Selective Enrichment Broth Research Articles

Growth of Yersinia enterocolitica and Y. pseudotuberculosis in Yersinia selective enrichment broth according to Ossmer

The growth of Yersinia enterocolitica and Yersinia pseudotuberculosis in Yersinia enrichment broth according to Ossmer (YSEO) was investigated. Y. enterocolitica reached a higher concentration than Y. pseudotuberculosis but both always exceeded 106CFU/ml. The medium may be useful for the detection of both species in foods.

Phenotypic traits and comparative detection methods of vaginal carriage of Group B streptococci and its associated micro-biota in term pregnant Saudi women

Group B streptococcus (GBS) is a significant cause of serious infections in neonates and the composition of the vaginal micro-biota also affect neonatal outcome. To investigate vaginal carriage of Group B streptococcus in 217 term Saudi pregnant women, the direct antigen detection method in selective enrichment broth, Todd-Hewitt broth with 8 µg/ml gentamicin and 15 µg/ml nalidixic acid, using 2 commercial kits was used and compared to Centers for Disease Control and Prevention, CDC-gold standard method where following enrichment conventional means for identify­ing GBS is through isolation on subculture to blood agar plates, which requires 72 h before a final result is achieved. Phenotypic traits of Group B streptococcus serotypes distribution, antibiotic susceptibility patterns, biofilm formation and its associated vaginal micro-biota were also studied. The CDC-gold standard method positivity rate of Group B streptococcus was 23%, with no significant correlation with any of the studied demographic factors. In comparison, direct antigen detection method in boiled selective enrichment broth growth revealed 52.2 and 100% sensitivity and 100% specificity after growth amplification for 4 and 18 h incubation, respectively. Of 50 Group B streptococcus isolates, serotype II was the most predominant (42%), followed by serotype IV (16%). No resistance to ampicillin, cefepime, ceftriaxone or vancomycin was found, but 10, and 6%, of the strains were resistant to erythromycin and clindamycin, respectively. Overall 54% of Group B streptococcus strains were biofilm producers, but (100%) among serotype Ib, III, or V. Of several coexistent, Escherichia coli and Enterococcus feacalis were the most frequently Group B streptococcus -associated organisms, but with no statistical correlation. Key words: Group B Streptococcus, GBS-serotypes, antibiotics susceptibility, GBS-biofilm, vaginal micro-biota, Escherichia coli, Enterococcus feacalis.

A combined enrichment/polymerase chain reaction based method for the routine screening of Streptococcus agalactiae in pregnant women.

Group B Streptococcus (GBS) is the most common cause of life-threatening infection in neonates. Guidelines from CDC recommend universal screening of pregnant women for rectovaginal GBS colonization. The objective of this study was to compare the performance of a combined enrichment/PCR based method targeting the atr gene in relation to culture using enrichment with selective broth medium (standard method) to identify the presence of GBS in pregnant women. Rectovaginal GBS samples from women at ≥36 weeks of pregnancy were obtained with a swab and analyzed by the two methods. A total of 89 samples were evaluated. The prevalence of positive results for GBS detection was considerable higher when assessed by the combined enrichment/PCR method than with the standard method (35.9% versus 22.5%, respectively). The results demonstrated that the use of selective enrichment broth followed by PCR targeting the atr gene is a highly sensitive, specific and accurate test for GBS screening in pregnant women, allowing the detection of the bacteria even in lightly colonized patients. This PCR methodology may provide a useful diagnostic tool for GBS detection and contributes for a more accurate and effective intrapartum antibiotic and lower newborn mortality and morbidity.

Evaluation of the Impact of Direct Plating, Broth Enrichment, and Specimen Source on Recovery and Diversity of Methicillin-Resistant Staphylococcus aureus Isolates among HIV-Infected Outpatients

We compared recovery of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from nasal and groin swab specimens of 600 HIV-infected outpatients by selective and nonselective direct plating and broth enrichment. Swabs were collected at baseline, 6-month, and 12-month visits and cultured by direct plating to mannitol salt agar (MSA) and CHROMagar MRSA (CM) and overnight broth enrichment with subculture to MSA (broth). MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the Panton-Valentine leukocidin. At each visit, 13 to 15% of patients were colonized with MRSA and 30 to 33% were colonized with methicillin-susceptible S. aureus (MSSA). Broth, CM, and MSA detected 95%, 82%, and 76% of MRSA-positive specimens, respectively. MRSA recovery was significantly higher from broth than CM (P ≤ 0.001) or MSA (P ≤ 0.001); there was no significant difference in recovery between MSA and CM. MSSA recovery also increased significantly when using broth than when using MSA (P ≤ 0.001). Among specimens collected from the groin, broth, CM, and MSA detected 88%, 54%, and 49% of the MRSA-positive isolates, respectively. Broth enrichment had a greater impact on recovery of MRSA from the groin than from the nose compared to both CM (P ≤ 0.001) and MSA (P ≤ 0.001). Overall, 19% of MRSA-colonized patients would have been missed with nasal swab specimen culture only. USA500/Iberian and USA300 were the most common MRSA strains recovered, and USA300 was more likely than other strain types to be recovered from the groin than from the nose (P = 0.05).

Relationship between the culturability of stressed Listeria monocytogenes cells in non-selective and selective culture media and the cellular esterase activity measured by solid phase cytometry

Solid phase cytometry in conjunction with fluorescent probe was applied to rapidly quantify cellular esterase activity of Listeria monocytogenes cells. Viability of cells stressed by several treatments (starvation, NaCl, lactic acid and peracetic acid) was assessed simultaneously by their esterase activity estimated by fluorescence intensity and by their ability to multiply in liquid and solid, non-selective and selective, culture media. It was determined that cell physiological state has a significant impact on the cellular fluorescence intensity which was very dependent on the stress suffered by cells. No general relationship was observed between the bacterial populations observed by cytometry and the populations able to grow on culture media. The link between the cell culturability in non-selective and selective media and the esterase activity was always dependent on the stress suffered. Nevertheless, it was also established that solid phase cytometry is an efficient, sensitive and accurate tool to characterize the ability of non-selective and selective enrichment broths to allow the repair of stressed L. monocytogenes cells by examining the increase in the fraction of the most esterase active cells during the course of resuscitation.

Comparação do isolamento microbiológico e da reação em cadeia da polimerase no diagnóstico de salmonelose em bezerros infectados experimentalmente com Salmonella Typhimurium

The efficiency of microbiological culture and polymerase chain reaction (PCR) for detection of Salmonella Typhimurium is compared in fecal samples of Holstein calves experimentally infected with 109 CFU of Salmonella Typhimurium. Seventy-two fecal samples were analyzed by microbiological culture and PCR associated with selenite cystine (SC) and Muller-Kauffmann tethrationate (TMK) selective enrichment broths. Regardless of the selective enrichment broth, the microbiological culture was significantly better than PCR for detection of positive samples of Salmonella Typhimurium. The selective enrichment broths SC and TMK had no effect on the efficiency of the microbiological culture. The SC broth was the best option as selective enrichment associated to PCR.

Rapid detection of Salmonella sp. by means of a combination of selective enrichment broth and MALDI-TOF MS

The identification of Salmonella sp. in stool samples usually takes 2 days when employing routine procedures. Fast approaches are necessary in order to shorten the analysis time. The aim of this work was the development of a rapid procedure for the detection of Salmonella sp. from clinical stool samples. Spiked stool samples were cultured in selective selenite enrichment broth. Identifications were directly performed from the liquid broth by the MALDI Biotyper. After the evaluation of this method, the same procedure was applied to clinical samples. Coevally, the samples were streaked on Hektoen agar and single colonies were analyzed by the MALDI Biotyper. For comparison, the liquid broth was plated according to the standard laboratory procedure. A total of 4,847 samples were analyzed for Salmonella sp. In total, 108 Salmonella sp.-positive samples were identified; 66 of these were identified after the streaking of stool samples on Hektoen agar and subsequent MALDI Biotyper analysis of Salmonella sp. suspicious colonies. These and a further 34 samples were detected as Salmonella sp.-positive directly from the selenite enrichment broth on day one. Eight Salmonella sp.-positive samples were not detected before plating of the selenite broth and subsequent MALDI Biotyper analysis on day two. The combination of MALDI Biotyper analysis and selective selenite enrichment broth identification delivers positive results for the majority of the samples already after one day.

In vitro evaluation of the performance of Granada selective enrichment broth for the detection of group B streptococcal colonization

A broth for the screening of group B streptococcal (GBS) carriage during pregnancy is about to be introduced. Simulating conditions in everyday practice, we have compared the sensitivity of this Granada tube broth (GT) with that of classical Amies transport medium (AT) in vitro. A total of 1,485 GT and 1,485 AT were tested with 33 well-characterized GBS strains in three different concentrations, five different incubation times, and three different temperatures. After initial incubation at room temperature (RT) or 4°C, GT were placed at 37°C. GT were scored for the presence of orange pigment. GT and AT were subcultured on blood agar (BA). Pigment was observed in 98% of GT incubated at 37°C. GBS could be cultured in 91%, 73%, and 55% of GT incubated at 37°C, RT, or 4°C, respectively. For AT, these percentages were only 20% at 37°C, 52% at RT, and 59% at 4°C. When GT initially incubated at RT or 4°C were subsequently incubated at 37°C, the sensitivity improved significantly. We conclude that GT is a more sensitive GBS transport and culture medium than the conventional method, especially for low inocula and prolonged transport/incubation times. GT does not exclude the presence of GBS, and should always be incubated at 37°C and subcultured on solid agar for optimal sensitivity.

Evaluation of a Rapid Culture-Based Screening Test for Detection of Methicillin Resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.

飼料中のサルモネラ検査に用いる選択増菌培地の検討

飼料中のサルモネラ検査に用いる選択増菌培地を比較し，RVとRVSは，飼料公定法のSCの代替培地として有用と判断した．1) RVとRVSに接種する前増菌培養液量は，選択増菌培地液量の1/100, 1/10いずれでも，各選択増菌培養液中のサルモネラ菌数は同レベルであり，サルモネラの分離を妨げるほどの競合菌の影響はなかった．2) 前増菌培養では，サルモネラのみを接種した場合に，サルモネラは108 cfu/ml 程度に増殖したが，大腸菌群が夾雑すると106 cfu/ml程度に増殖が抑えられた．3) 選択増菌培養では，SC,RVおよびRVSにサルモネラを接種した場合に，サルモネラは108 cfu/ml 程度に増殖し，HTTでは107 cfu/ml 程度に抑えられた．一方，サルモネラ以外の大腸菌群が夾雑した場合でも，選択増菌培養液中の総大腸菌群は107～108 cfu/ml となり，その大部分をサルモネラが占めた．4) 陽性試料による選択増菌培地の性能比較では，23検体中13検体(56.5%)がサルモネラ陽性と判定され，HTT, SC, RVおよびRVSの個別結果はすべて一致した．陰性と判定された10検体はDSE法でもサルモネラは検出されなかった．5) 飼料公定法の選択増菌培地とRVまたはRVSを用いた場合のサルモネラ検出結果は，RV比較用試料で2.1%(6/288検体)が陽性，RVS比較用試料で3.0%(12/404検体)が陽性と判定された．各選択増菌培地の個別結果に有意差はなかった(p<0.05).

Comparison of scpB gene and cfb gene polymerase chain reaction assays with culture on Islam medium to detect Group B Streptococcus in pregnancy

Purpose: The purpose of the current study was to evaluate two low-costing PCR assays for rapid detection of Group B Streptococcus (GBS) in comparison to a pigment-based culture method. Materials and Methods: One-hundred and fifty vaginal swabs were collected from pregnant women at 35–40 weeks of gestation. Vaginal swabs were inoculated in selective enrichment broth medium, and examined using Islam medium, cfb PCR and scpB PCR assays. The demographic data were analysed to identify independent predictors of GBS colonization (age and gravidity), with GBS status as the dependent variable. Results: There was a significant association of age and gravidity with GBS colonization. GBS was detected in 25.3% of isolates by Islam medium, in 30.6% by using the cfb PCR assay and in 30% by using the scpB PCR assay. Conclusion: older pregnant women (≥30 years) and multigravida (>3 pregnancies) are at higher risk of GBS colonization. Both scpB-gene and cfb-gene-based PCR methods are highly sensitive techniques (100% sensitivity) compared to culture method. However, the specificities of the scpB and cfb PCR assays were 93.75 and 92.85%, respectively.

A multipathogen selective enrichment broth for simultaneous growth ofSalmonella entericaserovar Enteritidis,Staphylococcus aureus, andListeria monocytogenes

A selective enrichment broth (SSL) was formulated to allow concurrent growth of 3 prominent food-borne pathogens: Salmonella enterica serovar Enteritidis, Staphylococcus aureus, and Listeria monocytogenes. Nalidixic acid, lithium chloride, and potassium tellurite were added as the selective agents, while sodium pyruvate and mannitol were employed as the supplemented elements. In the individual growth trial, the target pathogens were capable of growing in SSL to as high as 7-8 log(10) colony-forming units (CFU)/mL after 24 h incubation at 37 degrees C when being inoculated at 50-100 CFU/mL. In the simultaneous growth trial, the 3 combined target pathogens showed similar growth rates. The results show that SSL could support the successful simultaneous enrichment of 3 pathogens; however, SSL inhibited the growth of nontarget bacteria. In the artificial contaminated raw beef and ready-to-eat chicken, a high recovery of these 3 target pathogens was obtained in SSL. Finally, Salmonella Enteritidis, Staphylococcus aureus, and L. monocytogenes were detected from 710 suspicious food samples by SSL with real-time PCR, and no false-positive or -negative results were reported. In summary, SSL has been shown to be a suitable broth for the simultaneous detection of the 3 prominent food-borne pathogens by multipathogen detection on a single-assay platform.

Avaliação da reação em cadeia da polimerase e do isolamento bacteriológico convencional na detecção de Salmonella Dublin em amostras de fezes de bezerros infectados experimentalmente

The efficiency of the Polymerase Chain Reaction (PCR) combined with selective enrichment broth was compared with the standard microbiological techniques for detection of Salmonella Dublin in fecal samples of 10 to 15-days-old Holstein calves, experimentally infected with 108 CFU of Salmonella Dublin. Seventy-six fecal samples were analyzed using PCR associated with selenite cystine (SC) and Muller-Kauffmann tetrathionate (TMK) broths and standard microbiological techniques. Regardless of the selective enrichment broth used, the standard microbiological techniques were significantly better than PCR in detection of positive samples of Salmonella Dublin. However, the simultaneous use of both techniques provided detection of a larger number of positive samples. The SC broth was the best option as selective enrichment in both techniques.

A Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection ofSalmonellaspp.,Escherichia coliO157, andListeria monocytogenesin Meat Products

To achieve an effective detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products, a multiplex real-time polymerase chain reaction (PCR) coupled with a multipathogen enrichment strategy was developed in this study. Pathogen-specific DNA sequences in the invA, rfbE, and hlyA genes were employed to design primers and TaqMan probes for identifying Salmonella spp., E. coli O157, and L. monocytogenes, respectively. An internal amplification control (IAC) utilizing a novel DNA sequence from human adenovirus was incorporated into the multiplex PCR assay to indicate false-negative results. Concurrent amplifications of multiple targets and IAC were thoroughly evaluated and optimized to minimize PCR competitions. Combined with a multipathogen enrichment in a selective enrichment broth for Salmonella, Escherichia, and Listeria (SEL), the multiplex real-time PCR assay was able to simultaneously detect all of the three organisms in artificially contaminated ground beef at a detection sensitivity of <18 CFU/10 g ground beef. Applying the assay to 26 retail meat samples including beef, chicken, turkey, and pork revealed that 12 samples were positive for one of the organisms and 3 samples were positive for two of the organisms after a 20-h enrichment in SEL. The remaining meat samples tested negative for all of the organisms by only showing amplification of the IAC. These results were confirmed by traditional culture methods testing for each individual species. Taken together, the multiplex real-time PCR assay combined with multipathogen enrichment is a rapid and reliable method for effectively screening single or multiple pathogen occurrences in various meat products.

Evaluation of the Xpert MRSA assay for rapid detection of methicillin-resistant Staphylococcus aureus from nares swabs of geriatric hospitalized patients and failure to detect a specific SCCmec type IV variant

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.

Direct Ertapenem Disk Screening Method for Identification of KPC-Producing Klebsiella pneumoniae and Escherichia coli in Surveillance Swab Specimens

Klebsiella pneumoniae carbapenemase (KPC) production in Gram-negative bacilli is an increasing problem worldwide. Rectal swab surveillance is recommended as a component of infection prevention programs, yet few screening methods are published. We compared detection of KPC-producing Klebsiella pneumoniae and Escherichia coli in surveillance specimens by 2 methods: (i) inoculation of swabs in tryptic soy broth containing 2 microg/ml imipenem followed by plating to MacConkey agar (MAC) (method 1) and (ii) streaking swabs on MAC onto which a 10-microg ertapenem disk was then placed (method 2). Simulated rectal swab specimens of challenge isolates from a collection of well-characterized K. pneumoniae and E. coli strains and salvage rectal swab specimens collected from patients at 4 different health care facilities over a 7-month period were tested. The gold-standard comparator was bla(KPC) PCR testing of isolates. Method 1 detected 4/9 (44%) KPC-positive challenge isolates. By method 2, 9/9 KPC-positive challenge isolates exhibited zones of inhibition of < or = 27 mm; all KPC-negative isolates exhibited zones of inhibition greater than 27 mm. The sensitivity and specificity of method 1 for detection of KPC-positive K. pneumoniae and E. coli in 149 rectal swab specimens were 65.6% (95% confidence interval [CI], 46.8% to 80.8%) and 49.6% (95% CI, 40.3% to 58.9%), respectively. With method 2, a zone diameter of < or = 27 mm had a sensitivity of 97.0% (95% CI, 82.5% to 99.8%) and specificity of 90.5% (95% CI, 83.3% to 94.9%) for detection of KPC in rectal swab specimens. Direct ertapenem disk testing is simpler, more sensitive, and more specific than selective broth enrichment with imipenem for detection of KPC-producing K. pneumoniae and E. coli in surveillance specimens.

A multipathogen selective enrichment broth for simultaneous growth of Salmonella spp., Vibrio parahaemolyticus, and Vibrio cholerae

A selective enrichment broth (SVV) was formulated to allow concurrent growth of Salmonella spp., V. parahaemolyticus, and V. cholerae. Potassium tellurite and sodium citrate were added as the inhibitors, while glucose, mannitol, anhydrous sodium sulfite and sodium pyruvate were employed as the growth-promoters. When mixed in equal or varied proportions, the target pathogens in SVV had a great accumulation (10(5)-10(8) CFU/ml) and effectively inhibited the growth of competitive microflora. In the artificially contaminated samples, a high recovery of these 3 target pathogens was obtained in SVV. Finally, Salmonella spp., V. parahaemolyticus, and V. cholerae were detected from 608 suspicious food samples by SVV with real-time PCR, and no false-positive or -negative results were reported. In summary, SVV has been shown to be a suitable broth for the simultaneous detection of the 3 pathogens by multipathogen detection on a single-assay platform.

A selective broth enrichment combined with real‐time nuc‐mecA‐PCR in the exclusion of MRSA

We analyzed the performance of a selective enrichment broth combined with Taqman-based real-time duplex nuc-mecA-PCR to expedite the screening of methicillin-resistant Staphylococcus aureus (MRSA). We found the broth to be able to select MRSA strains (oxacillin MIC range 4-256 microg/ml) from MSSA strains. A total of 31 MRSA strains were found from 1250 clinical samples screened. The nuc-mecA-PCR was positive from all enrichment broths containing MRSA. From the remaining 1219 samples negative for MRSA on culture/subculture, 138 samples were nuc+/mecA+ in PCR. The sensitivity of the test was 93.5%, specificity 88.6%, positive predictive value 17.3%, and negative predictive value 99.8% as compared to culture. Thus, with this method, the negative MRSA results can be reliably reported within 24-48 h from sampling. The method is a practical additional alternative to those already described for the same purpose.

Intrafamilial Transmission of Methicillin-ResistantStaphylococcus aureus1

Intrafamilial Transmission of Methicillin-Resistant<i>Staphylococcus aureus</i>1

Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts

Direct and sensitive PCR detection of contaminant microflora in cocoa extracts is affected by the quality of the template DNA. This study compares the efficacy of five different commercial DNA extraction methods, selective enrichment broths and use of glycolitic enzymes to obtain quality DNA for PCR detection of both fungi and bacteria in artificially inoculated cocoa extract samples. PCR-based methods were applied to detect contaminant microflora in cocoa extracts using as model organisms: Aspergillus nidulans, Bacillus subtilis, Escherichia coli and Salmonella enterica. The quality of the extracted DNA was assessed in terms of PCR inhibitor content with results indicating that the HighPure PCR template (Roche) kit was the best methodology under the conditions assayed. PCR protocols using this commercial kit and a combination of glycolitic enzymes and enrichment procedures gave a detection limit of 100 conidia/g and 100 cfu/g for filamentous fungi and bacteria, respectively. The selected extraction and PCR procedures were also tested to assess their suitability for detecting filamentous fungi and bacteria on an industrial scale. They were sensitive enough to detect fungal and bacterial contaminants within the legally required limits. The results obtained with the molecular approach were in agreement with those of standard microbiological tests but require a considerably shorter analysis time. Thus, the molecular approach provides a sensitive and rapid alternative to check for microbial contamination in cocoa extracts.