Biofilm Formation and Antimicrobial Susceptibility of E. coli Associated With Colibacillosis Outbreaks in Broiler Chickens From Saskatchewan.

The global poultry industry has grown to the extent that the number of chickens now well exceeds the number of humans on Earth. Escherichia coli infections in poultry cause significant morbidity and economic losses for producers each year. We obtained 94 E. coli isolates from 12 colibacillosis outbreaks on Saskatchewan farms and screened them for antimicrobial resistance and biofilm formation. Fifty-six isolates were from broilers with confirmed colibacillosis, and 38 isolates were from healthy broilers in the same flocks (cecal E. coli). Resistance to penicillins, tetracyclines, and aminoglycosides was common in isolates from all 12 outbreaks, while cephalosporin resistance varied by outbreak. Most E. coli were able to form biofilms in at least one of three growth media (1/2 TSB, M63, and BHI broth). There was an overall trend that disease-causing E. coli had more antibiotic resistance and were more likely to form biofilms in nutrient-rich media (BHI) as compared to cecal strains. However, on an individual strain basis, there was no correlation between antimicrobial resistance and biofilm formation. The 21 strongest biofilm forming strains consisted of both disease-causing and cecal isolates that were either drug resistant or susceptible. Draft whole genome sequencing indicated that many known antimicrobial resistance genes were present on plasmids, with disease-causing E. coli having more plasmids on average than their cecal counterparts. We tested four common disinfectants for their ability to kill 12 of the best biofilm forming strains. All disinfectants killed single cells effectively, but biofilm cells were more resistant, although the difference was less pronounced for the disinfectants that have multiple modes of action. Our results indicate that there is significant diversity and complexity in E. coli poultry isolates, with different lifestyle pressures affecting disease-causing and cecal isolates.

Purification of the Bacterial Amyloid "Curli" from Salmonella enterica Serovar Typhimurium and Detection of Curli from Infected Host Tissues.

Microbiologists are learning to appreciate the importance of "functional amyloids" that are produced by numerous bacterial species and have impacts beyond the microbial world. These structures are used by bacteria to link together, presumably to increase survival, protect against harsh conditions, and perhaps to influence cell-cell communication. Bacterial functional amyloids are also beginning to be appreciated in the context of host-pathogen interactions, where there is evidence that they can trigger the innate immune system and are recognized as non-self-molecular patterns. The characteristic three-dimensional fold of amyloids renders them similar across the bacterial kingdom and into the eukaryotic world, where amyloid proteins can be undesirable and have pathological consequences. The bacterial protein curli, produced by pathogenic Salmonella enterica and Escherichia coli strains, was one of the first functional amyloids discovered. Curli have since been well characterized in terms of function, and we are just starting to scratch the surface about their potential impact on eukaryotic hosts. In this manuscript, we present step-by-step protocols with pictures showing how to purify these bacterial surface structures. We have described the purification process from S. enterica, acknowledging that the same method can be applied to E. coli. In addition, we describe methods for detection of curli within animal tissues (i.e., GI tract) and discuss purifying curli intermediates in a S. enterica msbB mutant strain as they are more cytotoxic than mature curli fibrils. Some of these methods were first described elsewhere, but we wanted to assemble them together in more detail to make it easier for researchers who want to purify curli for use in biological experiments. Our aim is to provide methods that are useful for specialists and non-specialists as bacterial amyloids become of increasing importance.

Synergistic activity of tetrasodium EDTA, ethanol and chlorhexidine hydrochloride against planktonic and biofilm cells of clinically relevant pathogens.

OBJECTIVES: Biofilms associated with implantable medical devices and wounds are clinically relevant, often requiring repeated use of antibiotics without success. A search for non-antibiotic antimicrobial and antibiofilm solutions is warranted, in line with antimicrobial stewardship. Our study aimed to evaluate the broad-spectrum antimicrobial efficacy of tetrasodium EDTA, ethanol and chlorhexidine hydrochloride (HCl) alone and in combination against clinically relevant planktonic and biofilm cells of bacterial and fungal pathogens. METHODS: MICs and MBCs were determined for tetrasodium EDTA, ethanol and chlorhexidine HCl against planktonic cells of test pathogens. The MBEC Assay® biofilm inoculator device was used to evaluate the biofilm eradication ability of test antimicrobials alone and in combination against clinically relevant pathogens. The checkerboard microbroth dilution assay was performed to analyze the synergism between test antimicrobials. RESULTS: Against planktonic cells, the combination of tetrasodium EDTA with ethanol or chlorhexidine HCl resulted in synergistic to indifferent activity, with no antagonism observed. Against mature biofilms, all combinations were synergistic. The MBEC of each test antimicrobial was decreased from 4- to -64-fold when used in combination as compared to when agents were used alone. We optimised the concentration of antimicrobials to achieve rapid eradication of pre-formed biofilms. A triple combination of 3% tetrasodium EDTA, 20% ethanol and 2.5 µg/mL chlorhexidine HCl completely eradicated 48-h-old biofilms of all test strains within 2 h. CONCLUSION: All three antimicrobial agents can be used together for prevention and treatment of biofilms and biofilm-related infections. The observed in vitro efficacy should be tested further through in vivo and clinical studies.

Deciphering the Antibacterial Mode of Action of Alpha-Mangostin on Staphylococcus epidermidis RP62A Through an Integrated Transcriptomic and Proteomic Approach.

Background: Alpha-mangostin (α-MG) is a natural xanthone reported to exhibit rapid bactericidal activity against Gram-positive bacteria, and may therefore have potential clinical application in healthcare sectors. This study sought to identify the impact of α-MG on Staphylococcus epidermidis RP62A through integrated advanced omic technologies. Methods: S. epidermidis was challenged with sub-MIC (0.875 µg/ml) of α-MG at various time points and the differential expression pattern of genes/proteins were analyzed in the absence and presence of α-MG using RNA sequencing and LC-MS/MS experiments. Bioinformatic tools were used to categorize the biological processes, molecular functions and KEGG pathways of differentially expressed genes/proteins. qRT-PCR was employed to validate the results obtained from these analyses. Results: Transcriptomic and proteomic profiling of α-MG treated cells indicated that genes/proteins affected by α-MG treatment were associated with diverse cellular functions. The greatest reduction in expression was observed in transcription of genes conferring cytoplasmic membrane integrity (yidC2, secA and mscL), cell division (ftsY and divlB), teichoic acid biosynthesis (tagG and dltA), fatty-acid biosynthesis (accB, accC, fabD, fabH, fabI, and fabZ), biofilm formation (icaA) and DNA replication and repair machinery (polA, polC, dnaE, and uvrA). Those with increased expression were involved in oxidative (katA and sodA) and cellular stress response (clpB, clpC, groEL, and asp23). The qRT-PCR analysis substantiated the results obtained from transcriptomic and proteomic profiling studies. Conclusion: Combining transcriptomic and proteomic methods provided comprehensive information about the antibacterial mode of action of α-MG. The obtained results suggest that α-MG targets S. epidermidis through multifarious mechanisms, and especially prompts that loss of cytoplasmic membrane integrity leads to rapid onset of bactericidal activity.

Inhibitory effect of α-mangostin on Acinetobacter baumannii biofilms - an in vitro study.

The present study was designed to investigate the anti-biofilm potential of alpha-mangostin (α-MG) against Acinetobacter baumannii (AB). The biofilm inhibitory concentration (BIC) of α-MG against AB was found to be 2 µg ml-1. α-MG (0.5, 1 and 2 µg ml-1) exhibited non-bactericidal concentration-dependent anti-biofilm activities against AB. However, α-MG failed to disintegrate the mature biofilms of AB even at a 10-fold increased concentration from its BIC. Results from qRT-PCR and in vitro bioassays further demonstrated that α-MG downregulated the expression of bfmR, pgaA, pgaC, csuA/B, ompA, bap, katE, and sodB genes, which correspondingly affects biofilm formation and its associated virulence traits. The present study suggests that α-MG exerts its anti-biofilm property by interrupting initial biofilm formation and the cell-to-cell signaling mechanism of AB. Additional studies are required to understand the mode of action responsible for the anti-biofilm property.

Inhibitory efficacy of geraniol on biofilm formation and development of adaptive resistance in Staphylococcus epidermidis RP62A.

PURPOSE: The current study has been designed to delineate the efficacy of geraniol (GE) on biofilm formation in Staphylococcus epidermidis as well as the effect of subinhibitory concentrations of GE on the development of adaptive resistance. METHODOLOGY: Biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of GE against S. epidermidis. Microscopic observation of biofilms and extracellular polymeric substance (EPS), slime and cell surface hydrophobicity (CSH) production were also studied to support the antibiofilm potential of GE. In addition, S. epidermidis was examined for its adaptive resistance development upon continuous exposure of GE at its subinhibitory concentrations.Results/Key findings. The MIC of GE against S. epidermidis was 512 µg ml-1. Without hampering the growth of the pathogen, GE at its sub-MICs (50, 100, 150 and 200 µg ml-1) exhibited a dose-dependent increase in antibiofilm activity. The minimal biofilm inhibitory concentration (MBIC) of GE was found to be 200 µg ml-1 with a maximum biofilm inhibition of 85 %. Disintegrated biofilm architecture, reduced EPS, slime and CSH production validated the antibiofilm efficacy of GE. Although the action of GE on preformed biofilm is limited, a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and live/dead cell staining method revealed reduction in the viability (47 %) of biofilm inhabitants at 2×MIC concentration. Sequential exposure of S. epidermidis to the sub-MICs of GE resulted in poor development of adaptive resistance with diminished biofilm formation. CONCLUSION: The present study highlights the potential of GE as a suitable candidate for the control of biofilm-mediated S. epidermidis infections.

In vitro activity of alpha-mangostin in killing and eradicating Staphylococcus epidermidis RP62A biofilms.

Alpha-mangostin (α-MG) has been reported to be an effective antibacterial agent against planktonic cells of many Gram-positive bacteria. However, the antibiofilm potency of α-MG remains unexplored till date. In this study, the antibiofilm and mature biofilm eradication ability of α-MG against Staphylococcus epidermidis RP62A (ATCC 35984) biofilms were evaluated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of α-MG against S. epidermidis RP62A were found to be 1.25 and 5 µg/mL, respectively. α-MG exhibited a phenomenal concentration dependent rapid bactericidal activity (>4-log reduction within 5 min). In a multi-passage resistance analysis using S. epidermidis, no development of resistance to α-MG as well as antibiotics was observed in its habituation. α-MG at its 1/2 MIC effectively inhibited the initial biofilm formation of S. epidermidis, which was further confirmed through scanning electron microscopic (SEM) analysis that portrayed a lucid reduction in the aggregation and the spread of biofilm. The crystal violet staining and viable cell quantification results confirmed the eradication of preformed immature and mature biofilms of S. epidermidis by α-MG in a concentration dependent manner. Besides, the biofilm eradication ability was also confirmed through SEM and live/dead BacLight staining using confocal laser scanning microscopy (CLSM). Thus, the present study exemplifies that α-MG could plausibly assist to eliminate biofilm infections associated with multidrug-resistance staphylococci.

Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach.

The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100µg/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25µg/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.

Cyclic dipeptide cyclo(l-leucyl-l-prolyl) from marine Bacillus amyloliquefaciens mitigates biofilm formation and virulence in Listeria monocytogenes.

This study was intentionally focused on cyclo(l-leucyl-l-prolyl) (CLP), a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant foodborne pathogen,Listeria monocytogenes(LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 µg mL(-1) CLP at sub-MICs (64 128, 256 µg mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface).In vitrobioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data ofin vivoassay usingCaenorhabditis eleganssignified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in extracellular polymeric substance, autoaggregation, hydrophobicity index and extracellular DNA of the CLP-treated LM cells. Collectively, this study unveils the antivirulence efficacy of CLP against the Gram-positive foodborne pathogen and the strainBacillus amyloliquefaciensaugurs well to be a promising probiotic in controlling infections associated with LM.