Enrichment Broth Cultures Research Articles

Rapid, sensitive, and validated method for detection of Salmonella in food by an enrichment broth culture – Nested PCR combination assay

A rapid nested PCR assay for detection of Salmonella from food was developed. The sensitivity of the assay developed was comparable to the traditional culture based methods with an advantage in reduction of assay time. The assay procedure with artificially contaminated samples was able to detect as low as 4CFU Salmonella/25g of food samples (sprout, carrot, cucumber and poultry meat). With two synthetic primers of 26 mer TS11 and 25 mer TS4, a 1.2kb fragment was amplified which served as a template for amplification of final 375bp product using TS11 and TS5 primers. No non-specific amplification from the native microbial flora of food samples was observed. The reaction generates a single band specific to Salmonella which allows the analyst to interpret data at ease and without any confusion. Enriched broth serves as template for the reaction which removes labour intensive DNA isolation procedures. In case of artificially contaminated samples, 6h enriched lactose broth can serve as template. However, for market samples where the organisms are under environmental stress, it is desirable to use template from Rappaport Vasiliadis medium. The assay also employes internal amplification control, which is amplified into a 300bp fragment and thus serves as positive control for the reaction and any possibility of false negative due to inhibitory action of food components on PCR reaction can be ruled out.

A capillary polymerase chain reaction for Salmonella detection from poultry meat

In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.

Use of Pooled Samples for the Detection of Salmonella in Feces by Polymerase Chain Reaction

Many epidemiological studies of Salmonella rely on conventional bacteriological culture methods to detect Salmonella in fecal samples. These culture-based methods are inefficient for epidemiological studies in populations with a low prevalence of Salmonella. The objective of this study was to optimize a protocol that uses pooled Salmonella enrichment broth cultures of bovine feces and polymerase chain reaction (PCR) for the detection of the invA gene of Salmonella in feces. In one field trial, 196 animals were sampled, and all samples were tested by culture, invA PCR on individual samples, invA PCR on pools of 5 samples, and BAX PCR on individual samples. All assays showed a high agreement on individual samples (kappa > or = 0.75). The invA PCR was run on each of 40 pools and detected 19 of 22 culture-positive pools. In another field trial, 152 samples were taken from 4 dairies, and the invA PCR was performed on pools of 5 samples in addition to bacteriological culture of individual samples. Salmonella was detected in 5 of the 32 pools (7 total positive samples) by both PCR and culture. One pool was PCR-positive but culture-negative. Pooling did not dramatically affect the performance of the invA PCR; most of the culture-positive samples were detected, including all of the samples when there were 4 or more Salmonella colonies on the agar plate. Based on these field trials, invA PCR on pooled samples appears to be an efficient method of Salmonella detection as long as Salmonella loads are not extremely low.

2004 Lowbury Lecture: the Western Australian experience with vancomycin-resistant enterococci – from disaster to ongoing control

The first hospital outbreak of a vancomycin-resistant enterococcus (VRE) in Western Australia (WA) started in the Royal Perth Hospital in July 2001 and initially involved the Intensive Care Unit (ICU) and the Nephrology and Dialysis Units. The outbreak was caused by vancomycin-resistant Enterococcus faecium (VREF) of the vanB genotype. Pulsed-field gel electrophoresis and plasmid analysis of the isolates demonstrated a single-strain outbreak. Despite the isolation of carriers and implementation of all the additional precautions recommended to control VRE, VREF spread rapidly. Two months after the index patient was detected, the epidemic strain had spread to 22 wards and units and one outpatient unit (Satellite Dialysis). Four patients were infected and 64 were colonized. A Hospital VRE Executive Group, which included the Chief Executive and Directors of Clinical Services and Nursing, was formed to eradicate the outbreak and to prevent the epidemic strain from becoming endemic in the hospital. The WA Department of Health agreed to provide substantial extra funding to enable the hospital to use expensive enhanced infection control practices, as follows. Control was handicapped by the slowness of conventional laboratory methods, which took four to five days to identify VRE and allowed environmental contamination and nosocomial transmission to occur before carriers were detected and isolated. A laboratory procedure to make rapid provisional identification of VRE within 30-48h was developed by performing multiplex polymerase chain reaction (PCR) for vanA and vanB genes directly on 24-h selective enrichment broth cultures. On average, four rectal swabs, each collected on separate days, were needed to detect >90% of carriers. In total, 1977 ward contacts were screened after discharge from hospital and 54 (2.73%) were found to be carrying VREF. The electronic labelling and active follow-up of ward contacts resulted in a significant number of carriers being detected who otherwise posed a risk of initiating further outbreaks in hospital if they were re-admitted. The outbreak was terminated after five months and the cost of the enhanced infection control practices was 2,700 000 Australian dollars (1,000,000 pounds sterlings). Ongoing control has been facilitated by targeted active surveillance cultures: on admission to high-risk units (ICU, Burns, Nephrology, Haematology, Bone Marrow Transplant Unit), on transfer out of the ICU to other hospital units, by monthly screening of patients regularly attending Dialysis Units, and by opportunistic laboratory screening of inpatient faecal specimens submitted for Clostridium difficile culture and toxin. Vigilance needs to be maintained as the epidemic strain of VREF remains in the Perth community. Ward contacts of the first outbreak have caused small outbreaks in two hospitals, and seven to 19 sporadic new carriers have been detected annually since the first outbreak. The key elements of the VRE control programme are as follows: To date, this programme has prevented VRE from becoming established in any WA hospital.

Comparison of rapid intrapartum screening methods for group B streptococcal vaginal colonization

Objective. To compare optical immunoassay (OIA) and rapid polymerase-chain reaction (PCR) with enrichment broth culture for intrapartum detection of vaginal group B streptococcal (GBS) colonization.Methods. Paired vaginal swabs from 315 consecutive term pregnant women at the time of presentation for delivery to a university medical center were tested for GBS by OIA, PCR, and culture. Sensitivity, specificity, and positive and negative predictive values were calculated.Results. Vaginal colonization was identified by culture in 56 subjects (17.8%). The sensitivity of OIA (7.1%, 95% confidence interval 5.1–9.5%) was significantly less than that of unenhanced rapid PCR (62.5%, 95% CI 48.5–74.8%).Conclusions. Neither PCR nor OIA is sufficiently sensitive for intrapartum detection of vaginal GBS colonization. Rapid PCR is more sensitive, but further improvements in technique to increase sensitivity will be necessary if PCR is to have a useful role in the management of women at time of presentation for delivery.

Use of the Roche LightCycler Strep B assay for detection of group B Streptococcus from vaginal and rectal swabs.

The results for a real-time PCR assay, using the LightCycler Strep B analyte-specific reagents (Roche Diagnostics Corporation, Indianapolis, Ind.), were compared to a direct plate method combined with a broth enrichment culture method for detection of group B streptococcus colonization in pregnant women. Two separate evaluations were conducted using two different automated nucleic extraction instruments, the MagNA Pure LC instrument (Roche Diagnostics Corporation) and the lower-capacity MagNA Pure Compact instrument (Roche Diagnostics Corporation). The sensitivities, specificities, and positive and negative predictive values for the different evaluation methods were as follow: for the LightCycler Strep B assay with MagNA Pure LC, 100, 97, 90, and 100%, respectively; for the LightCycler Strep B assay with MagNA Pure Compact, 92.5, 99, 97, and 97.5%, respectively. The LightCycler Strep B assay combined with either MagNA Pure LC or MagNA Pure Compact extraction is a suitable method for detecting group B streptococcus colonization in pregnant women. An advantage of the LightCycler assay over culture is the considerably reduced turnaround time for results.

Prevalence of Acinetobacter baumannii and other Acinetobacter spp. in faecal samples from non-hospitalised individuals

In total, 226 individuals from the community were investigated for faecal carriage of Acinetobacter spp. by broth enrichment culture, followed by growth on blood agar and/or Leeds Acinetobacter Medium (LAM). Acinetobacter baumannii was isolated on both LAM and blood agar from one of 100 specimens in the UK and one of 126 specimens in The Netherlands. The predominant species were Acinetobactor johnsonii and genomic sp. 11, which were cultured from 22 and five specimens, respectively. A. baumannii did not seem to be widespread in the faecal flora of individuals in the community.

Comparison of Three PCR Primer Sets for Identification of vanB Gene Carriage in Feces and Correlation with Carriage of Vancomycin-Resistant Enterococci: Interference by vanB -Containing Anaerobic Bacilli

We assessed the sensitivities and specificities of three previously described PCR primers on enrichment broth cultures of feces for the accurate detection of fecal carriage of vancomycin-resistant enterococci (VRE). In addition, we investigated specimens that were vanB PCR positive but VRE culture negative for the presence of other vanB-containing pathogens. Feces from 59 patients (12 patients carrying vanB Enterococcus faecium strains and 47 patients negative for VRE carriage) were cultured for 36 h in aerobic brain heart infusion (BHI) broth, anaerobic BHI (AnO(2)BHI) broth, or aerobic Enterococcosel (EC) broth. DNA was extracted from the cultures and tested for the presence of vanB by using the PCR primers of Dutka-Malen et al. (S. Dutka-Malen, S. Evers, and P. Courvalin, J. Clin. Microbiol. 33:24-27, 1995), Bell et al. (J. M. Bell, J. C. Paton, and J. Turnidge, J. Clin. Microbiol. 36:2187-2190, 1998), and Stinear et al. (T. P. Stinear, D. C. Olden, P. D. R. Johnson, J. K. Davies, and M. L. Grayson, Lancet 357:855-856, 2001). The sensitivity (specificity) of PCR compared with the results of culture on BHI, AnO(2)BHI, and EC broths were 67% (96%), 50% (94%), and 17% (100%), respectively, with the primers of Dutka-Malen et al.; 92% (60%), 92% (45%), and 92% (83%), respectively, with the primers of Bell et al.; and 92% (49%), 92% (43%), and 100% (51%) respectively, with the primers of Stinear et al. The primers of both Bell et al. and Stinear et al. were significantly more sensitive than those of Dutka-Malen et al. in EC broth (P = 0.001 and P < 0.001, respectively). The poor specificities for all primer pairs were due in part to the isolation and identification of six anaerobic gram-positive bacilli, Clostridium hathewayi (n = 3), a Clostridium innocuum-like organism (n = 1), Clostridium bolteae (n = 1), and Ruminococcus lactaris-like (n = 1), from five fecal specimens that were vanB positive but VRE culture negative. All six organisms were demonstrated to contain a vanB gene identical to that of VRE. VanB-containing bowel anaerobes may result in false-positive interpretation of PCR-positive fecal enrichment cultures as VRE, regardless of the primers and protocols used.

Comparison of optical immunoassay and polymerase-chain reaction for Group B streptococcal rapid intrapartum screening

The management of Group B streptococcus (GBS) in pregnancy remains a challenge despite revised treatment guidelines. The purpose of this study was to compare the efficacy of rapid optical immunoassay (OIA) and rapid polymerase-chain reaction (PCR) screening techniques to standard enrichment broth culture for detection of GBS colonization in pregnant women during labor.

Detection of enterohaemorrhagic Escherichia coli in patients attending hospital in Melbourne, Australia

The objectives of this study were (i) to determine the prevalence of enterohaemorrhagic Escherichia coli (EHEC) in adults and children with diarrhoea attending hospital in Melbourne, and (ii) to evaluate diagnostic assays for the detection of EHEC. EHEC were sought in 860 faecal samples (655 from children) using direct plating, a cytotoxicity assay and an enzyme immunoassay for Shiga toxin (Stx), and PCR for virulence-associated genes of EHEC. EHEC were isolated from 14 of 858 (1.6%) faecal samples (excluding repeat isolates from one patient). Isolation rates in children (1.7%) and adults (1.5%) were similar. EHEC was detected 2.5 times more frequently in samples that contained blood, but this was not statistically significant. EHEC isolates were heterogeneous in terms of serotype and virulence profile, although all produced EHEC haemolysin. Of the screening assays used, direct plating on EHEC agar, assays for Stx in MacConkey broth inoculated with faeces, and detection of the genes for Stx and EHEC haemolysin were highly sensitive and specific. EHEC are an infrequent cause of diarrhoea in Melbourne. EHEC can readily be isolated from faeces by screening enrichment broth cultures for Stx using PCR or enzyme immunoassay, followed by isolation of the bacteria on EHEC agar.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) in screening swabs by direct culture on a solid screening medium and broth enrichment culture separately and in combination

Patient and staff contacts of methicillin-resistant Staphylococcus aureus (MRSA) carriers were screened during a hospital outbreak over a 45 day period from 16 February – 1 April 2001 inclusive. Screening swabs were collected from patient nostrils, perineum, hands and throat. Wounds, ulcers and skin lesions were swabbed when present. An urethral swab and urine were collected when an indwelling urinary catheter was present. Screening swabs were collected from staff nostrils and hands. Broken skin areas were also swabbed.Swabs were individually plated onto methicillin aztreonam mannitol salt agar (MAMSA) and incubated in air at 36°C for 20-24 hours. All the swabs were placed in 10mL of selective enrichment broth consisting of nutrient broth containing methicillin 4mg/L, aztreonam 2mg/L and sodium chloride 25g/L in a single 30mL container and incubated at 36°C for 20-24 hours. The broth culture was resuspended by manual shaking and one microlitre was plated onto MAMSA and incubated in air at 36°C for 20-24 hours.A total of 2,130 sets of screening swabs were collected; 1,358 from patients and 772 from staff. Direct culture alone and enrichment culture alone each detected MRSA in 312 sets.Direct culture detected MRSA in some sets of swabs in which MRSA was missed by enrichment culture and vice versa. The combination of direct and enrichment cultures detected MRSA in 446 sets, which was 134 sets more than either method alone. As direct culture and enrichment culture each detected MRSA in only 70% of the sets of screening swabs compared with the combination of both methods, it is concluded that the laboratory should use both methods for processing MRSA screening swabs.

Occurrence of Clostridia in Commercially Available Spices, Spice Mixtures and Herbs

The occurrence of clostridia was investigated in a total of 100 commercially available samples of spices, spice mixtures and herbs. Clostridia were isolated from 47 (47%) samples, andClostridium perfringenswas detected in 24 (24%) samples.Clostridium botulinumwas not found. None of theC. perfringensisolates tested demonstrated the enterotoxin gene. The incidence of detection was higher with the enrichment broth culture detection method using cooked meat medium than with the agar plate method or pouch method. In several cases of two or more samples of the same spice (basil, cardamon, chili powder, coriander, curry powder, garam masala, garlic, nutmeg, oregano, paprika, red pepper, thyme, turmeric and white pepper), detection of clostridia differed by maker. Moreover, in the samples of curry powder, garam masala, paprika, turmeric and white pepper, detection of clostridia was different in those from the same maker.

Role of fecal incontinence in contamination of the environment with vancomycin-resistant enterococci

Background: We tested the hypothesis that patients with vancomycin-resistant Enterococcus (VRE) stool colonization who are continent of feces contaminate the environment less frequently than patients who are colonized and incontinent. Methods: We prospectively examined the frequency of environmental VRE contamination in the rooms of 15 patients who were continent and 15 who were incontinent and VRE-colonized. Broth-enrichment cultures of bed rails, bedside table, and call buttons were performed at baseline, and 2 and 5 days after environmental disinfection. The numbers of VRE colonies isolated after directly plating environmental swabs onto agar were compared for the continent and incontinent groups. Results: The percentages of patients with 1 or more positive environmental cultures for VRE were not significantly different for the groups of patients who were continent and incontinent at baseline (60% vs 73%, P =.45) or 2 days after disinfection (60% vs 80%, P =.24). The numbers of VRE colonies isolated by direct plating were not significantly different for the continent and incontinent groups (P =.42). Conclusions: Environmental contamination occurs frequently in the rooms of patients who are continent, and those who are incontinent and VRE-colonized. Our findings suggest that similar infection control measures should be implemented for patients who are continent and incontinent. (Am J Infect Control 2003;31:221-5.)

Laboratory support for prevention of perinatal group B streptococcal disease: commentary on the new guidelines on screening for group B streptococci during pregnancy

New guidelines from Centers for Disease Control and Prevention (CDC) used data from recent large studies as the basis for recommending a 35- to 37-week enrichment broth culture of a vaginal-anal swab to screen for group B streptococcal colonization in pregnant women. Various selective and enrichment media have been used to detect the organism. It is imperative that microbiologists perform screening cultures as outlined in the guideline, or validate their approach if different. Along with positive screening cultures, the guideline states that detection of any group B streptococci in urine is grounds for intrapartum prophylaxis; implementation of this recommendation will be difficult. Risk factors as the determinant of whether to give prophylaxis should be reserved for those without prenatal screening cultures. Use of PCR is promising. Despite increasing rates of clindamycin resistance, clindamycin is recommended for penicillin-allergic patients. Therefore, group B streptococcal isolates should be tested for susceptibility to clindamycin and erythromycin.

Rapid detection of Salmonella from poultry by real-time polymerase chain reaction with fluorescent hybridization probes.

Detection of Salmonella by bacteriologic methods is known to be time consuming. Therefore, we have developed a real-time probe-specific polymerase chain reaction (PCR) to rapidly detect Salmonella invA gene-based PCR products from chicken feces and carcasses by a fluorescence resonance energy transfer assay. The sensitivity and the specificity of this system were determined as 3 colony-forming units ml(-1) and 100%, respectively. Overnight tetrathionate broth enrichment cultures of chicken feces and carcass samples were used in template preparation for PCR. Also, a standard bacteriology was performed (National Poultry Improvement Plan-U.S. Department of Agriculture, Bacteriological Analytical Manual-Food and Drug Administration Center for Food Safety and Applied Nutrition) for confirmation. Seventy-two cloacal swab, 147 intestine, and 50 carcass (neck) samples were examined. Thirteen (8.8%) and 25 (17%) of the intestinal samples were found to harbor Salmonella by bacteriology and PCR, respectively. Forty-five of 50 (90%) carcass samples were Salmonella positive by both methods. Salmonella was not detected from cloacal swab samples. Results indicate that this assay has the potential for use in routine monitoring and detection of Salmonella in infected flocks and carcasses.

RAPID TEST FOR DIAGNOSIS OF ENTEROHEMORRHAGIC E. COLI O157:H7 IN HUMAN STOOL SAMPLES

A new rapid test platform for the direct detection of E. coli O157:H7 in stool samples of infected patients was compared with the current standard methods. The new test, DIAPRO FAST‐Q®, used biochip ICEflo ®technology that provided results within 20 min. Twenty‐one stool samples from patients infected with E. coli O157:H7 or of unknown status were studied. Using the DIAPRO FAST‐Q® method, within 20 min, we confirmed positive results for the seven known E. coli O157:H7 samples. While the standard culture method gave rise to only four positives in the remaining 14 unknown samples, the DIAPRO FAST‐Q® detected E. coli O157:H7 in eight of these samples, which was confirmed subsequently by broth enrichment culture method.

Serogroup specific single and multiplex PCR with pre-enrichment culture and immuno-magnetic bead capture for identifying strains of D. nodosus in sheep with footrot prior to vaccination

The identification of Dichelobacter nodosus present in a flock is a prerequisite to specific (autogenous) vaccination. Current methods of identification of the serogroup present in a population requires that the organisms be isolated, identified visually in mixed culture on streak plates, subcultured to purify and subjected to antigenic analysis. This process takes at least 3 to 4 weeks. This study describes the development of a simple and rapid serogroup specific PCR test forD. nodosus . A common forward primer was designed from the conserved amino-terminal region of the fimbrial gene (fimA) and 9 (A–I) serogroup specific reverse primers were designed from the carboxy-terminal regions offimA of the different serogroups. To verify the specificity within D. nodosus, each specific primer pair was tested in PCR against 18 serogroups/serotypes (prototypes) and found to be specific for all the serotypes within the homologous serogroups. Eighty four other bacterial strains, either commonly occurring in sheep or found in the environment of sheep, and including organisms related taxonomically to D. nodosus, were used to check the specificity of these assays. They were found to be specific for D. nodosus as none of the 84 bacterial stains reacted. These primers detected 1pg of purified chromosomal DNA, or 50–100 cells of D. nodosus in crude lysates. Sensitivity was markedly improved when an immuno-magnetic capture was employed. Single tube multiplex PCRs were tested with different combinations of common forward primer and groups of 3, 4 or 5 reverse primers chosen so that amplicon size for each reaction product was different. These were able to amplify DNA of isolates from all the relevant serogroups included in the reactions. These tests were evaluated with samples taken directly from lesions of footrot, either directly or preceded by DNA purification, immuno-magnetic capture, enrichment broth culture and culture on hoof agar media. Of these methods only PCR on mixed colonies from 4-day-old cultures on 4% hoof agar media yielded results of practical value.

Occurrence of Clostridia in Commercially Available Curry Roux.

The occurrence of clostridia was investigated in a total of 60 commercially available curry roux samples. Clostridia were isolated from 37 (62%) samples, and Clostridium perfringens was isolated from 7 (12%) samples. The isolates of C. perfringens did not produce enterotoxin. The frequency of occurrence was higher by the enrichment broth culture detection method than by the agar plate or pouch method. These findings suggest that enrichment broth culture is necessary for the detection of clostridia.

Detection on surfaces and in Caco-2 cells of Campylobacter jejuni cells transformed with new gfp, yfp, and cfp marker plasmids.

We have developed two sets of Campylobacter shuttle vectors containing either the gfp (green fluorescent protein), yfp (yellow fluorescent protein), or cfp (cyan fluorescent protein) reporter gene. In one set, the reporter gene is fused to a consensus Campylobacter promoter sequence (P(c)). The other set contains a pUC18 multicloning site upstream of the reporter gene, allowing the construction of transcriptional fusions using known promoters or random genomic fragments. C. jejuni cells transformed with the P(c) fusion plasmids are strongly fluorescent and easily visualized on chicken skin, on plant tissue, and within infected Caco-2 cells. In each C. jejuni strain tested, these plasmids were maintained over several passages in the absence of antibiotic selection. Also, in many C. jejuni strains, >91% of the cells transformed with the P(c) fusion plasmids remained fluorescent after several days. Experiments with yellow fluorescent and cyan fluorescent C. jejuni transformants suggest that aggregates containing two or more strains of C. jejuni may be present in an enrichment broth culture. Colonies arising from these aggregates would be heterologous in nature; therefore, isolation of a pure culture of C. jejuni, by selecting single colonies, from an environmental sample may not always yield a single strain.

Occurrence of clostridia in glass bottled foods

Forty-one marketed samples of imported and domestic glass bottled foods were tested for clostridia contamination. It was detected in nine (22%) samples. Clostridia were isolated from fish sauce (Nam pla, Nuoc-mam), dressing, mustard, hot and sour soup mix (Tom Yum), mysids boiled down in soy, and salmon flakes. The origin of all clostridia positive samples was Asia. Clostridium botulinum and C. perfringens were not detected. The frequency of occurrence was higher by enrichment broth culture detection methods than by agar plate or pouch methods. These findings suggest that the number of bacteria in most of these clostridia positive food samples is very low, and the use of enrichment methods for detection of clostridia is essential.