Specific Bacterial Cells Research Articles

Resolution of Chronic Bacterial Prostatitis with Bacteriophage-antibiotic Therapy

Chronic bacterial prostatitis is a commonly diagnosed genitourinary infection that presents significant challenges both in diagnosis and treatment. In an upcoming era of antibiotic resistance, and limited therapeutic options it becomes imperative to revise current guidelines and to provide more effective treatment strategies. At the Eliava Phage Therapy Center (Tbilisi, Georgia) we utilize bacteriophage therapy as an alternative approach against chronic bacterial infections. Bacteriophages, viruses that target and lyse specific bacterial cells, can be used as a stand-alone treatment or in conjunction with antibiotics. We present a case report of a patient with prostatitis caused by Escherichia coli infection, who prior to addressing our clinic, has been receiving antibiotic therapy without any positive effect. Our approach of combined use of antibiotics and phages was successful not only in complete clinical improvement but also in total bacterial eradication. This outcome shows the potential of bacteriophage therapy as a valuable adjunct to conventional antibacterials in the management of prostatitis.

Estimation of the In-Vitro Synergistic Effect of Vancomycin with Bacteriophage and its Endolysin on Iraqi Local Isolates of Enterococcus faecalis

Enterococcus faecalis is known to cause many serious infections with an upsurge in the development of antibiotic resistant strains, especially vancomycin. Lytic bacteriophage and its endolysin have the potential to affect and lyse specific bacterial cells making them a possible alternative therapy to antibiotic resistant strains. The study aimed to investigate the synergistic effect of phage/ phage cocktail/ endolysin with vancomycin on E. faecalis isolated from urine samples. The antibioresistant profile 17 E. faecalis was done with an emphasis on the sensitivity to vancomycin. The lytic phages were isolated from the local environment. The infectivity and the coverage rate for the phage and phage cocktail were calculated. The enzyme of the lytic phage was isolated by chromatography. The synergistic effect was tested by turbidity reduction assay to evaluate the growth reduction level. It had been found that 6/17 E. faecalis isolates were resistant to vancomycin, 7/17 isolates were intermediate, and 4/17 were susceptible. Three lytic phages were identified with an infectivity rate of 100%, 76.4%, and 88.2%. The phage cocktail yielded 100% rate of infectivity and 91.6 % coverage rate. The reduction in the growth levels of the 17 E.faecalis after treatment with phage / phage cocktail/ vancomycin/ mixture of vancomycin and phage cocktail was effective. The synergistic effect of vancomcyin together with the phage cocktail significantly reduced the growth of 70.6% of the isolates. The antibacterial activity of vancomycin plus endolysin treatment was revealed in a time dependent manner. Thus, the synergistic effect of vancomycin with phage/ endoloysin illustrated an effective method to treat the antibiotic-resistant strains of E. faecalis to overcome this emerging clinical problem.

Propagation, Purification, and Characterization of Bacteriophages for Phage Therapy.

Phage therapy is an alternative approach to combat bacterial infections. In this approach, bacteriophages are used as antimicrobial agents due to their properties to infect specific bacterial cells, to propagate inside their hosts, and to lyse host cell to release progeny phages. However, to introduce bacteriophages to clinical or veterinary practice, it is necessary to construct a large library of precisely characterized phages. Therefore, in this chapter, methods for propagation, purification, and microbiological characterization of bacteriophages are presented in the light of their potential use in phage therapy. Isolation of newly discovered bacteriophages from different habitats is also described as it is a preliminary assessment of their efficacy in combating bacterial biofilms and in the treatment of bacterial infections in a simple insect model-Galleria mellonella.

Saponin treatment for eukaryotic DNA depletion alters the microbial DNA profiles by reducing the abundance of Gram-negative bacteria in metagenomics analyses.

Background: Recent advances in microbiome sequencing techniques have provided new insights into the role of the microbiome on human health with potential diagnostic implications. However, these developments are often hampered by the presence of a large amount of human DNA interfering with the analysis of the bacterial content. Nowadays, extensive scientific literature focuses on eukaryotic DNA depletion methods, which successfully remove host DNA in microbiome studies, even if a precise assessment of the impact on bacterial DNA is often missing. Methods: Here, we have investigated a saponin-based DNA isolation protocol commonly applied to different biological matrices to deplete the released host DNA. Results: The bacterial DNA obtained was used to assess the relative abundance of bacterial and human DNA, revealing that the inclusion of 2.5% wt/vol saponin allowed the depletion of most of the host's DNA in favor of bacterial DNA enrichment. However, shotgun metagenomic sequencing showed inaccurate microbial profiles of the DNA samples, highlighting an erroneous increase in Gram-positive DNA. Even the application of 0.0125% wt/vol saponin altered the bacterial profile by depleting Gram-negative bacteria, resulting in an overall increase of Gram-positive bacterial DNA. Conclusion: The application of the saponin-based protocol drastically changes the detection of the microbial composition of human-related biological specimens. In this context, we revealed that saponin targets not only host cells but also specific bacterial cells, thus inducing a drastic reduction in the profiling of Gram-negative bacterial DNA.

The phosphotransferase system gene ptsH affects persister formation in Klebsiella pneumoniae by regulating cyclic adenosine monophosphate levels

Klebsiella pneumoniae is one of the most common opportunistic pathogens causing hospital- and community-acquired infections. Antibiotic resistance in K. pneumoniae has emerged as a major clinical and public health threat. Persisters are specific antibiotic-tolerant bacterial cells. Studies on the mechanism underlying their formation mechanism and growth status are scarce. Therefore, it is urgent to explore the key genes and signalling pathways involved in the formation and recovery process of K. pneumoniae persisters to enhance the understanding and develop relevant treatment strategies. In this study, we treated K. pneumoniae with a lethal concentration of levofloxacin. It resulted in a distinct plateau of surviving levofloxacin-tolerant persisters. Subsequently, we obtained bacterial samples at five different time points during the formation and recovery of K. pneumoniae persisters to perform transcriptome analysis. ptsH gene was observed to be upregulated during the formation of persisters, and down-regulated during the recovery of the persisters. Further, we used CRISPR-Cas9 to construct ΔptsH, the ptsH-knockout K. pneumoniae strain, and to investigate the effect of ptsH on the persister formation. We observed that ptsH can promote the formation of persisters, reduce accumulation of reactive oxygen species, and enhance antioxidant capacity by reducing cyclic adenosine monophosphate (cAMP) levels. To the best of our knowledge, this is the first study to report that ptsH plays a vital role in forming K. pneumoniae persisters. This study provided important insights to further explore the mechanism underlying the formation of K. pneumoniae persisters and provided a potential target for treating infection with K. pneumoniae persisters.

Moisture-enabled hydrovoltaic power generation with milk protein nanofibrils

Biologic materials have been shown to be good candidates for moisture-enabled hydrovoltaic power generation, but only those from specific bacterial cells have been explored. Herein, we report a moist-electric generator (PN-MEG) based on protein nanofibrils from milk β-lactoglobulin. The as-developed device can generate an open-circuit voltage of up to 0.65 V and a short-current of 2.9 µA. This device can generate a maximum power density of 38.88 μW·cm−2, which is one order of magnitude larger than those of previously reported analogous devices. The superior performance of this device is attributed to the remarkable hydrophilicity, high ionization and surface-to-volume ratio of protein nanofibrils. Density functional theory (DFT) calculations revealed that carboxyl groups were the favorable active sites in protein nanofibrils for binding water molecules. This study provides a novel perspective for common protein nanofibrils and paves the way for developing high-performance hydrovoltaic devices from biological materials.

Evaluation of Bacteriophage-Antibiotic Combination Therapy for Biofilm-Embedded MDR Enterococcus faecium.

Multidrug-resistant (MDR) Enterococcus faecium is a challenging pathogen known to cause biofilm-mediated infections with limited effective therapeutic options. Lytic bacteriophages target, infect, and lyse specific bacterial cells and have anti-biofilm activity, making them a possible treatment option. Here, we examine two biofilm-producing clinical E. faecium strains, daptomycin (DAP)-resistant R497 and DAP-susceptible dose-dependent (SDD) HOU503, with initial susceptibility to E. faecium bacteriophage 113 (ATCC 19950-B1). An initial synergy screening was performed with modified checkerboard MIC assays developed by our laboratory to efficiently screen for antibiotic and phage synergy, including at very low phage multiplicity of infection (MOI). The data were compared by one-way ANOVA and Tukey (HSD) tests. In 24 h time kill analyses (TKA), combinations with phage-DAP-ampicillin (AMP), phage-DAP-ceftaroline (CPT), and phage-DAP-ertapenem (ERT) were synergistic and bactericidal compared to any single agent (ANOVA range of mean differences 3.34 to 3.84 log10 CFU/mL; p < 0.001). Furthermore, phage-DAP-AMP and phage-DAP-CPT prevented the emergence of DAP and phage resistance. With HOU503, the combination of phage-DAP-AMP showed the best killing effect, followed closely by phage-DAP-CPT; both showed bactericidal and synergistic effects compared to any single agent (ANOVA range of mean differences 3.99 to 4.08 log10 CFU/mL; p < 0.001).

A multi-targeted nanoconjugate for light-driven therapy of chronic wounds

The pathogenic bacteria-induced diabetic ulcer becomes a life-threatening miscellaneous disease and remains a pivotal clinical challenge due to poor healing. To overcome the obstacle, we developed a non-antibiotic strategy based on a three-dimensional multi-targeted nanostructure with near-infrared laser induced photothermal therapy for promoting the healing of diabetic wound infected by Pseudomonas aeruginosa and Staphylococcus aureus. This nanoconjugate was prepared by integrating gold nanorods decorated with bacterial affinitive molecules phenylboronic acid and pH-induced charge-convertible tertiary-amine group. In addition to the binding with the cell wall of bacteria from phenylboronic acid motifs, a dedicated charge conversion of the nanoconjugate from negative charge to positive charge in the acidic microenvironment of bacterial infectious sites further provides robust targeting to negatively charged bacteria. This nanoconjugate could target and kill specific bacterial cells using photothermal ablation. Following excitation by near-infrared light, gold nanorods release energy through nonradioactive decay pathways, locally generating heat that efficiently kills targeted bacterial cells and finally accelerating the wound healing of diabetic rats by down-regulating the expression of proinflammatory factors. Collectively, this nanostructure evolved targeting strategy with the photothermal property is expected to be other kind of treatment programs for repairing chronic wounds of diabetic ulcers infected by multiple bacteria.

Using Phage as A Highly Specific Antibiotic Alternative Against Methicillin Resistance Staphylococcus aureus (MRSA)

Misuse of antibiotics in humans and animals often leads to the development ofmultidrug resistance (MDR)bacteria. Resistance can occur within a few years of novel antibiotics being introduced. Lytic bacteriophage (phage) are a kind of virus that undergo a cyclical lifestyle wherein they infect and replicate through the use of a bacterial host cell and cause cell lysis. Phage recognizes specific receptors on their host cell to attach to, insert their DNA and take over their host's molecular machinery. These receptors only exist on the surface of specific bacterial host cells and are often not present on other non-specific bacteria and not especially on the surfaces of eukaryotic cells. The mechanisms by which phage can destroy bacteria are different from antibiotics; phage can lyse MDR resistant bacteria without being affected by hydrolytic enzymes or ribosomal variations that's mean unlike drug resistance mechanism which bacteria can destroy the drug before can inter the bacteria cell, the phage not effected with such enzyme.

The Filter-Feeding Ciliates Colpidium striatum and Tetrahymena pyriformis Display Selective Feeding Behaviours in the Presence of Mixed, Equally-Sized, Bacterial Prey

This study examined whether two ciliates could discriminate between equally-sized bacterial prey in mixture and if so, how selectivity might benefit the ciliate population. Live Klebsiella aerogenes, K. ozaenae and Escherichia coli, expressing different coloured fluorescent proteins, were cultured in such a way as to provide populations containing equally-sized cells (to prevent size-selective grazing taking place) and these prey were fed to each ciliate in 50:50 mixtures. Colpidium striatum selected K. aerogenes over K. ozaenae which itself was selected over E. coli. Tetrahymena pyriformis showed no selectivity between K. aerogenes and E. coli but K. aerogenes was selected over K. ozaenae while E. coli was not. This apparent selection of K. aerogenes over K. ozaenae was sustained in ciliate populations with different feeding histories and when K. aerogenes comprised only 20% of the prey mixture, suggesting possible optimal foraging behaviour. The metabolic benefits for selecting K. aerogenes were identified as possibly being an increase in cell biovolume and yield for C. striatum and T. pyriformis, respectively. The mechanism by which these ciliates selected specific bacterial cells in mixture is currently unknown but the use of live fluorescent bacteria, in prey mixtures, offers an exciting avenue for further investigation of selective feeding by protozoa.

Rapid, Absolute, and Simultaneous Quantification of Specific Pathogenic Strain and Total Bacterial Cells Using an Ultrasensitive Dual-Color Flow Cytometer

This paper describes a rapid and sensitive strategy for the absolute and simultaneous quantification of specific pathogenic strain and total bacterial cells in a mixture. A laboratory-built compact, high-sensitivity, dual channel flow cytometer (HSDCFCM) was modified to enable dual fluorescence detection. A bacterial cell mixture comprising heat-killed pathogenic Escherichia coli E. coli O157:H7 and harmless E. coli DH5alpha was used as a model system. Pathogenic E. coli O157:H7 cells were selectively labeled by red fluorescent probe via antibody-antigen interaction, and all bacterial cells were stained with membrane-permeable nucleic acid dye that fluoresces green. When each individual bacterium passes through the interrogating laser beam, E. coli O157:H7 emits both red and green fluorescence, while E. coli DH5alpha exhibits only green fluorescence. Because the fluorescence burst generated from each individual bacterial cell was easily distinguished from the background, accurate enumeration and consequently absolute quantification were achieved for both pathogenic and total bacterial cells. By using this strategy, accurate counting of bacteria at a density above 1.0 x 10(5) cells/mL can be accomplished with 1 min of data acquisition time after fluorescent staining. Excellent correlation between the concentrations measured by the HSDCFCM and the conventional plate-counting method were obtained for pure-cultured E. coli O157:H7 (R(2) = 0.9993) and E. coli DH5alpha (R(2) = 0.9998). Bacterial cell mixtures with varying proportions of E. coli O157:H7 and E. coli DH5alpha were measured with good ratio correspondence. We applied the established approach to detecting artificially contaminated drinking water samples; E. coli O157:H7 of 1.0 x 10(2) cells/mL were accurately quantified upon sample enrichment. It is believed that the proposed method will find wide applications in many fields demanding bacterial identification and quantification.

Combined capillary electrophoresis and DNA‐fluorescence in situ hybridization for rapid molecular identification of Salmonella Typhimurium in mixed culture

CE, long a staple in analytical chemistry for molecular separations, has recently been adapted for separating heterogeneous mixtures of microbial cells based on intrinsic differences in cell morphology and surface charge. In this application, CE enables effective separations of both relatively broad categories of cells, as well as of more similar cell types. As a phenotypic approach, CE may be less applicable to certain populations, including those comprised of pleiomorphic cells or chain-forming cells, where differences in cell size, shape, or chain length may lead to broad, "unfocusable" distributions in cell surface charge. At the other end of the spectrum, closely related species having similar surface charge profiles may not be separable via CE alone. Successful combination of microbial CE with a compatible method for generating cell-specific signals could address these limitations, increasing the diagnostic power of this approach. Fluorescence in situ hybridization (FISH) is a rapid molecular technique for fluorescence-based labeling of whole target cells. In this work, we combined a simple CE-based presence/absence test with FISH to develop a bacterial detection assay having an additional "layer" of molecular specificity. Using this approach, we were able to differentiate Salmonella Typhimurium from Escherichia coli in mixed populations via CE. Both hybridizations and CE run times were short (10-15 min), bacterial populations were highly focused ( approximately 2-3 s peak width) and there was no need for a posthybridization wash step. As few as three injected cells of S. Typhimurium were detected against a background of approximately 300 injected E. coli cells, suggesting the possibility for single-cell detection of pathogens using this technique. This proof of concept study highlights the potential of CE-FISH as a promising new tool for molecular detection of specific bacterial cells within mixtures of closely related, physiologically inseparable populations.

The control of root-knot nematodes (Meloidogyne spp.) by Pseudomonas oryzihabitans and its immunological detection on tomato roots

Abstract Pseudomonas oryzihabitans, a bacterium associated with the entomopathogenic nematode Steinernema abbasi, was evaluated for its potential to colonise roots and thereby control a field population of root-knot nematodes. Immunological techniques were developed to detect root colonisation of P. oryzihabitans on tomato roots using a specific polyclonal antibody raised against vegetative bacterial cells. In vitro, bacterial cell filtrates were also shown significantly to inhibit juveniles hatching. In a glasshouse pot experiment, there were 22 and 82% fewer females in roots of plants treated with suspensions containing 103 and 106 cells ml−1 of P. oryzihabitans, respectively. In addition, there were significantly fewer egg masses produced; however, the numbers of eggs per egg mass did not differ significantly. The relationship between root colonisation and nematode control is discussed.

Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri.

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species.

Development of a direct in situ PCR method for detection of specific bacteria in natural environments.

We applied HNPP (2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate) to direct in situ PCR for the routine detection of specific bacterial cells at the single-cell level. PCR was performed on glass slides with digoxigenin-labeled dUTP. The digoxigenin-labeled PCR products were detected with alkaline phosphatase-labeled antidigoxigenin antibody and HNPP which was combined with Fast Red TR. A bright red fluorescent signal was produced from conversion to HNP (dephosphorylated form) by alkaline phosphatase. We used the ECOL DNA primer set for amplification of ribosomal DNA of Escherichia coli to identify cells specifically at the single-cell level in a bacterial mixture. High-contrast images were obtained under an epifluorescence microscope with in situ PCR. By image analysis, E. coli cells in polluted river water also were detected.

Multiparametric flow cytometry (FCM): An approach to detect specific bacterial cells in heterologous environments

Multiparametric flow cytometry (FCM): An approach to detect specific bacterial cells in heterologous environments

The construction of a nopaline-inducible marker system for rapid detection of Agrobacterium strains

An inducible marker system suitable for Agrobacterium tumefaciens was constructed to enable detection and enumeration of specific bacterial cells introduced into soil. A BamHI cassette carrying the catechol 2,3-dipxygenase (C230) gene, tdnC, fused to the nopaline-inducible Agrobacterium promoter Pi 2[noc] was constructed. This cassette was introduced into the broad host range vector pDSK5019 resulting in plasmid pTVNC2. Inducible C230 activity was observed in an A. tumefaciens strain carrying the plasmid pTVNC2 when nopaline was present. Colonies of bacteria tagged with the system could easily be identified by spraying agar plates containing nopaline with catechol, which is converted to the bright yellow compound 2-hydroxymuconic semialdehyde. The inducible tdnC cassette has also been introduced into the BamHI site of the transposon Tn5 carried by the pSUP1011 suicide vector which can be used as a delivery system for the stable introduction of the inducible marker into the chromosome of target cells.

Localisation and immunological properties of a 24-kDa surface protein of Haemophilus ducreyi

The cell wall and outer structures of Haemophilus ducreyi bacteria were investigated. The 24-kDa outer protein from two strains was purified with an SDS-PAGE preparative continuous-elution electrophoresis cell. The protein was further characterised by SDS-PAGE and immunoblotting, and the immunological properties were investigated by ELISA. Localisation on the bacterial surface was investigated by immuno-electron-microscopy with a polyclonal antiserum raised against the purified protein. A triple-laminar cell wall typical of gram-negative bacteria, close cellular contact between bacterial cells and outer blebs were seen on thin sections. An additional high mol. wt band of c. 165 kDa was seen when not treated by heating to 100 degrees C. A high density fibrilla-like material was detected on the bacterial cell and in the environment by negative staining and immuno-electron-microscopy with antisera specific for the 24-kDa protein. The surface localisation of the 24-kDa protein was confirmed by an ELISA technique with the specific antiserum and whole bacterial cells as antigen. The presence of antibodies to the 24-kDa protein was demonstrated in antisera to 13 strains of H. ducreyi, indicating antigenic identity or within-species cross-reactivity. Low titres of antibodies to this protein were also detected in 19 antisera raised against different strains of gram-negative bacteria, indicating cross-reactivity with other species. Antibody response to the 24-kDa protein in rabbits immunised subcutaneously with live bacteria resulted in a secondary IgG response. Of 28 sera from patients with culture-verified chancroid, 26 manifested high titres of IgG antibodies to the 24-kDa protein, thus indicating the involvement of this antigen in the disease process in man.

Bacterial Concentration Fluorescence Immunoassay (BCFIA) for the Detection of Periodontopathogens in Plaque.

A bacterial concentration fluorescence immunoassay (BCFIA) was developed to rapidly detect periodontopathic bacteria in human plaque samples. The BCFIA utilized fluorescent-tagged monoclonal antibodies (MAbs) directed against the lipopolysaccharide of selected Gram-negative bacteria. Microorganisms identified in plaque using the BCFIA included Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. The immunoassay procedure involved combining a patient's plaque sample with a species-specific fluorescein isothiocyanate-labeled MAb and then incubating the mixture in a specialized microtiter plate allowing the MAb to bind to its homologous bacteria. Bound and unbound fluorescent-tagged MAbs were separated by filtration and total bound bacterial fluorescence was determined with a fluorimeter. The relative number of a bacterial species in a given plaque sample was estimated by reference to a standard curve carried through the BCFIA. The BCFIA had a lower detection limit of near 104 specific bacterial cells in a mixed bacterial preparation or plaque sample. When compared to cultivable flora procedures in detecting the 4 periodontopathogens, the BCFIA had high levels of statistical sensitivity, 97% to 100%, while statistical specificity ranged between 57% and 92%. There was a 71% to 82% agreement between BCFIA and DNA probe methodology in detecting periodontopathogens in plaque. The BCFIA, when compared to cultivable flora, offers the advantage of evaluating both live and dead bacterial cells in plaque. This may in part, if not fully, explain the lower specificity values of the BCFIA when compared to cultivable flora. Screening plaque samples for periodontopathic bacteria is considerably faster and results in a greater frequency of detection with BCFIA than cultivable flora based methods. J Periodontol 1992; 63:1093-1101.

Applied Significance of Polyvalent Bacteriophages

A bacterial virus or bacteriophage is an exogenous submicroscopic particle capable of multiplication only inside specific bacterial cells .Viral reproduction, however, is unique because typical viral particles cannot be found inside infected host cells for some finite period immediately following infection. This eclipse period perhaps constitutes the only useful criterion for differentiating between large viruses and strict intracellular parasites such as rickettsia. The best-known bacterial viruses are Escherichia coli , and until recently, the T-even coliphages held the attention of the vast majority of the microbial virologists. The chapter emphasizes the diversity of bacteriophage behaviour and accordingly chooses examples from a variety of microbial genera.