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Understanding bacteria and challenges in microbiology

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Understanding bacteria and challenges in microbiology
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In 2020 we celebrate 75 years of the anniversary of our founding with a year of activities dedicated to demonstrating the impact of microbiologists’ past, present and future – bringing together and empowering communities that help shape the future of microbiology. We are launching new collections of digital content throughout the anniversary year. The second digital hub is 'Understanding bacteria and the challenges in microbiology', which will explore novel antimicrobial strategies, the world of biofilms and bacteria in industry.

Collection Contents

1 - 20 of 26 results

    • Antibiofilm activity in the culture supernatant of a marine sp. bacterium

      In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit sp. II2003 biofilm formation and the sp. IV2006 strain was identified as producing a strong antibiofilm activity. The sp. IV2006 culture supernatant (SN) inhibited sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SN had no effect on the sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid–base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SN and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SN exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as species that are pathogenic for fish, and human pathogens in both the medical environment, such as and , and in the food industry, such as . Thus, a wide range of applications could be envisaged for the SN compounds, both in aquaculture and human health.

    • Dental floss impregnated with povidone-iodine coated with Eudragit L-100 as an antimicrobial delivery system against periodontal-associated pathogens

      Periodontitis is among the most widespread oral bacterial diseases affecting 15-20% of the world population.

      . This study aimed to develop dental floss impregnated with povidone-iodine (PVP-I) as an antimicrobial delivery system against periodontopathogenic bacteria in a planktonic form and within biofilms.

      Identical lengths of dental floss impregnated with PVP-I formulations were placed on agar along with previously grown periodontal pathogens. The bioactivity of the dental floss was investigated by response-surface methodology. In order to explore the antibacterial activity of the selected formulation and the potential application in the prevention and treatment of plaque-caused diseases such as periodontitis and caries, the antibacterial and anti-biofilm activity of the selected PVP-I formulation against pathogenic bacteria were investigated.

      The results indicated that the coating formulation containing Eudragit L-100 2.90 %, PVP-I 24.58 % and PEG 400 3.73 % had antimicrobial activity for all pathogens. The mechanism of this formulation involved disruption of bacterial cell membranes. Moreover, this formulation inhibited the formation of oral pathogenic biofilms.

      It was concluded that Eudragit L-100 and PVP-I-coated dental floss represented a potential therapeutic agent to prevent periodontal diseases and dental caries and exhibited non-toxicity to periodontal ligament cells.

    • Furanone quorum-sensing inhibitors with potential as novel therapeutics against

      Micro-organisms use quorum sensing (QS), a cell density-dependent process, to communicate. This QS mode of interchange leads to the production of a variety of virulence factors, co-ordination of complex bacterial behaviours, such as swarming motility, degradation of host tissue and biofilm formation. QS is implicated in numerous human infections and consequently researchers have sought ways of effectively inhibiting the process in pathogenic bacteria. Two decades ago, furanones were the first class of chemical compounds identified as QS inhibitors (QSIs). is a ubiquitous organism, capable of causing a wide range of infections in humans, including eye and ear infections, wound infections and potentially fatal bacteraemia and thus novel treatments against this organism are greatly needed. This review provides a brief background on QS and the use of furanones as QSIs. Based on the effectiveness of action, both and we will explore the use of furanones as potential antimicrobial therapeutics and conclude with open questions.

    • activities of telithromycin against biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin

      biofilms are difficult to treat and the effect of telithromycin treatment is still unclear.

      This study aimed to explore the effect of telithromycin against biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin.

      Eight methicillin-susceptible and eight methicillin-resistant isolates (MSSA and MRSA, respectively) were used for this study. Biofilm biomasses were detected by crystal violet staining and the adherent cells in the established biofilms were quantified by determination of colony-forming units (c.f.u.). The RNA levels of biofilm formation-related genes were determined by RT-qPCR.

      Telithromycin [8× minimum inhibitory concentration (MIC)] eradicated more established biofilms than azithromycin or clindamycin in the four MSSA isolates, and eliminated the established biofilms of six MRSA isolates more effectively than vancomycin or daptomycin. Telithromycin (8× MIC) killed more adherent cells in the established biofilms than azithromycin or clindamycin in the six MSSA isolates, and killed more adherent cells than vancomycin in all eight MRSA isolates. Daptomycin also showed an excellent effect on the adherent cells of MRSA isolates, with similarresults to telithromycin. The effect of a subinhibitory concentration of telithromycin (1/4× MIC) was significantly superior to that of azithromycin or clindamycin, inhibiting the biofilm formation of six MSSA isolates and seven MRSA isolates more effectively than vancomycin or daptomycin. The RNA levels of , , , and decreased when treated with telithromycin (1/4× MIC).

      Telithromycin is more effective than azithromycin, clindamycin, vancomycin, or daptomycin against biofilms.

    • Analysis of CRISPR gene drive design in budding yeast

      Control of biological populations remains a critical goal to address the challenges facing ecosystems and agriculture and those posed by human disease, including pests, parasites, pathogens and invasive species. A particular architecture of the CRISPR/Cas biotechnology – a gene drive – has the potential to modify or eliminate populations on a massive scale. Super-Mendelian inheritance has now been demonstrated in both fungi and metazoans, including disease vectors such as mosquitoes. Studies in yeast and fly model systems have developed a number of molecular safeguards to increase biosafety and control over drive systems , including titration of nuclease activity, anti-CRISPR-dependent inhibition and use of non-native DNA target sites. We have developed a CRISPR/Cas9 gene drive in that allows for the safe and rapid examination of alternative drive designs and control mechanisms. In this study, we tested whether non-homologous end-joining (NHEJ) had occurred within diploid cells displaying a loss of the target allele following drive activation and did not detect any instances of NHEJ within multiple sampled populations. We also demonstrated successful multiplexing using two additional non-native target sequences. Furthermore, we extended our analysis of ‘resistant’ clones that still harboured both the drive and target selection markers following expression of Cas9; mutation or NHEJ-based repair could not explain the majority of these heterozygous clones. Finally, we developed a second-generation gene drive in yeast with a guide RNA cassette integrated within the drive locus with a near 100 % success rate; resistant clones in this system could also be reactivated during a second round of Cas9 induction.

    • Sub-MIC effects of a proline-rich antibacterial peptide on clinical isolates of

      is one of the most important nosocomial pathogens, mainly due to its ability to accumulate antibiotic-resistances and to persist in the hospital environment – characteristics related to biofilm production. It is well-known that is inhibited by the proline-rich peptide Bac7(1-35), but its putative effects at sub-MICs were never considered.

      We examined the sub-MIC effect of Bac7(1-35) on the growth rate, resistance induction and some features linked to virulence.

      Growth kinetics in the presence of sub-MICs of Bac7(1-35) were evaluated spectrophotometrically. Peptide uptake was quantified by cytometric analysis. The ability of Bac7(1-35) to interfere with biofilm production was investigated by the crystal violet method and confocal microscopy. Bacterial motility was observed at the interphase between a layer of a semi-solid medium and the polystyrene bottom of a Petri dish. The induction of resistance was evaluated after serial passages with sub-MICs of the peptide.

      Although the MIC of Bac7(1-35) was between 2–4 µM for all tested strains, its effect on the growth rate at sub-MICs was strain-dependent and correlated with the amount of peptide internalized by each strain. Sub-MICs of Bac7(1-35) induced a strongly strain-dependent effect on biofilm formation and reduced motility in almost all strains, but interestingly the peptide did not induce resistance.

      Bac7(1-35) is internalized into and is able to inhibit biofilm formation and bacterial motility, without inducing resistance. This study stresses the importance of considering possible effects that antimicrobials could have at sub-MICs, mimicking a common condition during antibiotic treatment.

    • Trying to treat the untreatable: experimental approaches to clear rabies virus infection from the CNS

      Rabies virus causes an invariably fatal encephalitis following the onset of clinical disease. Despite the availability of safe and effective vaccines, the clinical stages of rabies encephalitis remain untreatable, with few survivors being documented. A principal obstacle to the treatment of rabies is the neurotropic nature of the virus, with the blood–brain barrier size exclusion limit rendering the delivery of antiviral drugs and molecules to the central nervous system inherently problematic. This review focuses on efforts to try and overcome barriers to molecule delivery to treat clinical rabies and overviews current progress in the development of experimental live rabies virus vaccines that may have future applications in the treatment of clinical rabies, including the attenuation of rabies virus vectors through either the duplication or mutation of existing genes or the incorporation of non-viral elements within the genome. Rabies post-infection treatment (PIT) remains the holy grail of rabies research.

    • Expression of adhesin genes and biofilm formation among clinical isolates from patients with antibiotic-associated haemorrhagic colitis

      Biofilm formation and resistance to last-line antibiotics have restricted chemotherapy options toward infection eradication.

      Fifty isolates were collected from patients with antibiotic-associated haemorrhagic colitis (AAHC). Antibiotic susceptibility tests were conducted and phenotypic biofilm formation was assessed using microtitre tissue plate (MTP) assay. PCR was employed to amplify the adhesins, extended-spectrum -lactamases (ESBLs), carbapenemase and colistin resistance genes. The expression of adhesin genes was evaluated using quantitative real-time PCR (RT-qPCR).

      The previous antibiotic consumption and hospitalization (<0.05) and older ages (=0.0033) were significantly associated with AAHC. None of the isolates produced biofilm strongly, but 70% of them produced moderate-level biofilm. The (12/14), the (8/14 MIC =4 µg ml ) and (5/14) and (4/14) genes were predominant, three of which harbouring all the genes. The expression of (0.023) and (0.011) was significantly different between multidrug-resistant and susceptible isolates. Furthermore, moderately biofilm producer isolates significantly exhibited higher expression of (=.0117), (=0.002) and (=0.020) genes compared to biofilm non-producers. No significant difference regarding gene expression was observed among ESBL alleles.

      Bacterial attachment by adhesins and biofilm formation among extensive drug-resistant isolates hinder the efficient infection eradication. Hence, control and surveillance studies should be performed and other therapeutic auspicious approaches must be taken into account against AAHC, biofilm formation and drug resistance spread. Furthermore, previous antibiotic consumption and long-term hospitalization should be controlled.

    • activity of mecillinam and nitroxoline against – re-purposing old antibiotics in the multi-drug resistance era

      In 2018, the European Centre for Disease Prevention and Control reported the first cases of extensively drug-resistant infections in Europe. Seeking new options for antimicrobial therapy we investigated the susceptibility of to nitroxoline (NIT) and mecillinam (MCM), both of which are currently only indicated to treat uncomplicated urinary tract infections. Clinical isolates with non-susceptibility to penicillin from two German medical centres were included ( =27). Most isolates were also non-susceptible to a range of other anti-gonococcal antimicrobials (cefotaxime, ciprofloxacin, azithromycin, tetracycline). All isolates were further characterized by multi-locus sequence typing. MICs of penicillin and cefotaxime were determined by agar gradient diffusion. Production of penicillinase was tested by cefinase disk test. Susceptibility of MCM was investigated by agar dilution, NIT by agar dilution and disk diffusion. Penicillin MICs ranged from 0.125 to 64 mg l and MICs of cefotaxime ranged from < 0.016 to 1 mg l . Five isolates were penicillinase-producers. MICs of MCM ranged from 16 to > 128 mg l whereas MICs of NIT ranged from 0.125 to 2 mg l . NIT disk diffusion (median zone diameter 32 mm) correlated well with results from agar dilution. We demonstrated excellent activity of NIT against clinical isolates with non-susceptibility to standard anti-gonococcal antibiotics. MCM activity was unsatisfactory. Correlation of agar dilution and disk diffusion in NIT susceptibility testing is an important aspect with potential clinical implications.

    • Microbiological and real-time mechanical analysis of and dual-species biofilm

      In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm development and properties. Still, very little is known about how the coexistence of multiple organisms impact the multispecies biofilm properties. In this study, we examined the behaviour of a dual-species biofilm at the air–liquid interface composed by two environmental bacteria: and a phenazine mutant of . Study of the planktonic and biofilm growths for each species revealed that grew faster than and no bactericidal effect from was detected, suggesting that the growth kinetics could be the main factor in the dual-species biofilm composition. To validate this hypothesis, the single- and dual-species biofilm were characterized by biomass quantification, microscopy and rheology. Bacterial counts and microscale architecture analysis showed that both bacterial populations coexist in the mature pellicle, with a dominance of . Real-time measurement of the dual-species biofilms' viscoelastic (i.e. mechanical) properties using interfacial rheology confirmed that was the main contributor of the biofilm properties. Evaluation of the dual-species pellicle viscoelasticity at longer time revealed that the biofilm, after reaching a first equilibrium, created a stronger and more cohesive network. Interfacial rheology proves to be a unique quantitative technique, which combined with microscale imaging, contributes to the understanding of the time-dependent properties within a polymicrobial community at various stages of biofilm development. This work demonstrates the importance of growth kinetics in the bacteria competition for the interface in a model dual-species biofilm.

    • Antimicrobial peptides prevent bacterial biofilm formation on the surface of polymethylmethacrylate bone cement

      Purpose. Antibiotic-loaded polymethylmethacrylate-based bone cement has been implemented in orthopaedics to cope with implant-related infections associated with the formation of bacterial biofilms. In the context of emerging bacterial resistance to current antibiotics, we examined the efficacy of short antimicrobial peptide-loaded bone cement in inhibiting bacterial adhesion and consequent biofilm formation on its surface.

      Methodology. The ability of α-helical antimicrobial peptides composed of 12 amino acid residues to prevent bacterial biofilm [methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis , Pseudomonas aeruginosa and Escherichia coli ] formation on the surface of model implants made from polymethylmethacrylate-based bone cement was evaluated by colony-forming unit (c.f.u.) counting of bacteria released by sonication from the biofilms formed on their surfaces. The biofilms on model implant surfaces were also visualized by light microscopy after staining with tetrazolium dye (MTT) and by scanning electron microscopy.

      Results. When incorporated in the implants, these peptides caused a mean reduction in the number of bacterial cells attached to implants’ surfaces (by five orders of magnitude), and 88 % of these implants showed no bacterial adhesion after being exposed to growth media containing various bacteria.

      Conclusion. The results showed that the antibiofilm activity of these peptides was comparable to that of the antibiotics, but the peptides exhibited broader specificity than the antibiotics. Given the rapid development of antibiotic resistance, antimicrobial peptides show promise as a substitute for antibiotics for loading into bone cements.

    • Introducing BAIT (Biofilm Architecture Inference Tool): a software program to evaluate the architecture of oral multi-species biofilms

      Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate (‘placebo’) or stannous fluoride (SnF). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF (1000, 3439 or 10 000 p.p.m. Sn) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

    • Cinnamaldehyde disrupts biofilm formation and swarming motility of Pseudomonas aeruginosa

      Bacterial biofilms can cause serious health care complications associated with increased morbidity and mortality. There is an urge to discover and develop new biofilm inhibitors from natural products or by modifying natural compounds or understanding the modes of action of existing compounds. Cinnamaldehyde (CAD), one of the major components of cinnamon oil, has been demonstrated to act as an antimicrobial agent against a number of Gram-negative and Gram-positive pathogens, including Pseudomonas aeruginosa, Helicobacter pylori and Listeria monocytogenes. Despite the mechanism of action of CAD against the model organism P. aeruginosa being undefined, based on its antimicrobial properties, we hypothesized that it may disrupt preformed biofilms of P. aeruginosa. The minimum inhibitory concentration (MIC) of CAD for planktonic P. aeruginosa was determined to be 11.8 mM. Membrane depolarization assays demonstrated disruption of the transmembrane potential of P. aeruginosa. CAD at 5.9 mM (0.5 MIC) disrupted preformed biofilms by 75.6 % and 3 mM CAD (0.25 MIC) reduced the intracellular concentrations of the secondary messenger, bis-(3′–5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), which controls P. aeruginosa biofilm formation. The swarming motility of P. aeruginosa was also reduced by CAD in a concentration-dependent manner. Collectively, these findings show that sub-MICs of CAD can disrupt biofilms and other surface colonization phenotypes through the modulation of intracellular signalling processes.

    • Diagnostics and antibiotic therapy for spondylodiscitis

      Spondylodiscitis as a rare disease has increased in recent years; there are only a few recommendations regarding the diagnostics and treatment. The objective of this retrospective study was to characterise these patients and identify factors for a favourable healing process – with an emphasis on antibiotic therapy.

      Findings/treatment regimens for all patients with spondylodiscitis treated at a tertiary care hospital in 2012/2013 were recorded. The patients were classified into two groups, depending on the healing process.

      Fifty-seven patients met the clinical/radiological criteria for spondylodiscitis. The main symptom was massive back pain, combined with a moderate CRP elevation (median 96 mg l), and occasionally fever (39 %). CT-guided aspiration – in addition to blood cultures – improved the detection rate for the causative pathogen from 49 to 82 %. Thirty patients had a favourable clinical course; 25 had an unfavourable clinical course (prolonged clinical course, recurrence, death). Pathogen-specific antibiotic therapy had a decisive effect on the clinical course: initial high-dose intravenous therapy for at least 14 days was associated significantly more often with a favourable clinical course (90 vs 30 % after 24 months, <0.001). Fourteen days after the start of treatment, marked pain reduction and a CRP reduction of at least 50 % were good prognostic parameters and markers of effective antibiotic therapy.

      Pathogen detection and establishment of an optimal antibiotic regimen are cornerstones of successful conservative therapy for spondylodiscitis. Targeted initial intravenous therapy for at least 14 days with a bactericidal antibiotic leads to a better clinical course.

    • Efficient bio-production of citramalate using an engineered strain

      Citramalic acid is a central intermediate in a combined biocatalytic and chemocatalytic route to produce bio-based methylmethacrylate, the monomer used to manufacture Perspex and other high performance materials. We developed an engineered strain and a fed-batch bioprocess to produce citramalate at concentrations in excess of 80 g l in only 65 h. This exceptional efficiency was achieved by designing the production strain and the fermentation system to operate synergistically. Thus, a single gene encoding a mesophilic variant of citramalate synthase from CimA3.7, was expressed in to convert acetyl-CoA and pyruvate to citramalate, and the and genes were deleted. By using a bioprocess with a continuous, growth-limiting feed of glucose, these simple interventions diverted substrate flux directly from central metabolism towards formation of citramalate, without problematic accumulation of acetate. Furthermore, the nutritional requirements of the production strain could be satisfied through the use of a mineral salts medium supplemented only with glucose (172 g l in total) and 1.4 g l yeast extract. Using this system, citramalate accumulated to 82±1.5 g l, with a productivity of 1.85 g l h and a conversion efficiency of 0.48 g g . The new bioprocess forms a practical first step for integrated bio- and chemocatalytic production of methylmethacrylate.

    • Colworth prize lecture 2016: exploiting new biological targets from a whole-cell phenotypic screening campaign for TB drug discovery

      is the aetiological agent of tuberculosis (TB) and is the leading bacterial cause of mortality and morbidity in the world. One third of the world’s population is infected with TB, and in conjunction with HIV represents a serious problem that urgently needs addressing. TB is a disease of poverty and mostly affects young adults in their productive years, primarily in the developing world. The most recent report from the World Health Organisation states that 8 million new cases of TB were reported and that ~1.5 million people died from TB. The efficacy of treatment is threatened by the emergence of multi-drug and extensively drug-resistant strains of . It can be argued that, globally, is the single most important infectious agent affecting mankind. Our research aims to establish an academic-industrial partnership with the goal of discovering new drug targets and hit-to-lead new chemical entities for TB drug discovery.

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