Selective Broth Culture Research Articles

Selective culture enrichment and sequencing of feces to enhance detection of antimicrobial resistance genes in third-generation cephalosporin resistant Enterobacteriaceae.

Metagenomic sequencing of fecal DNA can usefully characterise an individual's intestinal resistome but is limited by its inability to detect important pathogens that may be present at low abundance, such as carbapenemase or extended-spectrum beta-lactamase producing Enterobacteriaceae. Here we aimed to develop a hybrid protocol to improve detection of resistance genes in Enterobacteriaceae by using a short period of culture enrichment prior to sequencing of DNA extracted directly from the enriched sample. Volunteer feces were spiked with carbapenemase-producing Enterobacteriaceae and incubated in selective broth culture for 6 hours before sequencing. Different DNA extraction methods were compared, including a plasmid extraction protocol to increase the detection of plasmid-associated resistance genes. Although enrichment prior to sequencing increased the detection of carbapenemase genes, the differing growth characteristics of the spike organisms precluded accurate quantification of their concentration prior to culture. Plasmid extraction increased detection of resistance genes present on plasmids, but the effects were heterogeneous and dependent on plasmid size. Our results demonstrate methods of improving the limit of detection of selected resistance mechanisms in a fecal resistome assay, but they also highlight the difficulties in using these techniques for accurate quantification and should inform future efforts to achieve this goal.

Mathematical Modelling of Beauveria Bassiana

The isolate of white muscardine, Beauveria bassiana was isolated from soil using SDAY media. The experiment was studied under submerged culture in selective broth at varied interval of days, in view of dry weight of hyphal biomass. In this presentation we want to introduce a mathematical growth model of Beauveria bassiana and the perspective of stochastic analysis of this fungus and compare with experimental data.

Detection of Listeria monocytogenes from selective enrichment broth using MALDI–TOF Mass Spectrometry

Conventional methods used for primary detection of Listeria monocytogenes from foods and subsequent confirmation of presumptive positive samples involve prolonged incubation and biochemical testing which generally require four to five days to obtain a result. In the current study, a simple and rapid proteomics-based MALDI-TOF MS approach was developed to detect L. monocytogenes directly from selective enrichment broths. Milk samples spiked with single species and multiple species cultures were incubated in a selective enrichment broth for 24h, followed by an additional 6h secondary enrichment. As few as 1 colony-forming unit (cfu) of L. monocytogenes per mL of initial selective broth culture could be detected within 30h. On applying the same approach to solid foods previously implicated in listeriosis, namely chicken pâté, cantaloupe and Camembert cheese, detection was achieved within the same time interval at inoculation levels of 10cfu/mL. Unlike the routine application of MALDI-TOF MS for identification of bacteria from solid media, this study proposes a cost-effective and time-saving detection scheme for direct identification of L. monocytogenes from broth cultures.This article is part of a Special Issue entitled: Trends in Microbial Proteomics. Globally, foodborne diseases are major causes of illness and fatalities in humans. Hence, there is a continual need for reliable and rapid means for pathogen detection from food samples. Recent applications of MALDI-TOF MS for diagnostic microbiology focused on detection of microbes from clinical specimens. However, the current study has emphasized its use as a tool for detecting the major foodborne pathogen, Listeria monocytogenes, directly from selective enrichment broths. This proof-of-concept study proposes a detection scheme that is more rapid and simple compared to conventional methods of Listeria detection. Very low levels of the pathogen could be identified from different food samples post-enrichment in selective enrichment broths. Use of this scheme will facilitate rapid and cost-effective testing for this important foodborne pathogen.

Rapid detection of Salmonella enterica in food of animal origins collected from Riyadh, King Saudi Arabia

The present study is aimed to investigate Salmonella species in food of animal origin collected from Riyadh, King Saudi Arabia (KSA) using conventional methods and polymerase chain reaction (PCR), targeting fimA gene specific for members of genus Salmonella. Salmonellaisolation revealed 20 Salmonella serovars (8%) out of 250 examined samples. Nine strains (6, 92%) were recovered from 130 examined minced meats and 5 Salmonella strains (8.33%) were recovered from 60 local frozen chickens. Moreover, 6 Salmonella strains (10%) were isolated from 60 examined local chicken cuts. PCR using selective broth culture, Rappaport-Vassiliadis (RV) was used for the detection of different Salmonella species, targeting the fimAgene. All samples revealed positive results with bacteriological examination were positive by PCR-RV, and amplification of 120 bp fragments specific for fimA gene were observed, in addition, to 4 samples (1.6%) previously identified as negative samples with bacteriological examination were positive with PCR using the two primer pairs. The results revealed that the PCR-RV using primers specific for fimA gene could detect more positive samples ofSalmonella species than conventional methods for rapid detection of food borne pathogens. Key words: Salmonella species, fimA gene, polymerase chain reaction- Rappaport- Vassiliadis (PCR-RV), frozen chickens, minced meats.

Detection of Group D Salmonellae Including Salmonella Enteritidis in Eggs by Polymyxin-Based Enzyme-Linked Immunosorbent Assay

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9–specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

Carriage of group B streptococcus in pregnant women from Oxford, UK

Objective: To investigate asymptomatic vagino-rectal carriage of group B streptococcus (GBS) in pregnant women. Methods: Women in the final trimester of pregnancy were recruited. A single vagino-rectal swab was taken,...

New DNA-based PCR approaches for rapid real-time detection and prevention of group B streptococcal infections in newborns and pregnant women.

Group B streptococci (GBS) are an important cause of neonatal sepsis and meningitis, and maternal infection. Although the pathogenesis of GBS infection is not well understood, several virulence factors have been identified. Two prevention strategies have been proposed: chemoprophylaxis and immunoprophylaxis. Implementation of selective intrapartum chemoprophylaxis on the basis of either screening or risk assessment has led to a substantial decrease in the morbidity and mortality of GBS disease in both mothers and infants. Penicillin remains the antibiotic of choice with no reported resistant GBS so far, whereas resistance of 10-20% of GBS to erythromycin and clindamycin has been reported in North America. Chemoprophylaxis based on screening requires optimal detection methods for GBS, which involve selective broth culture of combined vaginal and anal samples. Other conventional methods are useful for rapid identification of heavily colonised women, but are unreliable for the detection of light GBS colonisation because of poor sensitivity. GBS-specific polymerase chain reaction (PCR) assays using real-time PCR coupled with fluorescence-labelling technology offer powerful tools for sensitive and specific, yet rapid (less than 1 h), detection of GBS directly from clinical specimens at the time of delivery. The application of these assays to the current prevention strategies will simplify the prevention practice and rationalise the use of antibiotics. Immunoprophylaxis relies on the development of new vaccines against GBS, and active research is being conducted in this area.

The Promise of PCR for Detecting Group B Streptococcus Colonization

Infectious Diseases| October 01 2000 The Promise of PCR for Detecting Group B Streptococcus Colonization AAP Grand Rounds (2000) 4 (4): 35. https://doi.org/10.1542/gr.4-4-35 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation The Promise of PCR for Detecting Group B Streptococcus Colonization. AAP Grand Rounds October 2000; 4 (4): 35. https://doi.org/10.1542/gr.4-4-35 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search nav search search input Search input auto suggest search filter All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: microbial colonization, polymerase chain reaction, streptococcus group b Source: Bergeron MG, Ke D, Menard C, et al. Rapid detection of group B streptococci in pregnant women at delivery. N Engl J Med. 2000;343:175–179. Bergeron et al prospectively evaluated the speed and accuracy of a polymerase chain reaction (PCR) assay to detect group B streptococcus (GBS)-specific DNA as compared to the standard selective broth culture method to identify colonization in pregnant women at the time of delivery. Vaginal, anal, and combined vaginal and anal specimens were obtained from 112 pregnant women in Quebec, Canada. Thirty-three of the women (29.5%) had combined vaginal and anal cultures that were positive for GBS. Together, the 2 PCR assays detected GBS in 32 of the 33 women. Notably, 39% of the colonized women would have been missed had only vaginal swabs been collected. Compared with cultures, both PCR assays had a sensitivity of 97%, a specificity of 100%,... You do not currently have access to this content.

Use of an enrichment broth cultivation-PCR combination assay for rapid diagnosis of swine erysipelas.

We have previously described the creation by Tn916 mutagenesis of avirulent transposition mutants from a highly virulent strain of Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas. In this study, we cloned a 2.2-kb DNA fragment which flanked the Tn916 insertion in an avirulent mutant (strain 33H6) and evaluated the possibility that this region could be used for the specific detection of E. rhusiopathiae. According to the sequences of this region, oligonucleotide primers were designed to amplify a 937-bp fragment of the E. rhusiopathiae chromosome by PCR. The specificity of the PCR was investigated by analyzing 64 strains of Erysipelothrix species and 27 strains of other genera different from Erysipelothrix. A 937-bp DNA fragment could be amplified from all E. rhusiopathiae strains tested, and no amplification was observed by using DNAs from the other species tested. To make a rapid and definite diagnosis of swine erysipelas in slaughterhouses, we developed an enrichment broth cultivation-PCR combination assay, which used a commercially available DNA extraction kit, to identify E. rhusiopathiae in the specimens from swine with arthritis. After samples were enriched in selective broth culture, detection of E. rhusiopathiae was tested by either conventional methods or the PCR. Of 102 samples tested, 15 samples were positive by conventional methods and 12 of the 15 samples were positive by the PCR. The detection limit of the PCR was 10(3) CFU per reaction mixture for the PCR-positive samples. These results indicate that this PCR technique could be used as a first-line screening technique for the specific detection of E. rhusiopathiae in specimens.

Preincubation of Cervical Swabs in Lim Broth Improves Performance of ICON Rapid Test for Detection of Group B Streptococci

Objective: The purpose of this study was to determine whether an enrichment method would improve the performance of an enzyme imunoassay test, the ICON Strep B, for detection of group B streptococci (GBS) in vaginoperineal swabs. Methods: The study was done in 3 phases. First, in 250 maternity patients, 2 swabs per patient were tested simultaneously by an overnight selective broth culture method (Lim broth) and the ICON assay. Forty-five (18%) specimens were positive for GBS by culture. The ICON assay detected only 2 (4%) of the positives. Second, in 391 maternity patients, a single swab was cultured as above. However, during the overnight incubation of the Lim broth, 0.5 ml aliquots were removed and tested by ICON assay at 4, 6, 8, 10, and 12 h post-incubation. Seventy-two specimens (18%) were positive by culture. The ICON assay detected 20% of the positives at 4 h, 46% at 6 h, 70% at 8 h, 94% at 10 h, and 100% at 12 h post-incubation. Third, 97 high-risk patients with the diagnosis of preterm labor (PTL)/or preterm premature rupture of the membranes (PPROM) were sampled. Three specimens per patient were obtained: a single swab that was cultured as before and 2 double swabs, of which 1 was tested directly using the ICON test and the other was placed directly in Lim broth and incubated overnight. The aliquots of broth were tested by the ICON assay at 2, 4, 6, and 8 h post-incubation. Twenty-four specimens were positive by culture. Results: The direct ICON test detected only 4 (17%) of the positives. The ICON assay performed on the enriched samples detected 4% of the positives at 2 h, 21% at 4 h, 58% at 6 h, and 100% at 8 h post-incubation. Conclusions: These data indicate that the ICON assay may be used with 100% sensitivity and specificity to detect GBS-colonized high-risk mothers within 8 h if the initial sample size is doubled and the enrichment broth is used in the performance of the ICON assay.

Research Note: Evaluation of Direct Plating for Detecting Salmonella enteritidis in Pools of Egg Contents

The effectiveness of direct plating of incubated egg contents pools onto selective agar media for detecting contamination by Salmonella enteritidis (SE) was evaluated in comparison to several broth enrichment strategies. Pools of mixed liquid whole egg were inoculated with very small number of SE cells, incubated for 4 days at 25 C, and sampled by direct plating or both enrichment culture. Methods involving broth nonselective (pre-enrichment) and selective broth culture steps detected a significantly higher percentage of contaminated egg pools than did direct plating, but required 48 h longer to provide results. Methods involving direct selective broth enrichment, without prior pre-enrichment, did not perform significantly better than direct plating. Direct plating appears to provide a relatively rapid and inexpensive method for detecting SE in egg pools, although greater sensitivity is attainable with more intensive methods.