scholarly journals A Novel Propidium Monoazide-Based PCR Assay Can Measure Viable Uropathogenic E. coli in Vitro and in Vivo

2021 ◽  
Author(s):  
Albert Lee ◽  
Olivia Lamanna ◽  
Kenji Ishida ◽  
Elaise Hill ◽  
Michael H Hsieh
Keyword(s):  
In Vivo Studies ◽  
Propidium Monoazide ◽  
Urine Sediment ◽  
E Coli ◽  
Persistent Symptoms ◽  
Specific Pcr ◽  
Viable Bacteria ◽  
Live Bacteria ◽  
Buffer Control

Background: Polymerase chain reaction (PCR) is an important means by which to study the urine microbiome and is emerging as possible alternative to urine cultures to identify pathogens that cause urinary tract infection (UTI). However, PCR is limited by its inability to differentiate DNA originating from viable, metabolically active versus non-viable, inactive bacteria. This drawback has led to concerns that urobiome studies and PCR-based diagnosis of UTI are confounded by the presence of relic DNA from non-viable bacteria in urine. Propidium monoazide(PMA) dye can penetrate cells with compromised cell membranes and covalently bind to DNA, rendering it inaccessible to amplification by PCR. Although PMA has been shown to differentiate between non-viable and viable bacteria in various settings, its effectiveness in urine has not been previously studied. We sought to investigate the ability of PMA to differentiate between viable and non-viable bacteria in urine. Methods: Varying amounts of viable or non-viable uropathogenic E. coli(UTI89) or buffer control were titrated with mouse urine. The samples were centrifuged to collect urine sediment or not centrifuged. Urine samples were incubated with PMA and DNA cross-linked using blue LED light. DNA was isolated and uidA gene-specific PCR was performed. For in vivo studies, mice were inoculated with UTI89, followed by ciprofloxacin treatment or no treatment. After the completion of ciprofloxacin treatment, an aliquot of urine was plated on non-selective LB agar and another aliquot was treated with PMA and subjected to uidA-specific PCR. Results: PMAs efficiency in excluding DNA signal from non-viable bacteria was significantly higher in bacterial samples in phosphate-buffered saline (PBS, dCT=13.69) versus bacterial samples in unspun urine (dCT=1.58). This discrepancy was diminished by spinning down urine-based bacterial samples to collect sediment and resuspending it in PBS prior to PMA treatment. In 3 of 5 replicate groups of UTI89-infected mice, no bacteria grew in culture; however, there was PCR amplification of E. coli after PMA treatment in 2 of those groups. Conclusion: We have successfully developed PMA-based PCR methods for amplifying DNA from live bacteria in urine. Our results suggest that non-PMA bound DNA from live bacteria can be present in urine, even after antibiotic treatment. This indicates that viable but non-culturable E. coli can be present following treatment of UTI, and may explain why some patients have persistent symptoms but negative urine cultures following UTI treatment.

scholarly journals Effects of Bacterial CLPB Protein Fragments on Food Intake and PYY Secretion

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2223
Author(s):  
Manon Dominique ◽  
Nicolas Lucas ◽  
Romain Legrand ◽  
Illona-Marie Bouleté ◽  
Christine Bôle-Feysot ◽  
...  
Keyword(s):  
Food Intake ◽  
Peptide Yy ◽  
Molecular Mimicry ◽  
Potential Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
E Coli ◽  
In Vivo Effects

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague–Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

scholarly journals Once-daily gentamicin therapy for experimental Escherichia coli meningitis.

1997 ◽  
Vol 41 (1) ◽  
pp. 49-53 ◽  
Author(s):  
A Ahmed ◽  
M M París ◽  
M Trujillo ◽  
S M Hickey ◽  
L Wubbel ◽  
...  
Keyword(s):  
In Vivo Studies ◽  
Bacterial Killing ◽  
Once Daily ◽  
E Coli ◽  
Aminoglycoside Therapy ◽  
Gentamicin Concentration ◽  
Gentamicin Therapy ◽  
The Impact

In vitro and in vivo studies have demonstrated that the bacteriologic efficacy of once-daily aminoglycoside therapy is equivalent to that achieved with conventional multiple daily dosing. The impact of once-daily dosing for meningitis has not been studied. Using the well-characterized rabbit meningitis model, we compared two regimens of the same daily dosage of gentamicin given either once or in three divided doses for 24 or 72 h. The initial 1 h mean cerebrospinal fluid (CSF) gentamicin concentration for animals receiving a single dose (2.9 +/- 1.7 micrograms/ml) was threefold higher than that for the animals receiving multiple doses. The rate of bacterial killing in the first 8 h of treatment was significantly greater for the animals with higher concentrations in their CSF (-0.21 +/- 0.19 versus -0.03 +/- 0.22 log10 CFU/ml/h), suggesting concentration-dependent killing. By 24h, the mean reduction in bacterial titers was similar for the two regimens. In animals treated for 72 h, no differences in bactericidal activity was noted for 24, 48, or 72 h. Gentamicin at two different dosages was administered intracisternally to a separate set of animals to achieve considerably higher CSF gentamicin concentrations. In these animals, the rate of bacterial clearance in the first 8 h (0.52 +/- 0.15 and 0.58 +/- 0.15 log10 CFU/ml/h for the lower and higher dosages, respectively) was significantly greater than that in animals treated intravenously. In conclusion, there is evidence of concentration-dependent killing with gentamicin early in treatment for experimental E. coli meningitis, and once-daily dosing therapy appears to be at least as effective as multiple-dose therapy in reducing bacterial counts in CSF.

scholarly journals Comparison of Escherichia coli Strains Recovered from Human Cystitis and Pyelonephritis Infections in Transurethrally Challenged Mice

1998 ◽  
Vol 66 (7) ◽  
pp. 3059-3065 ◽  
Author(s):  
David E. Johnson ◽  
C. Virginia Lockatell ◽  
Robert G. Russell ◽  
J. Richard Hebel ◽  
Michael D. Island ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Virulence Factors ◽  
Bacterial Infections ◽  
Clinical Symptoms ◽  
Epidemiological Studies ◽  
In Vivo Studies ◽  
E Coli ◽  
Tract Infections

ABSTRACT Urinary tract infection, most frequently caused byEscherichia coli, is one of the most common bacterial infections in humans. A vast amount of literature regarding the mechanisms through which E. coli induces pyelonephritis has accumulated. Although cystitis accounts for 95% of visits to physicians for symptoms of urinary tract infections, few in vivo studies have investigated possible differences between E. coli recovered from patients with clinical symptoms of cystitis and that from patients with symptoms of pyelonephritis. Epidemiological studies indicate that cystitis-associated strains appear to differ from pyelonephritis-associated strains in elaboration of some putative virulence factors. With transurethrally challenged mice we studied possible differences using three each of the most virulent pyelonephritis and cystitis E. coli strains in our collection. The results indicate that cystitis strains colonize the bladder more rapidly than do pyelonephritis strains, while the rates of kidney colonization are similar. Cystitis strains colonize the bladder in higher numbers, induce more pronounced histologic changes in the bladder, and are more rapidly eliminated from the mouse urinary tract than pyelonephritis strains. These results provide evidence that cystitis strains differ from pyelonephritis strains in this model, that this model is useful for the study of the uropathogenicity of cystitis strains, and that it would be unwise to use pyelonephritis strains to study putative virulence factors important in the development of cystitis.

scholarly journals Smart Sensing Fabrics for Live Bacteria Detection

Proceedings ◽  
2018 ◽  
Vol 2 (13) ◽  
pp. 916
Author(s):  
Amparo Ferrer-Vilanova ◽  
Kristina Ivanova ◽  
María Díaz-González ◽  
Yasmine Alonso ◽  
Gonzalo Guirado ◽  
...  
Keyword(s):  
Colour Change ◽  
Cotton Fabrics ◽  
The Self ◽  
Bacterial Metabolism ◽  
Bacteria Detection ◽  
Antibacterial Compounds ◽  
E Coli ◽  
Smart Textile ◽  
Viable Bacteria ◽  
Live Bacteria

A smart textile for live bacteria detection of antimicrobial hospital tissues is here proposed. The capacity to detect viable bacteria is based on the use of Prussian Blue (PB) as electrochromic compound, with a clear reversible change of colour from PB to Prussian White (PW) after reduction from a bacterial metabolism process. PB nanoparticles are incorporated to polyester cotton fabrics by ultrasonic deposition. After performing different tests with bacterial samples of E. coli and S. aureus, a full colour change of the textiles was observed. These smart textiles will allow to determine the self-life of the antibacterial compounds as well to improve the control of hospital infections.

scholarly journals In vitro antibiotic sensitivity and therapeutic efficacy of experimental salmonellosis, colibacillosis and pasteurellosis in broiler chickens

1970 ◽  
Vol 2 (2) ◽  
pp. 99-102 ◽  
Author(s):  
MA Rahman ◽  
MA Samad ◽  
MB Rahman ◽  
SML Kabir
Keyword(s):  
Broiler Chickens ◽  
Pasteurella Multocida ◽  
Antibiotic Sensitivity ◽  
Morbidity And Mortality ◽  
In Vivo Studies ◽  
Penicillin G ◽  
E Coli ◽  
Therapeutic Trials

Avian salmonellosis (AS), avian colibacillosis (AC) and avian pasteurellosis (AP) have been recognized as important bacterial diseases in poultry associated with morbidity and mortality in Bangladesh. The causative agents of these three diseases were isolated (5 isolates / disease) from dead chickens submitted for diagnosis at the BRAC Poultry Disease Diagnostic Centre, Gazipur during the period from January to December 2002. Five isolates of each of the Salmonella pullorum, Escherichia coli and Pasteurella multocida were evaluated against eight antibiotic containing disc which included ciprofloxacin, gentamicin, ampicillin, chloramphenicol, erythromycin, tetracycline, cephradine and penicillin G. Erythromycin in S. pullorum and Ciprofloxacin both in the E. coli and P. multocida were found highest sensitive, gentamicin, chloramphenicol, cephradine were found moderately sensitive to S. pullorum, gentamicin, tetracycline, erythromycin and ampicillin were found moderately sensitive to E. coli, and gentamicin ampicillin, cephradine and penicillin G were moderately sensitive to P. multocida. Therapeutic trials against experimentally produced S. pullorum, E. coli and P. multocida infection in three groups of broiler chickens showed that cephradine against S. pullorum and ciprofloxacin against both in E. coli and P. multocida were found highly effective both in vitro and in vivo studies, therefore, cephradine against salmonellosis and ciprofloxacin against colibacillosis and pasteurellosis are effective drugs of choice which could be used to control morbidity and mortality in poultry caused by these diseases.Key words: antibiotic sensitivity; salmonellosis; colibacillosis; pasteurellosis, broiler chickensdoi: 10.3329/bjvm.v2i2.2538Bangl. J. Vet. Med. (2004). 2 (2): 99-102

scholarly journals Dra/AfaE Adhesin of Uropathogenic Dr/Afa+Escherichia coli Mediates Mortality in Pregnant Rats

2005 ◽  
Vol 73 (11) ◽  
pp. 7597-7601 ◽  
Author(s):  
K. Wroblewska-Seniuk ◽  
R. Selvarangan ◽  
A. Hart ◽  
R. Pladzyk ◽  
P. Goluszko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Maternal Mortality ◽  
Double Mutant ◽  
Lethal Effect ◽  
In Vivo Studies ◽  
Sprague Dawley ◽  
Pregnant Rats ◽  
E Coli

ABSTRACT Escherichia coli bearing adhesins of the Dr/Afa family frequently causes urogenital infections during pregnancy in humans and has been associated with mortality in pregnant rats. Two components of the adhesin, Dra/AfaE and Dra/AfaD, considered virulence factors, are responsible for bacterial binding and internalization. We hypothesize that gestational mortality caused by Dr/Afa+ E. coli is mediated by one of these two proteins, Dra/AfaE or Dra/AfaD. In this study, using afaE and/or afaD mutants, we investigated the role of the afaE and afaD genes in the mortality of pregnant rats from intrauterine infection. Sprague-Dawley rats, on the 17th day of pregnancy, were infected with the E. coli afaE + afaD and afaE afaD + mutants. The clinical E. coli strain (afaE + afaD +) and the afaE afaD double mutant were used as positive and negative controls, respectively. The mortality rate was evaluated 24 h after infection. The highest maternal mortality was observed in the group infected with the afaE + afaD + strain, followed by the group infected with the afaE + afaD strain. The mortality was dose dependent. The afaE afaD double mutant did not cause maternal mortality, even with the highest infection dose. The in vivo studies corresponded with the invasion assay, where the afaE + strains were the most invasive (afaE + afaD strain > afaE + afaD + strain), while the afaE mutant strains (afaE afaD + and afaE afaD strains) seemed to be noninvasive. This study shows for the first time that the afaE gene coding for the AfaE subunit of Dr/Afa adhesin is involved in the lethal outcome of gestational infection in rats. This lethal effect associated with AfaE correlates with the invasiveness of afaE + E. coli strains in vitro.

scholarly journals Influence of the Sodium Salt of 3α,7α-Dihydroxy-12-Oxo-5β-Cholanate on Antimicrobial Activity of Ampicillin In Vitro

2018 ◽  
Vol 44 (1) ◽  
pp. 6
Author(s):  
Ljiljana Suvajdžić ◽  
Slobodan Gigov ◽  
Aleksandar Rašković ◽  
Srđan Stojanović ◽  
Maja Bekut ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Sodium Salt ◽  
Antimicrobial Effect ◽  
In Vivo Studies ◽  
Bacterial Suspension ◽  
Commercial Preparation ◽  
E Coli ◽  
Colony Forming Units

Background: Multiple resistances to antibiotics are an emergent problem worldwide. Scientists intensively search for new substances with the antimicrobial potential or the mode to restore the activity of old-generation antibiotics. Ampicillin is the antibiotic with the expanded range of antimicrobial activity, but its use has decreased due to the poor absorption and highly developed resistance. In vivo studies showed that ampicillin has better absorption and bioavailability if combined with bile acid salts. The aim of this study was to examine antimicrobial effects of ampicillin alone and its combination with semisynthetic monoketocholic acid salt (MKH) in vitro.Materials, Methods & Results: In this study, commercial preparation of ampicillin and sodium salt of 3α,7α-dihydroxy-12oxo-5β-cholanate were used. Their effects were evaluated on Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium), obtained from urine specimens of dogs with clinically manifested cystitis. The first two investigated strains were ampicillin-sensitive, while E. faecium was resistant to ampicillin. Modified macrodilution method according to Clinical and Laboratory Standards Institute Guidelines (M7-A8) was performed. Bacterial suspension equivalent to 0.5 McFarland was prepared in saline, compared to the standard (Biomerieux) ad oculi. The density was checked spectrophotometrically at a wavelength of 625 nm and adjusted if necessary to the desired absorbance from 0.08 to 0.1. The resultant suspension was diluted 1:100 and inoculated in test tubes. Number of bacteria was counted on Petri plates using dilutions from 10-3 to 10-7 in order to obtain valid and countable plates. One hundred microliters of appropriate dilutions were aseptically plated in triplicate onto nutrient agar. Plates were incubated on 37°C for 72 h, under aerobic conditions. The number of colony forming units (CFU) was determined by direct counting. As a valid for enumeration, we took plates with 30 to 300 CFU. Percentage of killed bacteria for ampicillin was from 69.33-95.19% for E. coli, 87.1296.92% for E. faecalis and 7.20-33.30% for E. faecium. Ampicillin applied in the combination with MKH killed 99.99% to 100% of E. coli, 94.59% to 99.91% of E. faecalis and 31.73% to 64.76% of E. faecium. Mean percentage of killed bacteria for ampicillin was 81.93% for E. coli, 91.64% for E. faecalis, and 18.13% for E. faecium, while in combination with MKH percentage was 99.96% for E. coli, 98.23% for E. faecalis and 47.54% for E. faecium.Discussion: Results are presented as pharmacological minimal inhibitory concentration (MIC) values. Ampicillin was applied at the concentration higher than the therapeutic one, which could explain high MIC values for E. coli and E. faecalis. The combination of ampicillin with MKH showed the best improvement of antimicrobial effect on E. faecium (Δ = 29.41%), isolate that was resistant to ampicillin when applied alone. In all the investigated isolates, the combinations with MKH were more effective than ampicillin administered alone. It seems that MKH demonstrates a synergistic antimicrobial activity with ampicillin in vitro, which considerably decreases MIC values for all investigated isolates. These results implicate that MKH could restore the previous activity of ampicillin against some bacteria, which could be a significant benefit for clinical practice.

scholarly journals The primary step of biotin synthesis in mycobacteria

2020 ◽  
Vol 117 (38) ◽  
pp. 23794-23801
Author(s):  
Zhe Hu ◽  
John E. Cronan
Keyword(s):  
Mycobacterium Smegmatis ◽  
Deletion Mutant ◽  
Essential Role ◽  
In Vivo Studies ◽  
Pimelic Acid ◽  
Chronic Infections ◽  
Acid Moiety ◽  
E Coli

Biotin plays an essential role in growth of mycobacteria. Synthesis of the cofactor is essential forMycobacterium tuberculosisto establish and maintain chronic infections in a murine model of tuberculosis. Although the late steps of mycobacterial biotin synthesis, assembly of the heterocyclic rings, are thought to follow the canonical pathway, the mechanism of synthesis of the pimelic acid moiety that contributes most of the biotin carbon atoms is unknown. We report that theMycobacterium smegmatisgene annotated as encoding Tam, anO-methyltransferase that monomethylates and detoxifiestrans-aconitate, instead encodes a protein having the activity of BioC, anO-methyltransferase that methylates the free carboxyl of malonyl-ACP. TheM. smegmatisTam functionally replacedEscherichia coliBioC both in vivo and in vitro. Moreover, deletion of theM. smegmatis tamgene resulted in biotin auxotrophy, and addition of biotin toM. smegmatiscultures repressedtamgene transcription. Although its pathogenicity precluded in vivo studies, theM. tuberculosisTam also replacedE. coliBioC both in vivo and in vitro and complemented biotin-independent growth of theM. smegmatis tamdeletion mutant strain. Based on these data, we propose that the highly conserved mycobacterial tamgenes be renamedbioC.M. tuberculosisBioC presents a target for antituberculosis drugs which thus far have been directed at late reactions in the pathway with some success.

scholarly journals EspJ Is a Prophage-Carried Type III Effector Protein of Attaching and Effacing Pathogens That Modulates Infection Dynamics

2005 ◽  
Vol 73 (2) ◽  
pp. 679-686 ◽  
Author(s):  
Sivan Dahan ◽  
Siouxsie Wiles ◽  
Roberto M. La Ragione ◽  
Angus Best ◽  
Martin J. Woodward ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ex Vivo ◽  
Pathogen Transmission ◽  
Effector Proteins ◽  
In Vivo Studies ◽  
E Coli ◽  
Type Iii ◽  
Infection Dynamics ◽  
Enterocyte Effacement

ABSTRACT Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5′ end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission.

Sign in / Sign up

Export Citation Format

Share Document