scholarly journals Development of a Rapid PCR Assay Specific forStaphylococcus saprophyticus and Application to Direct Detection from Urine Samples

2000 ◽  
Vol 38 (9) ◽  
pp. 3280-3284 ◽  
Author(s):  
Francis Martineau ◽  
François J. Picard ◽  
Christian Ménard ◽  
Paul H. Roy ◽  
Marc Ouellette ◽  
...  
Keyword(s):  
Direct Detection ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Urine Samples ◽  
Urine Specimen ◽  
Clinical Microbiology Laboratory ◽  
Specimen Preparation ◽  
Specific Detection ◽  
Pcr Assay ◽  
Tract Infections

Staphylococcus saprophyticus is one of the most frequently encountered microorganisms associated with acute urinary tract infections (UTIs) in young, sexually active female outpatients. Conventional identification methods based on biochemical characteristics can efficiently identify S. saprophyticus, but the rapidities of these methods need to be improved. Rapid and direct identification of this bacterium from urine samples would be useful to improve time required for the diagnosis of S. saprophyticus infections in the clinical microbiology laboratory. We have developed a PCR-based assay for the specific detection ofS. saprophyticus. An arbitrarily primed PCR amplification product of 380 bp specific for S. saprophyticus was sequenced and used to design a set of S. saprophyticus-specific PCR amplification primers. The PCR assay was specific for S. saprophyticus when tested with DNA from 49 gram-positive and 31 gram-negative bacterial species. This assay was also able to amplify efficiently DNA from all 60 strains of S. saprophyticus from various origins tested. This assay was adapted for direct detection from urine samples. The sensitivity levels achieved with urine samples was 19 CFU with 30 cycles of amplification and 0.5 CFU with 40 cycles of amplification. This PCR assay for the specific detection of S. saprophyticus is simple and rapid (approximately 90 min, including the time for urine specimen preparation).

Isolation and Identification of Multidrug-Resistant Isolates of Uropathogenic Klebsiella spp in Dogs

2021 ◽  
Vol 3 (1) ◽  
pp. 6-12
Author(s):  
M Mustapha ◽  
P Goel
Keyword(s):  
Antimicrobial Agents ◽  
Bacterial Species ◽  
Multidrug Resistant ◽  
Urine Samples ◽  
Penicillin G ◽  
Isolation And Identification ◽  
Sensitivity Testing ◽  
Bacterial Organism ◽  
Tract Infections ◽  
Klebsiella Spp

The most widespread ailments in dogs are urinary tract infections (UTIs) caused by bacterial species. It is necessary to recognize the prevailing bacterial pathogens and their susceptibility to antimicrobial agents to effectively treat UTIs. The present study aimed to classify the bacterial organism that causes UTIs in dogs and their patterns of antimicrobial resistance. A total of 141 urine samples were collected from diseased dogs in Veterinary Clinical Complex LUVAS in Hisar, India. Culture, biochemical and sensitivity testing were performed for each of the urine samples based on standard method. Of the total 141 urine samples from dogs, 21 (14.9%) isolates were identified as Klebsiella spp. The isolates were found to be highly resistant to ampicillin (100%), penicillin G (100%), oxytetracycline (100%), enrofloxacin (85.7%), chloramphenicol (80.6%), ceftriaxone (76.2%) and cloxacillin (71.4%), while susceptibility was observed against gentamicin (100%), amikacin (100%) and neomycin (90.5%). In the current study, 19 out of 21 identified isolates were found to be multidrug-resistant. This study indicates that dogs in the study area are found to harbor highly resistant Klebsiella spp. Therefore, when deciding on the antibiotic regimen for UTIs cases, Vets should consider resistance profile of chosen antibacterial agents before usage in order to discourage dissemination of resistant organisms in the study area.

scholarly journals Antimicrobial Evaluation of Bacterial Isolates from Urine Specimen of Patients with Complaints of Urinary Tract Infections in Awka, Nigeria

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Perpetua A. Ekwealor ◽  
Malachy C. Ugwu ◽  
Ifeanyi Ezeobi ◽  
George Amalukwe ◽  
Belinda C. Ugwu ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Antimicrobial Susceptibility ◽  
Bacterial Species ◽  
Resistance Index ◽  
Diffusion Method ◽  
Urine Specimen ◽  
Anambra State ◽  
Tract Infections ◽  
Susceptibility Patterns

Urinary tract infections (UTIs) account for one of the major reasons for most hospital visits and the determination of the antimicrobial susceptibility patterns of uropathogens will help to guide physicians on the best choice of antibiotics to recommend to affected patients. This study is designed to isolate, characterize, and determine the antimicrobial susceptibility patterns of the pathogens associated with UTI in Anambra State Teaching Hospital, Amaku, Anambra State, Nigeria. Clean catch urine samples of inpatient and outpatient cases of UTI were collected and bacteriologically analyzed using standard microbiological procedures. Antibiogram was done by the Kirby-Bauer disc diffusion method. The most prevalent isolates wereS. aureus(28%),E.coli(24.6%), andS. saprophyticus(20%). The antibacterial activities of the tested agents were in the order of Augmentin < Ceftazidime < Cefuroxime < Cefixime < Gentamicin < Ofloxacin < Ciprofloxacin < Nitrofurantoin. It was found that all the organisms were susceptible in varying degrees to Nitrofurantoin, Ciprofloxacin, and Ofloxacin. It was also observed that all the bacterial species exceptStreptococcusspp. have a Multiple Antibiotic Resistance Index (MARI) greater than 0.2. For empiric treatment of UTIs in Awka locality, Nitrofurantoin, Ciprofloxacin, and Ofloxacin are the first line of choice.

scholarly journals Species-Specific and Ubiquitous-DNA-Based Assays for Rapid Identification of Staphylococcus aureus

1998 ◽  
Vol 36 (3) ◽  
pp. 618-623 ◽  
Author(s):  
Francis Martineau ◽  
François J. Picard ◽  
Paul H. Roy ◽  
Marc Ouellette ◽  
Michel G. Bergeron
Keyword(s):  
Staphylococcus Aureus ◽  
Genomic Library ◽  
Antibiotic Resistance Genes ◽  
Pcr Amplification ◽  
Rapid Identification ◽  
Clinical Microbiology Laboratory ◽  
Chromosomal Dna ◽  
Pcr Assay ◽  
Serious Infections ◽  
Species Specific

Staphylococcus aureus is the cause of serious infections in humans, including endocarditis, deep-seated abscesses, and bacteremia, which lead to toxic and septic shock syndromes. Rapid and direct identification of this bacterium specifically and ubiquitously directly from clinical specimens would be useful in improving the diagnosis of S. aureus infections in the clinical microbiology laboratory. A wide variety of kits based on biochemical characteristics efficiently identify S. aureus, but the rapidity and the accuracy of each of these methods combined with testing of clinically relevant antibiotic resistance genes need to be improved. On the basis of hybridization assays with randomly selected clones from an S. aureus genomic library, we have identified a chromosomal DNA fragment which is specific for S. aureus and which detected all 82 S. aureus isolates tested. This 442-bp fragment was sequenced and was used to design a set of PCR amplification primers. The PCR assay was also specific and ubiquitous for the identification from bacterial cultures of 195 clinical strains of S. aureus isolated from a variety of anatomical sites and obtained from hospitals throughout the world. The PCR assay that we have developed is simple and can be performed in about 1 h. This DNA-based test provides a novel diagnostic tool for the diagnosis of S. aureus infections.

scholarly journals An alternative for urine cultures: Direct identification of uropathogens from urine by MALDI-TOF MS

2020 ◽  
Author(s):  
Arzu Akşit İlki ◽  
Sevim Özsoy ◽  
Gulşen Gelmez ◽  
Burak Aksu ◽  
Güner Söyletir
Keyword(s):  
Flow Cytometry ◽  
Antibiotic Susceptibility ◽  
Clinical Microbiology ◽  
Urine Samples ◽  
Clinical Microbiology Laboratory ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Susceptibility Tests ◽  
Tract Infections ◽  
Tof Ms

AbstractUrinary tract infections are one of the most common bacterial infections and rapid diagnosis of the infection is essential for appropriate antibiotic therapy. The goal of our study was to identify urinary pathogens directly by MALDI-TOF MS and to perform antibiotic susceptibility tests in order to shorten the period spent for culturing.Urine samples submitted for culture to the Clinical Microbiology Laboratory were enrolled in this study. Urine samples were screened for leukocyte and bacteria amount by flow cytometry. Samples with bacterial load of 106–107/mL were tested directly by MALDI-TOF MS and antibiotic susceptibility tests (AST) were performed.In total, 538 positive urine samples were evaluated in our study. MALDI-TOF MS identified the microorganism directly from the urine sample in 91.8% of these samples and the concordance rate of conventional identification and direct detection was 95.8% for Gram-negatives at the genus and species level. Escherichia coli (n:401) was the most frequently isolated microorganism, followed by Klebsiella pneumoniae (n:57). AST results were generated for 111 of these urine samples and the concordance was 90% and 87% for E. coli and K. pneumoniae, respectively.Our results showed that screening of urine samples with flow cytometry to detect positive samples and identification of uropathogens directly by MALDI-TOF MS with an accuracy of over 90% can be a suitable method particularly for Gram-negative bacteria in clinical microbiology laboratories.

Group-specific differentiation of Rhizobium from clover species by PCR amplification of 23S rDNA sequences

1998 ◽  
Vol 44 (11) ◽  
pp. 1102-1105
Author(s):  
Mesfin Tesfaye ◽  
F Brian Holl
Keyword(s):  
Root Nodule ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Dna Amplification ◽  
Specific Detection ◽  
Rdna Sequences ◽  
Rhizobium Spp ◽  
Polymerase Chain ◽  
Inoculation Group ◽  
Identification Tool

Two 20-bp primers that provide group-specific detection of Rhizobium spp. by polymerase chain reaction (PCR) have been used to differentiate strains that belong to different effectiveness groups within the Rhizobium-Trifolium cross-inoculation group. The target for DNA amplification was a 370-bp fragment of the 23S rDNA region. Analysis of additional root-nodule forming, as well as root-associated bacterial species by PCR-primer assay revealed that variability within this 20-bp segment of the 23S rDNA region may be widespread and provide an effective identification tool. Our data suggest that strains of Rhizobium isolated from the perennial clover Trifolium semipilosum may be phylogenetically more closely related to Rhizobium etli.Key words: Rhizobium, Trifolium, detection, PCR, rDNA.

scholarly journals Does cranberry have a role in catheter-associated urinary tract infections?

2017 ◽  
Vol 11 (11) ◽  
pp. E421-4 ◽  
Author(s):  
Dominique Thomas ◽  
Matthew Rutman ◽  
Kimberly Cooper ◽  
Andrew Abrams ◽  
Julia Finkelstein ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Bacterial Species ◽  
Signs And Symptoms ◽  
Urine Specimen ◽  
Indwelling Catheters ◽  
Resistance Patterns ◽  
Catheter Urine ◽  
Tract Infections

Introduction: Catheter-associated urinary tract infections (CA-UTIs) are a prevalent and costly condition, with very few therapeutic options. We sought to evaluate the efficacy of an oral cranberry supplement on CA-UTIs over a six-month period. Methods: Subjects with long-term indwelling catheters and recurrent symptomatic CA-UTIs were enrolled to take a once-daily oral cranberry supplement with 36 mg of the active ingredient proanthocyanidin (PACs). Primary outcome was reducing the number of symptomatic CA-UTIs. This was defined by ≥103 (cfu)/mL of ≥1 bacterial species in a single catheter urine specimen and signs and symptoms compatible with CA-UTI. Secondary outcomes included bacterial counts and resistance patterns to antibiotics.Results: Thirty-four patients were enrolled in the trial; 22 patients (mean age 77.22 years, 77.27% were men) completed the study. Cranberry was effective in reducing the number of symptomatic CA-UTIs in all patients (n=22). Resistance to antibiotics was reduced by 28%. Furthermore, colony counts were reduced by 58.65%. No subjects had adverse events while taking cranberry.Conclusions: The cranberry supplement reduced the number of symptomatic CA-UTIs, antibiotic resistances, and major causative organisms in this cohort. Larger, placebo-controlled studies are needed to further define the role of cranberry in CA-UTIs.

scholarly journals Characterization of vaginal microbial communities in adult healthy women using cultivation-independent methods

Microbiology ◽  
2004 ◽  
Vol 150 (8) ◽  
pp. 2565-2573 ◽  
Author(s):  
Xia Zhou ◽  
Stephen J. Bent ◽  
Maria G. Schneider ◽  
Catherine C. Davis ◽  
Mohammed R. Islam ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Dna Sequences ◽  
Phylogenetic Analyses ◽  
Bacterial Species ◽  
Pcr Amplification ◽  
Single Species ◽  
Rrna Gene ◽  
Caucasian Women ◽  
Tract Infections

The normal microbial flora of the vagina plays an important role in preventing genital and urinary tract infections in women. Thus an accurate understanding of the composition and ecology of the ecosystem is important to understanding the aetiology of these diseases. Common wisdom is that lactobacilli dominate the normal vaginal microflora of post-pubertal women. However, this conclusion is based on methods that require cultivation of microbial populations; an approach that is known to yield a biased and incomplete assessment of microbial community structure. In this study cultivation-independent methods were used to analyse samples collected from the mid-vagina of five normal healthy Caucasian women between the ages of 28 and 44. Total microbial community DNA was isolated following resuspension of microbial cells from vaginal swabs. To identify the constituent numerically dominant populations in each community 16S rRNA gene libraries were prepared following PCR amplification using the 8f and 926r primers. From each library, the DNA sequences of approximately 200 16S rRNA clones were determined and subjected to phylogenetic analyses. The diversity and kinds of organisms that comprise the vaginal microbial community varied among women. Species of Lactobacillus appeared to dominate the communities in four of the five women. However, the community of one woman was dominated by Atopobium sp., whereas a second woman had appreciable numbers of Megasphaera sp., Atopobium sp. and Leptotrichia sp., none of which have previously been shown to be common members of the vaginal ecosystem. Of the women whose communities were dominated by lactobacilli, there were two distinct clusters, each of which consisted of a single species. One class consisted of two women with genetically divergent clones that were related to Lactobacillus crispatus, whereas the second group of two women had clones of Lactobacillus iners that were highly related to a single phylotype. These surprising results suggest that culture-independent methods can provide new insights into the diversity of bacterial species found in the human vagina, and this information could prove to be pivotal in understanding risk factors for various infectious diseases.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

2007 ◽  
Vol 70 (2) ◽  
pp. 341-347 ◽  
Author(s):  
A. GONZÁLEZ ◽  
S. BOTELLA ◽  
R. M. MONTES ◽  
Y. MORENO ◽  
M. A. FERRÚS
Keyword(s):  
Multiplex Pcr ◽  
Water Samples ◽  
Direct Detection ◽  
Pcr Amplification ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Culture Methods ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Wastewater Samples

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

Evaluation of a SYBR Green Real-Time PCR Assay for Specific Detection of Pasteurella multocida in Culture and Tissue Samples from Sheep

2020 ◽  
Author(s):  
Jyoti Kumar ◽  
G. G. Sonawane ◽  
Fateh Singh ◽  
S. Jegaveera Pandian ◽  
Rajiv Kumar
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pasteurella Multocida ◽  
Bacterial Species ◽  
Sybr Green ◽  
Economic Losses ◽  
Conventional Pcr ◽  
Specific Detection ◽  
Pcr Assay ◽  
Tissue Samples

Pasteurella multocida is one of the bacterial species involved in cases of ovine respiratory complex that has been implicated to cause significant economic losses in sheep production system worldwide. The present study was undertaken with the aim of evaluating a SYBR Green dye based real time PCR assay targeting KMT1 gene for the detection of P. multocida. The analytical specificity and sensitivity of the PCR primers were evaluated. The test showed ten-fold more sensitivity than conventional PCR and detected down to 275.5 fg/ µl of genomic DNA concentration, equivalent to 100 copies of KMT1 gene of P. multocida. The real-time PCR was found to be specific for KMT1 gene of P. multocida, as no cross reactivity was detected with a variety of known bacterial isolates. A total of 52 ovine lung tissue samples were screened for P. multocida, which showed improved level of detection as compared to conventional PCR. It is concluded that, this assay may be used as a valuable diagnostic tool for the rapid and specific detection of P. multocida. By virtue of its high throughput format and its ability to accurately identify as well as quantify the bacterial DNA, the method may be useful in large scale epidemiological studies and clarification of pathogenesis.

scholarly journals Using PCR technique for diagnosis of bacterium Escherichia coli O157:H7 isolated from urine samples of humans and sheep

2014 ◽  
Vol 38 (2) ◽  
pp. 17-21
Author(s):  
Al-wgaa A. A.
Keyword(s):  
Young Children ◽  
Urine Samples ◽  
Escherichia Coli O157 ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
E Coli ◽  
Healthy Sheep ◽  
Tract Infections ◽  
Sheep Urine ◽  
Apparently Healthy

     The aims of the current study were to determine the percentages of E.coli O157:H7 in the urinary tract infections (UTIs) of humans and in the urine of apparently healthy sheep, in addition to determine the genotype of the isolates by PCR assay. Two hundred and twenty eight urine samples were collected from young children and adult patients both sexes suffering from UTIs during a period December 2012 to the end of March 2013. And randomly collected 75 urine sample from apparent healthy sheep both sexes that were slaughtered in AL Shoela, AL Rahmanea Slaughterhouse and College of Veterinary Medicine field in Baghdad the end of February to half of April 2013.   All urine samples were incubated aerobically at 37°C for 24-48 hrs on blood agar, MacConkey agar as well as special media  and the isolates were identified  by  biochemical tests, then the isolates were confirmed diagnosed  by PCR assay. The  results showed that  humans urine samples ,8 samples were  E. coli O157:H7 positive  isolates(3.50%) ,young children expressed (4) isolates of E.coli O157:H7 as compare with  those   in adult (4 isolates)  and the percentage of this isolates in females were (2.6%) as compare with those  in males (0.87%). Also the current study demonstrated that all  isolates culturing positive  serotype of E.coliO 157:H7 were positive by PCR assay .The genes of  eaeA ,hly and Stx2 were recorded in 7,8 and  2 serotype of E.coliO157:H7 respectively. The result revealed that 21(28%) out 75 sheep urine samples were E.coli positive isolates, 13 out 75 samples were E.coli O157:H7 positive isolates. The percentage of isolates from rams urine samples was (14.66%) as it compare to those samples of ewes (2.66%).The result also was recorded that 10,13,12,4 and 3 of bacterial isolates of sheep urine samples  were carried eaeA, hlyA, hlyA plasmid,Stx1 and  Stx2 genes respectively. In conclusion the healthy sheep in Baghdad city,  harbor  shiga -toxin producing  E.coliO157:H7 in their  urinary tracts and these organism induced UTIs  associated with  renal failure in the humans and carried the same virulent genes that  reported in the sheep isolates.

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