with various concentrations of the antibiotics was examined in order to estimate the least dose-response inhibitory concentration of and polymyxin B. Cell viability following antibiotic treatment was evaluated by removing the whole biofilm from the agar surface using a sterile loop and resuspending it in broth in 24-well plates to establish the lowest dose-response eradication concentration. The biofilm was significantly dispersed by minor homogenization using repeated pipetting. After that, plates were incubated at and each bacterial suspension was pipetted. Onto a 96-well plate in quadruplicates, and OD405 was assessed using a Tecan Infinite microplate reader. Visual inspection of turbidity and study of OD values were used to establish if the culture had been eradicated. MD-REC is the concentration at which OD values significantly decrease as compared to the mock-treated biofilm. Using a Tecan Infinite M1000 PRO microplate reader, spectrophotometry was used to record the fluorescence of Ebba680 attached to curli. Ebba680 fluorescence was measured using an area scan after the pre-treatment biofilm had grown and after antibiotic treatment. In GraphPad Prism 8, signals from columns were combined to measure both the antibiotics' antibiofilm and pro-biofilm effects. In order to exclude the antibiotic-containing cavities and Kirby-Bauer discs, signals from columns were eliminated. We collected the complete pretreatment biofilm using sterile 10 L loops to determine the CFU in the biofilms that grew on the agar surface throughout the 24 h incubation (VWR, Sweden). Each collected biofilm was resuspended in 1.5 ml Eppendorf tubes containing 1 ml PBS. Suspensions were homogenised by being passed through many times in order to remove any apparent clumps and scatter the biofilm's cell population. There was strong vortexing after that. We desired a large panel of representative agents from several antimicrobial classes when choosing antibiotics to be examined for their potential effects on biofilm, regardless of the repertory already in clinical use. The antibacterial agents we evaluated the efficacies of all antibiotic formulations using conventional microbroth dilution tests to ensure that they all had antibacterial activity as anticipated. According we utilised Mueller- Hinton broth-grown ISO strain E. coli in accordance with EUCAST criteria. The MIC values for all antibiotic formulations were produced as predicted as a result. We used Salmonella Enteritidis, a wild type strain known to exhibit typical biofilm morphologies linked to its expression of the ECM components curli and cellulose when grown on Luria Bertani agar plates under hypoosmotic conditions, as there are no standard strains defined for susceptibility testing of bacterial biofilms. Since this microbe and its assay settings for generating biofilms are significantly different from those used in the present EUCAST-guided AST approach, we began by examining step-by-step how the changing growing circumstances affected the antibiotic efficacies on this strain. We conducted microbroth dilution tests with bacteria cultivated from S. Enteritidis to determine the baseline MIC for each antibiotic. In the red channel, fluorescence of Ebba680 bound to curli showed this ECM component to form distally projecting radial ridge patterns and channel-like structures typical to biofilms by this strain Since this verified that Salmonella had formed a biofilm, we applied this analysis in all following experiments to ensure that bacteria had adopted a biofilm lifestyle before we initiated antibiotic exposure. Prior to antibiotic exposure, we also collected data of the status of the pre-treatment biofilms we employed an isogenic strain of Salmonella with a GFP-expressing plasmid that causes cells to glow under a fluorescent microscope for the fluorescence-based analysis. We inoculated from a S. Enterica culture using the described procedure onto Ebba680- supplemented LB agar without salt cast in the wells of 6-well plates. We used an automated microscope to determine if biofilm had developed after incubation at h. The ordered biofilm with a central core and an intermediate zone was visible by fluorescence imaging of the macrocolony, and the unique fluorescence from GFP-expressing bacteria varied in intensity relative to the total biofilm. The fluorescent pictures' analysis revealed that the biofilms had reached a diameter. Relative fluorescence units were used to measure the quantity of ECM using spectrophotometric recordings of curli-bound Ebba680 fluorescence. We started antimicrobial treatment using readily available Kirby-Bauer discs containing AMP, CXI, polymyxin B, RIF, CIP, ERY, TET, and GEN after determining the pre-treatment parameters of the biofilm. To imitate antimicrobial therapy, antibiotic-free discs were utilised. One disc was positioned in each well, 1 cm from the edge of the pre-treatment biofilm and on the agar surface. The antibiotics were then allowed to permeate from the disc through the agar and exercise their effects on the developing biofilm while the plates were incubated. We positioned the well plate in the automated microscope at the conclusion of incubation to view the morphology and measure the size of the treated biofilm. Additionally, using fluorescence, we calculated how much curli ECM was present. The in curls. The mocktreated biofilm showed uninterrupted development as evidenced by GFP and Ebba680 fluorescence. The biofilm became larger, the intermediate area grew larger, and the curli-rich ridge patterns and channel-like ECM structures grew longer. The diameter of the biofilm was Curli-bound Ebba680 fluorescence measured at 31822 RFU corresponded to AMP-treated biofilms, which had morphologies and growth patterns that were comparable to those of the mock-treated biofilm. With a diameter of 15.4 mm and curli-bound Ebba680 fluorescence, it was evident that the AMP-treated biofilm's size and curli ECM amount were comparable. Because this strain has a plasmid encoding AMP resistance for the selection of GFP-expressing bacteria, the lack of inhibitory effects of AMP was not unexpected Biofilms emerged after Comparing the mock treated to the CXI treated, the morphology was noticeably different. This fluorescence imaging Since this microbe and its assay settings for generating biofilms are significantly different from those used in the present EUCAST-guided AST approach, we began by examining step-by-step how the changing growing circumstances affected the antibiotic efficacies on this strain. We conducted microbroth dilution tests with bacteria cultivated from S. Enteritidis 3934 to determine the baseline MIC for each antibiotic. Salmonella often grows in biofilms. Since Luria Bertani agar is frequently used to culture Salmonella, we next examined the effectiveness of antibiotics on S. Enteritidis 3934 that was being grown on LB agar. RIF provided an unexpected outcome, as the switch to LB agar restored the 2-fold rise for ERY and the 4-fold increase for GEN.

Conclusion

Resistance to this drug other antibiotics just displayed variations in the assay's repeatability range rather than finally, using LB agar without salt at 28 °C, we carried out the E-test in the most favourable Salmonella biofilm-inducing circumstances. For polymyxin B and CIP, there were modest increases in MICs compared to the LB agar; for AMP, TET, and GEN, there were slight decreases; and for CXI and ERY, there were no changes. Fascinatingly, a study shown that under biofilm-inducing circumstances, bacteria become distinctly susceptible to RIF. Comparing the sensitivity to RIF to the liquid AST, it was much higher. It is necessary to make sure that bacteria from a planktonic inoculum have adopted the biofilm lifestyle before being exposed to an antibiotic in order to research an antibiotic's effectiveness on biofilm bacteria. To progress we developed a process based on our newly published optotracer-based semi-high throughput biofilm assay, an AST especially built for the Salmonella biofilm lifestyle. In this test, when the biofilm develops on agar supplemented with the optotracer, the formation of Salmonella biofilms may be seen in real-time by fluorescence imaging and spectroscopy. Reduced GFP signal in the region facing the antibiotic disc in the EbbaBiolight biofilm may indicate decreased bacterial vitality at this location. The decrease of the curli-rich intermediate zone when compared to mock treated biofilm was confirmed visually by quantification of curli ECM by Ebba680. This shows that CXI prevents the formation of both biofilms and the ECM. However, the diameter increased in comparison to the pretreatment biofilm, indicating that there is a delay between beginning and completion. Treatment with CXI and the start of noticeable effects. This lag time most likely corresponds to the time it takes for CXI to diffuse through the agar. We utilised a binary analysis to determine if the biofilm still included any viable cells after treatment. We harvested the whole biofilm and used it to inoculate new; antibiotic-free after incubation, the turbidity was plainly evidence of a healthy bacterial culture. All of this demonstrates that CXI did not successfully remove the biofilm and does not have biofilm-tidal effects. Biofilm treated with polymyxin B did not exhibit any discernible size decrease. The amount of the ECM was the same in mock treated biofilms and was shown as a curli-rich intermediate area with distally extending ridges and channel-like features. However, careful analysis of the red fluorescence from showed that along the biofilm's perimeter towards the Polymyxin B disc, new continuous wall structures had developed. These curli-rich, barrier-like structures show that Polymyxin B may generate a resistance response by increasing the synthesis of curli ECM rather than inhibiting the growth of biofilms per se. A lack of biofilm tidal RIF treatment, as indicated by the posttreatment bacterial viability test, resulted in a reduced biofilm size compared to the mock treated, with the reduction being most pronounced in the intermediate area facing the RIF disc. Although there were ridges and channels with distal projections, the fluorescence of the biofilm as a whole revealed less curli. Spectrophotometric recordings showing the curli-bound Ebba680 signal were used to confirm this.

References

1. Mandell LA, Wunderink RG, Anzueto et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis. 44, S27-S72 (2007).

Indexed at, Crossref, Google Scholar

2. Ibrahim EH, Sherman G, Ward S, et al. The influence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest. 118, 146-155 (2000).

Indexed at, Crossref, Google Scholar

3. Kollef MH, Sherman G, Ward S et al. Inadequate antimicrobial treatment of infections: a risk factor for hospital mortality among critically ill patients. Chest. 115, 462-474 (1999).

Indexed at, Crossref, Google Scholar

4. Tunkel AR, Hartman BJ, Kaplan SL et al. Practice guidelines for the management of bacterial meningitis. Clin Infect Dis. 39, 1267-1284 (2004).

Indexed at, Crossref, Google Scholar

5. Thompson RL, Wright AJ General Principles of antimicrobial therapy. Mayo Clin Proc. 73, 995-1006 (1998).

Indexed at, Crossref, Google Scholar

6. Drusano GL Human pharmacodynamics of beta-lactams, aminoglycosides and their combination. Scand J Infect Dis Suppl. 74, 235-248 (1990).

Indexed at, Crossref, Google Scholar

7. Baddour LM, Wilson WR, Bayer AS et al. Infective endocarditis: diagnosis, antimicrobial therapy, and management of complications: a statement for healthcare professionals from the Committee on Rheumatic Fever, Endocarditis, and Kawasaki Disease, Council on Cardiovascular Disease in the Young, and the Councils on Clinical Cardiology, Stroke, and Cardiovascular Surgery and Anesthesia, American Heart Association. Circulation. 111, e394-e434 (2005). 

Indexed at, Crossref, Google Scholar

8. Levison ME Pharmacodynamics of antimicrobial drugs. Infect Dis Clin North Am. 18, 451-465 (2004).

Indexed at, Crossref, Google Scholar

9. Saag M, Balu R, Phillips E et al. High sensitivity of human leukocyte antigen-b*5701 as a marker for immunologically confirmed abacavir hypersensitivity in white and black patients. Clin Infect Dis. 46, 1111-1118 (2008).

Indexed at, Crossref, Google Scholar

10. Crider KS, Cleves MA, Reefhuis J et al. Antibacterial medication use during pregnancy and risk of birth defects: National Birth Defects Prevention Study. Arch Pediatr Adolesc Med. 163, 978-985 (2009).

Indexed at, Crossref, Google Scholar

11. Czock D, Rasche FM Dose adjustment of ciprofloxacin in renal failure: reduce the dose or prolong the administration interval? Eur J Med Res. 10, 145-148 (2005).

Indexed at, Crossref, Google Scholar

12. Wagenlehner FM, Naber KG Fluoroquinolone antimicrobial agents in the treatment of prostatitis and recurrent urinary tract infections in men. Curr Infect Dis Rep. 7: 9-16 (2005).

Indexed at, Crossref, Google Scholar

13. Silverman JA, Mortin LI, Vanpraagh AD et al. Inhibition of daptomycin by pulmonary surfactant: in vitro modeling and clinical impact. J Infect Dis. 191, 2149-2152 (2005)

Indexed at, Crossref, Google Scholar

14. Tice AD, Rehm SJ, Dalovisio JR et al. Practice guidelines for outpatient parenteral antimicrobial therapy. Clin Infect Dis. 38, 1651-1672 (2004).

Indexed at, Crossref, Google Scholar

15. Begg EJ, Barclay ML, Kirkpatrick CM et al. The therapeutic monitoring of antimicrobial agents. Br J Clin Pharmacol. 52, 35S-43S (2001).

Indexed at, Crossref, Google Scholar