CFU Enumeration Research Articles

P06.09 Lactobacilluscrispatus inhibits growth ofgardnerella vaginalisandneisseria gonorrhoeaeon a porcine vaginal mucosa model

Introduction The vaginal microbiota affects susceptibility to bacterial vaginosis (BV) and sexually transmitted infections (STIs). BV is characterised by depletion of Lactobacillus spp., an overgrowth of anaerobes (usually dominated by Gardnerella vaginalis) and a pH > 4.5. BV is associated with an increased risk of acquiring STIs such as chlamydia and gonorrhoea. An ex vivo porcine vaginal mucosal model (PVM) was developed to explore the mechanistic role of Lactobacillus in affecting vaginal colonisation by G. vaginalis and Neisseria gonorrhoeae. Methods Explants (5 mm) of freshly collected PVM were placed in transwells over various media, including Lactobacillus culture supernatant, inoculated with bacteria and incubated under aerobic or anaerobic conditions. Colonised explants were processed for CFU enumeration and presence of biofilm (via confocal microscopy) at indicated times. Lactic acid produced by a clinical isolate of L. crispatus growing on PVM was also quantified. Results All isolates tested could colonise and grow on PVM. G. vaginalis and N. gonorrhoeae form biofilms on PVM. L. crispatus produces lactic acid on PVM and inhibits the growth of N. gonorrhoeae and G. vaginalis in a pH-dependent manner. Finally, L. crispatus produces a secreted factor that kills N. gonorrhoeae on PVM at an otherwise permissible pH. Conclusion These data demonstrate that PVM is a useful model for studying the interactions of commensal vaginal microbes with pathogens on the vaginal mucosa. The data confirm a role for lactic acid in inhibiting growth of G. vaginalis and N. gonorrhoeae. The discovery of an L. crispatus secreted factor that kills N. gonorrhoeae is intriguing and future work will identify this compound and explore its mechanism of action. Disclosure of interest statement Funding provided by the Office of the Vice President for Research, University of Minnesota and NIH grant U19AI084044. No pharmaceutical grants were received in the development of this study.

Comparison of real-time PCR assay and plate count for Lactobacillus paracasei enumeration in yoghurt

Lactobacillus paracasei is a mesophilic lactic acid bacterium technologically active in food fermentation. Culture-independent methods have rapidly been recognized as a valuable alternative to culture-dependent methods for lactic acid bacteria enumeration. In the present work, the efficacy of different protocols to extract DNA from yoghurt were compared, real-time PCR (qPCR) targeting tuf gene for L. paracasei enumeration was evaluated, and qPCR and plate counts of L. paracasei in yoghurt samples were compared. Total DNA concentrations from commercial yoghurts were higher using DNAzol method 2 than using the other tested methods. Standard curves presented suitable mean efficiency values of 91 % (pure L. paracasei strain CTT 7501), 95 % (pure L. paracasei strain FNU), and 103 % (yoghurt with L. paracasei strain FNU). Limit of detection is 3 log DNA copy number, corresponding to 2.78 log CFU, a suitable range of CFU enumeration for probiotic bacteria in yoghurt samples, considering that they should be present in large amounts. The L. paracasei (CFU) enumerated by qPCR were compared to culturable L. paracasei enumerated by plate counts at 7, 14, 21, and 28 days of yoghurt manufacture. Differences between qPCR and plate counts were observed only 28 days after yoghurt preparation, counts were similar at 7, 14, and 21 days. In conclusion, this qPCR assay is a useful and rapid tool to enumerate L. paracasei in yoghurt, although it does not distinguish dead and viable cells.

Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples

We propose a simple technique for bacterial and yeast cfu estimations from diverse samples with no prior idea of viable counts, designated as single plate-serial dilution spotting (SP-SDS) with the prime recommendation of sample anchoring (100 stocks). For pure cultures, serial dilutions were prepared from 0.1OD (100) stock and 20μl aliquots of six dilutions (101–106) were applied as 10–15 micro-drops in six sectors over agar-gelled medium in 9-cm plates. For liquid samples 100–105 dilutions, and for colloidal suspensions and solid samples (10% w/v), 101–106 dilutions were used. Following incubation, at least one dilution level yielded 6–60cfu per sector comparable to the standard method involving 100μl samples. Tested on diverse bacteria, composite samples and Saccharomyces cerevisiae, SP-SDS offered wider applicability over alternative methods like drop-plating and track-dilution for cfu estimation, single colony isolation and culture purity testing, particularly suiting low resource settings.

Antifungal activity of cathelicidin peptides against planktonic and biofilm cultures of Candida species isolated from vaginal infections

Vulvovaginal candidiasis (VVC) is a frequent gynecological condition caused by Candida albicans and a few non-albicans Candida spp. It has a significant impact on the quality of life of the affected women also due to a considerable incidence of recurrent infections that are difficult to treat. The formation of fungal biofilm may contribute to the problematic management of recurrent VVC due to the intrinsic resistance of sessile cells to the currently available antifungals. Thus, alternative approaches for the prevention and control of biofilm-related infections are urgently needed. In this regard, the cationic antimicrobial peptides (AMPs) of the innate immunity are potential candidates for the development of novel antimicrobials as many of them display activity against biofilm formed by various microbial species. In the present study, we investigated the in vitro antifungal activities of the cathelicidin peptides LL-37 and BMAP-28 against pathogenic Candida spp. also including C. albicans, isolated from vaginal infections, and against C. albicans SC5314 as a reference strain. The antimicrobial activity was evaluated against planktonic and biofilm-grown Candida cells by using microdilution susceptibility and XTT [2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide] reduction assays and, in the case of established biofilms, also by CFU enumeration and fluorescence microscopy. BMAP-28 was effective against planktonically grown yeasts in standard medium (MIC range, 2–32μM), and against isolates of C. albicans and Candida krusei in synthetic vaginal simulated fluid (MIC range 8–32μM, depending on the pH of the medium). Established 48-h old biofilms formed by C. albicans SC5314 and C. albicans and C. krusei isolates were 70–90% inhibited within 24h incubation with 16μM BMAP-28. As shown by propidium dye uptake and CFU enumeration, BMAP-28 at 32μM killed sessile C. albicans SC5314 by membrane permeabilization with a faster killing kinetics compared to 32μM miconazole (80–85% reduced biofilm viability in 90min vs 48h). In addition, BMAP-28 at 16μM prevented Candida biofilm formation on polystyrene and medical grade silicone surfaces by causing a >90% reduction in the viability of planktonic cells in 30min. LL-37 was overall less effective than BMAP-28 against planktonic Candida spp. (MIC range 4–≥64μM), and was ineffective against established Candida biofilms. However, LL-37 at 64μM prevented Candida biofilm development by inhibiting cell adhesion to polystyrene and silicone surfaces. Finally, Candida adhesion was strongly inhibited when silicone was pre-coated with a layer of BMAP-28 or LL-37, encouraging further studies for the development of peptide-based antimicrobial coatings.

Stable Expression of Lentiviral Antigens by Quality-Controlled Recombinant Mycobacterium bovis BCG Vectors.

The well-established safety profile of the tuberculosis vaccine strain, Mycobacterium bovis bacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCD transformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging both in vitro and in vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCD vaccine expressing HIV gp120 that retained stable full-length expression after 10(24)-fold amplification in vitro and following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >10(68)-fold amplification in vitro and induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCD lots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Vulnerability of Bacillus spores and of related genera to physical impaction injury with particular reference to spread-plating.

To examine whether bacterial spores are vulnerable to impaction injury during standard spread-plating or to other modes of physical impaction. Employing heat-challenged spores of Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Lysinibacillus, Paenibacillus and Brevibacillus spp. from day-4 to day-10 nutrient agar (NA) plates in 50% ethanol, plating the spore suspension to the extent of just drying the agar surface on fresh NA (50-60 s; SP-B) was tested in comparison with the spreader-independent approach of spotting-and-tilt-spreading (SATS), or a brief plating (<10 s; SP-A). Spore CFU was significantly reduced with SP-B in different organisms (23-40%) over SATS independent of the spore size. Comparing 4-, 7- and 10-day-old B. pumilus spores, the former two displayed significant CFU reduction in SP-B indicating a spore age-related effect. Continuous plating for 2-5 min showed a reduction in spore CFU in all organisms depending on plating duration. CFU reduction effect with SP-B was less manifest on refrigerated plates where no friction was experienced but acute on prewarmed and surface-dried plates. Spreader movement over agar surface subsequent to the exhaustion of free moisture proved highly detrimental to spores. A simulated plating study by plating the spores over a plastic film till drying showed a significant reduction in spore CFU. DAPI staining and glass bead-vortexing studies confirmed spore disruption through physical impaction. Bacterial spores are vulnerable to injury during spread-plating or with other forms of physical impaction with variable effects on different genotypes independent of the spore size but altered by spore age. Implications during spore CFU estimations employing spread-plating and during spore surveillance, and the recommendation of SATS as an easier and safer alternative for spore CFU enumeration.

Simple and rapid method to determine antimycobacterial potency of compounds by using autoluminescent Mycobacterium tuberculosis.

A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (∼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.

Experimental mouse model of tuberculous uveitis

Results The i.v. and i.n. challenged mice showed significant DTH response. The CFU enumeration in eye did not show any bacilli initially by 30th day however tubercle bacilli could be recovered from the eyes of i.v. infected mice by 45 day at the time of death. Histopathology of these eyes showed inflammation with cluster of lymphocytes. The RTPCR for IS6110 gene from the eyes of i.n. infected animals was positive at different doses whereas i.v. challenged mice showed variability. Furthermore, the cytokine analysis in infected tissue supernatants of both eyes and lung showed high Th1 response (IFN-g) in comparison to non-infected group.

Novel non-neuroleptic phenothiazines inhibit Mycobacterium tuberculosis replication

Phenothiazines are a commercially available class of psychotropic drugs known to show antituberculosis activity. At clinically relevant bactericidal doses, however, the psychotropic drugs produce undesirable side effects in addition to their neuroleptic properties. This study aimed to evaluate rationally designed novel phenothiazines as antimycobacterial drug candidates. Remodelling of psychotropic drugs by substitution of characteristic N-alkylamine side chains, important for CNS activity, with N-alkylsulphonates gave novel drug candidates, which were then tested for post-synaptic receptor binding affinity in a radioligand displacement assay. The bactericidal activities were screened using green fluorescent protein (GFP) microplate assays, and the efficacy of intracellular bacillus killing was evaluated by cfu enumeration. Of the four selected phenothiazine derivatives (PTZ3, PTZ4, PTZ31 and PTZ32) tested, PTZ31 displayed marginal serotonergic activity. The remaining three derivatives did not exhibit dopamine or serotonin receptor binding activity. In vitro results showed significant growth inhibition of virulent Mycobacterium tuberculosis with MICs of 12.5-25 mg/L. None of the phenothiazine derivatives displayed cytotoxicity in infected primary bone marrow-derived macrophages. Moreover, the phenothiazines showed significant antimycobacterial activity of between 40% and 60% against intracellular (ex vivo) M. tuberculosis. We demonstrate that structural modification of the phenothiazine core is possible in a manner that does not affect the ability of the phenothiazine derivatives to inhibit M. tuberculosis, but that abolishes undesirable dopamine and serotonin receptor binding.

Antibacterial responses of retinal Müller glia: production of antimicrobial peptides, oxidative burst and phagocytosis

BackgroundWe have previously shown that, in response to microbial infection, activated Müller glia secrete inflammatory cytokines/chemokines and exhibit antimicrobial properties. The aim of this study is to understand the mechanisms and the key components involved in this response.MethodsImmortalized human retinal Müller glia (MIO-M1 cells) were challenged with Staphylococcus (S) aureus, the leading cause of severe intraocular infection followed by RT2 profile PCR array analysis. The expression of human β-defensin 1 (HBD1), 2 (HBD2), 3 (HBD3), hepcidine and cathelicidin LL37 was checked by RT-PCR and quantified by Taqman® qPCR. The expression of AMPs was confirmed at protein level by dot-blot analysis. The production of ROS was measured by dicholoro-dihydro-fluorescein diacetate (DCFH-DA) staining by flow cytometry as well as fluorescence microscopy. The level of nitric oxide (NO) was measured by measuring a stable metabolite, nitrite using the Griess reagent. In vitro killing assay was performed by Live/Dead® BacLight™ staining as well as by dilution plating in suspension and adherent conditions following S. aureus infection. Phagocytosis was measured by CFU enumeration following infection.ResultsPCR array data showed that, in comparison to uninfected control cells, bacterial challenge significantly (> two-fold) induced the expression of 26 genes involved in cytokine/chemokine, antimicrobials, Toll-like receptor, apoptotic, and NF-κB signaling. RT-PCR analysis showed time-dependent increased expression of HBD1, HBD2, HBD3, LL-37, and hepcidin mRNA in bacteria-challenged Müller glia. The expression of these antimicrobial molecules was also increased at the protein level in the culture supernatant, as detected by dot-blot analysis. Additionally, the bacteria-stimulated Müller glia were found to produce reactive oxygen (ROS) and reactive nitrogen (RNS) species. In vitro, killing assays revealed that Müller glia exhibited bactericidal activity against S. aureus in both adherent and suspension cultures. Furthermore, our data demonstrated that Müller glia can phagocytize and kill the bacteria in a time-dependent manner.ConclusionsThese data suggest that retinal Müller glia behave like classical innate immune cells by producing a variety of antimicrobial molecules in response to bacterial challenge, suggesting their pivotal role in retinal innate defense.

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Evaluation of the respiratory route as a viable portal of entry for Salmonella in poultry via intratracheal challenge of Salmonella Enteritidis and Salmonella Typhimurium

Experimental and epidemiological evidence suggests that primary infection of Salmonella is by the oral-fecal route for poultry. However, the airborne transmission of Salmonella and similar enteric zoonotic pathogens has been historically neglected. Increasing evidence of Salmonella bioaerosol generation in production facilities and studies suggesting the vulnerabilities of the avian respiratory architecture together have indicated the possibility of the respiratory system being a potential portal of entry for Salmonella in poultry. Presently, we evaluated this hypothesis through intratracheal (IT) administration of Salmonella Enteritidis and Salmonella Typhimurium, as separate challenges, in a total of 4 independent trials, followed by enumeration of cfu recovery in ceca-cecal tonsils and recovery incidence in liver and spleen. In all trials, both Salmonella Enteritidis and Salmonella Typhimurium, challenged IT colonized cecae to a similar or greater extent than oral administration at identical challenge levels. In most trials, chickens cultured for cfu enumeration from IT-challenged chicks at same dose as orally challenged, resulted in an increase of 1.5 log higher Salmonella Enteritidis from ceca-cecal tonsils and a much lower dose IT of Salmonella Enteritidis could colonize ceca to the same extent than a higher oral challenge. This trend of increased cecal colonization due to IT challenge was observed with all trails involving week-old birds (experiment 2 and 3), which are widely considered to be more difficult to infect via the oral route. Liver-spleen incidence data showed 33% of liver and spleen samples to be positive for Salmonella Enteritidis administered IT (106 cfu/chick), compared with 0% when administered orally (experiment 2, trial 1). Collectively, these data suggest that the respiratory tract may be a largely overlooked portal of entry for Salmonella infections in chickens.

Immunotherapeutic potential of recombinant ESAT-6 protein in mouse model of experimental tuberculosis

Recent understanding of the pathogenesis of tuberculosis allows the possible application of immunotherapy for the treatment of tuberculosis. Therapies that would upregulate the host antimycobacterial immune response and/or attenuate T-cell suppressive and macrophage-deactivating cytokines may prove to be useful in the treatment of tuberculosis. ESAT6, 6-kDa early secreted antigenic target, is a potent protective antigen and is considered as major target for long-lived memory cells. In the present study the immunotherapeutic potential of ESAT-6 has been evaluated in mouse model of experimental tuberculosis. In the present study the ESAT-6 protein was cloned in Escherichia coli using pET23a(+) plasmid and purified by Ni2+-NTA chromatography. Further, the immunotherapeutic potential of the recombinant ESAT-6 (in terms of CFU enumeration in the target organs and histopathological analysis of lungs) was evaluated against experimental tuberculosis. The recombinant ESAT-6 with C-terminal histidine-tag and free N-terminus mimics the natural form of ESAT-6 has been successfully cloned and purified. The recombinant ESAT-6 protein adjuvanted with dimethyl dioctadecylammonium bromide (DDA) moderately reduced the bacterial load in the target organs of infected mice. Further, the formulation (ESAT-6-DDA) was able to act synergistically when given in combination with antituberculosis drugs. This recombinant ESAT-6 showed good immunotherapeutic potential against experimental tuberculosis and can be used as an adjunct to the conventional antituberculosis chemotherapy.

Fitness of acquired drug resistant Mycobacterium tuberculosis isolates from DOTS compliant patients

The increasing levels of multi drug resistant tuberculosis (MDRTB) in endemic regions are a cause of concern. When MDR strains exhibit higher fitness they threaten global tuberculosis control. Evaluation of factors other than the particular non-synonymous substitutions that contribute to the fitness of strains may have an important bearing on understanding the spread of the disease. The aim of the study was to determine the fitness of MDRTB strains from TB endemic region, acquiring drug resistance during the course of treatment. Paired isolates of different lineages (one that was drug resistant and one that was drug susceptible) from 5 patients, which acquired resistance during antibiotic treatment and bearing drug resistance mutations in the rpoβ and katG/inhA regions were mixed axenically. Fitness of Mycobacterium tuberculosis (M.tb) strains was evaluated using the difference in generation times of drug resistant and susceptible population obtained by enumeration of CFU. The drug resistant strains bore no significant fitness cost. The selection of specific allelic combinations in multiple drug resistance governing loci may attribute the enhanced/retained fitness of the drug resistant strains.

Noneluting enzymatic antibiofilm coatings.

We developed a highly efficient, biocompatible surface coating that disperses bacterial biofilms through enzymatic cleavage of the extracellular biofilm matrix. The coating was fabricated by binding the naturally existing enzyme dispersin B (DspB) to surface-attached polymer matrices constructed via a layer-by-layer (LbL) deposition technique. LbL matrices were assembled through electrostatic interactions of poly(allylamine hydrochloride) (PAH) and poly(methacrylic acid) (PMAA), followed by chemical cross-linking with glutaraldehyde and pH-triggered removal of PMAA, producing a stable PAH hydrogel matrix used for DspB loading. The amount of DspB loaded increased linearly with the number of PAH layers in surface hydrogels. DspB was retained within these coatings in the pH range from 4 to 7.5. DspB-loaded coatings inhibited biofilm formation by two clinical strains of Staphylococcus epidermidis. Biofilm inhibition was ≥98% compared to mock-loaded coatings as determined by CFU enumeration. In addition, DspB-loaded coatings did not inhibit attachment or growth of cultured human osteoblast cells. We suggest that the use of DspB-loaded multilayer coatings presents a promising method for creating biocompatible surfaces with high antibiofilm efficiency, especially when combined with conventional antimicrobial treatment of dispersed bacteria.

Evaluation of methods to assess the biofilm-forming ability of Listeria monocytogenes.

The contamination of ready-to-eat products with Listeria monocytogenes has been related to the presence of biofilms in production lines, as biofilms protect cells from chemical sanitizers. The ability of L. monocytogenes to produce biofilms is often evaluated using in vitro methodologies. This work aims to compare the most frequently used methodologies, including high-throughput screening methods based on microplates (crystal violet and the Calgary Biofilm Device) and methods based on CFU enumeration and microscopy after growth on stainless steel. Thirty isolates with diverse origins and genetic characteristics were evaluated. No (or low) correlations between methods were observed. The only significant correlation was found between the methods using stainless steel. No statistically significant correlation (P > 0.05) was detected among genetic lineage, serovar, and biofilm-forming ability. Because results indicate that biofilm formation is influenced by the surface material, the extrapolation of results from high-throughput methods using microplates to more industrially relevant surfaces should be undertaken with caution.

FdhTU-Modulated Formate Dehydrogenase Expression and Electron Donor Availability Enhance Recovery ofCampylobacter jejunifollowing Host Cell Infection

Campylobacter jejuni is a food-borne bacterial pathogen that colonizes the intestinal tract and causes severe gastroenteritis. Interaction with host epithelial cells is thought to enhance severity of disease, and the ability of C. jejuni to modulate its metabolism in different in vivo and environmental niches contributes to its success as a pathogen. A C. jejuni operon comprising two genes that we designated fdhT (CJJ81176_1492) and fdhU (CJJ81176_1493) is conserved in many bacterial species. Deletion of fdhT or fdhU in C. jejuni resulted in apparent defects in adherence and/or invasion of Caco-2 epithelial cells when assessed by CFU enumeration on standard Mueller-Hinton agar. However, fluorescence microscopy indicated that each mutant invaded cells at wild-type levels, instead suggesting roles for FdhTU in either intracellular survival or postinvasion recovery. The loss of fdhU caused reduced mRNA levels of formate dehydrogenase (FDH) genes and a severe defect in FDH activity. Cell infection phenotypes of a mutant deleted for the FdhA subunit of FDH and an ΔfdhU ΔfdhA double mutant were similar to those of a ΔfdhU mutant, which likewise suggested that FdhU and FdhA function in the same pathway. Cell infection assays followed by CFU enumeration on plates supplemented with sodium sulfite abolished the ΔfdhU and ΔfdhA mutant defects and resulted in significantly enhanced recovery of all strains, including wild type, at the invasion and intracellular survival time points. Collectively, our data indicate that FdhTU and FDH are required for optimal recovery following cell infection and suggest that C. jejuni alters its metabolic potential in the intracellular environment.

724 EVALUATION OF INFECTION AND INFLAMMATION IN THE SPINAL CORD-INJURED, NEUROGENIC BLADDER ANIMAL FOLLOWING ESCHERICHIA COLI URINARY TRACT INFECTION

You have accessJournal of UrologyPediatrics: Basic Research1 Apr 2012724 EVALUATION OF INFECTION AND INFLAMMATION IN THE SPINAL CORD-INJURED, NEUROGENIC BLADDER ANIMAL FOLLOWING ESCHERICHIA COLI URINARY TRACT INFECTION Rajeev Chaudhry, Yuping Tang, Zarine Balsara, Sherry Ross, and Patrick Seed Rajeev ChaudhryRajeev Chaudhry Durham, NC More articles by this author , Yuping TangYuping Tang Durham, NC More articles by this author , Zarine BalsaraZarine Balsara Durham, NC More articles by this author , Sherry RossSherry Ross Durham, NC More articles by this author , and Patrick SeedPatrick Seed Durham, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.809AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recurrent urinary tract infections cause significant morbidity in patients with neurogenic bladder (NB). Our lab has established an animal model of experimental Escherichia coli urinary tract infection (UTI) in the spinal cord-injured (SCI) rat. Using this model, we have studied the natural course of acute and chronic UTI in SCI versus spinal cord-intact rats and demonstrated enhanced susceptibility to UTI in SCI rats. We now address the consequences of early antibiotic therapy on UTI in the SCI-NB animal, hypothesizing that, despite abrogation of infection, SCI-NB rats have persistent elevated inflammation of the urinary tract. METHODS Rats underwent T10 spinal cord transection or sham surgery (spinal cord-intact). After a 14 day post-operative recovery, the SCI and sham animals were inoculated with 1.2 x 104 CFU and 1.1 x 107 CFU of the human cystitis E. coli isolate UTI89, respectively. Antibiotic treatment with enrofloxacin was initiated 48 hr post-infection and continued for 5 days. Urine samples were obtained by twice daily crede and plated for enumeration of CFU. Animals were subsequently sacrificed, and bladder and kidneys were harvested and processed for enumeration of CFU, and measurement of IL-6. RESULTS UTI was established in 83% and 100% of SCI and sham rats, respectively. Bacteriuria was cleared from all animals by 24 hr of antibiotic treatment. E. coli was recovered from bladder homogenates from 33% of SCI rats, indicating persistent bacterial reservoirs despite clearance of bacteriuria. No bacteria were isolated from kidney homogenates in any animals. Mean IL-6 levels in organ homogenates from SCI rats and sham rats after 5 days of antibiotics were 7.62 ± 1.71 vs. 4.23 ± 3.51 pg/g of bladder tissue, respectively (p=0.36), and 55.38 ± 10.50 vs. 14.58 ± 3.675 pg/g of kidney tissue, respectively (p=0.04). IL-6 levels were greater in the kidneys vs. bladders in SCI (p=<0.01), but not sham animals (p=0.2). CONCLUSIONS Despite early control and clearance of bacteriuria in experimental E. coli UTI, SCI animals have evidence of elevated IL-6 in kidneys as compared to sham animals. This may suggest persistence of inflammation in the upper urinary tract that may lead to chronic inflammatory changes. Future studies will address the consequences of chronic inflammation and recurrent infections. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e297 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Rajeev Chaudhry Durham, NC More articles by this author Yuping Tang Durham, NC More articles by this author Zarine Balsara Durham, NC More articles by this author Sherry Ross Durham, NC More articles by this author Patrick Seed Durham, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Importance of Micromonospora spp. as Colonizers of Cellulose in Freshwater Lakes as Demonstrated by Quantitative Reverse Transcriptase PCR of 16S rRNA

The relative abundance of micromonosporas in the bacterial communities inhabiting cellulose baits, water columns, and sediments of two freshwater lakes was determined by quantitative PCR (qPCR) of reverse-transcribed 16S rRNA. Micromonospora spp. were shown to be significant members of the active bacterial population colonizing cellulosic substrates in the lake sediment, and their increased prevalence with greater depth was confirmed by enumeration of CFU.

Plasmidic qnrA3 Enhances Escherichia coli Fitness in Absence of Antibiotic Exposure

The widespread presence of plasmid-mediated quinolone resistance determinants, particularly qnr genes, has become a current issue. By protecting DNA-gyrase from quinolones, Qnr proteins confer a low level quinolone resistance that is not sufficient to explain their emergence. Since Qnr proteins were hypothesized to act as DNA-binding protein regulators, qnr genes could have emerged by providing a selective advantage other than antibiotic resistance. We investigated host fitness of Escherichia coli isogenic strains after acquisition of the qnrA3 gene, inserted either alone onto a small plasmid (pBR322), or harbored on a large conjugative native plasmid, pHe96(qnrA3) found in a clinical isolate. The isogenic strains were derived from the susceptible E. coli CFT073, a virulent B2 group strain known to infect bladder and kidneys in a mouse model of pyelonephritis. In vitro experiments included growth analysis by automatic spectrophotometry and flow cytometry, and competitions with CFU enumeration. In vivo experiments included infection with each strain and pairwise competitions in absence of antimicrobial exposure. As controls for our experiments we used mutations known to reduce fitness (rpsL K42N mutation) or to enhance fitness (tetA deletion in pBR322). E. coli CFT073 transformed with pBRAM(PBR322-qnrA3) had significantly higher maximal OD than E. coli CFT073 transformed with pBR322 or pBR322ΔtetA, and in vivo competitions were more often won by the qnrA3 carrying strain (24 victories vs. 9 loss among 42 competitions, p = 0.001). In contrast, when pHe96(qnrA3) was introduced by conjugation in E. coli CFT073, it exerted a fitness cost shown by an impaired growth observed in vitro and in vivo and a majority of lost competitions (33/35, p<0.0001). In conclusion, qnrA3 acquisition enhanced bacterial fitness, which may explain qnr emergence and suggests a regulation role of qnr. However, fitness was reduced when qnrA3 was inserted onto multidrug-resistant plasmids and this can slow down its dissemination without antibiotic exposure.