Selective Enrichment Broth Research Articles

A New Method for Detection of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Using a Novel Chromogenic Agar

Arcobacter species are gram-negative rods that have been implicated in food- and waterborne illness. Although various cultural isolation methods have been proposed, the current procedures are unable to fully suppress the growth of background microbiota present in food samples, which inhibits Arcobacter isolation. The purpose of this study was to develop a selective enrichment broth and chromogenic plating medium to detect three Arcobacter species that have been recognized as emerging foodborne pathogens: Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The developed Nguyen-Restaino-Juárez (NRJ) Arcobacter detection system consists of a selective enrichment broth (NRJ-B) and a selective-differential plating medium (NRJ-M). The protocol of the detection method was determined by evaluating the growth of A. butzleri, A. cryaerophilus, and A. skirrowii under various temperatures (30, 35, and 42°C) and incubation (aerobic, microaerophilic, and anaerobic) conditions. Additionally, 47 Arcobacter strains and 39 non-Arcobacter strains were tested in inclusivity and exclusivity evaluations of NRJ-B and NRJ-M. Overall, the study determined that the optimal growth conditions of Arcobacter species using the NRJ Arcobacter detection system were aerobic incubation at 30°C. NRJ-B supported good growth of A. butzleri, A. cryaerophilus, and A. skirrowii while effectively suppressing the growth of non-Arcobacter strains after 48 h. Furthermore, NRJ-M yielded 97.8% inclusivity and 100.0% exclusivity using the tested strains and resulted in salmon-pigmented Arcobacter colonies (1.0 to 1.5 mm in diameter) after 72 h. The novel protocol is the first to develop a chromogenic plating medium for the isolation of Arcobacter species. This simple and accurate test method would greatly contribute to understanding the distribution of pathogenic Arcobacter species in food samples.

Periodic screening of donor faeces with a quarantine period to prevent transmission of multidrug-resistant organisms during faecal microbiota transplantation: a retrospective cohort study

On June 13, 2019, the US Food and Drug Administration issued a warning after transfer of faeces containing an extended-spectrum β-lactamase (ESBL)-producing Escherichia coli by faecal microbiota transplantation led to bacteraemia in two immunocompromised patients. Consequently, we evaluated the effectiveness of the faeces donor-screening protocol of the Netherlands Donor Faeces Bank, which consists of screening of donors for multidrug-resistant organisms every 3 months, combined with additional screening on indication (eg, after travelling abroad) and application of a quarantine period for all faecal suspensions delivered within those 3 months. We did a retrospective cohort study of data collected between Jan 1, 2015, and Oct 14, 2019, on the multidrug-resistant organism testing results of donor faeces. Additionally, we tested previously quarantined faecal suspensions approved for faecal microbiota transplantation between Dec 12, 2016, and May 1, 2019, for the presence of multidrug-resistant organisms using both aselective and selective broth enrichment media. Whole-genome sequencing with core-genome multilocus sequence typing (cgMLST) was done on all multidrug-resistant isolates. Among initial screenings, six (9%) of 66 tested individuals were positive for multidrug-resistant organisms and 11 (17%) of 66 tested individuals were positive for multidrug-resistant organisms at any timepoint. Multidrug-resistant organisms were detected in four (25%) of 16 active donors, who had a median donation duration of 268 days (IQR 92 to 366). Among all screening results, 14 (74%) of 19 detected multidrug-resistant organisms were ESBL-producing E coli. 170 (49%) of 344 approved faecal suspensions had corresponding research faeces aliquots available and were tested (from 11 active donors with a median of eight [IQR five to 26] suspensions per donor). No multidrug-resistant organisms were detected in the 170 approved faecal suspensions (one-sided 95% CI 0 to 1·7). cgMLST revealed that all multidrug-resistant organisms were genetically different. Healthy faeces donors can become colonised with multidrug-resistant organisms during donation activities. Our screening protocol did not result in approval of multidrug-resistant organism-positive faecal suspensions for microbiota transplantation. None.

Prevalence, risk factors and molecular characteristics of Shiga toxin-producing Escherichia coli in beef abattoirs in Gauteng, South Africa

A cross-sectional study was conducted to determine the prevalence, risk factors, and molecular characteristics of Shiga toxin-producing Escherichia coli (STEC) in 12 beef abattoirs in Gauteng province, South Africa. The relationship between STEC contamination and aerobic bacterial counts on carcasses at various stages of processing was investigated. Multiplex PCR was used to detect virulence genes in broth-enriched samples, to determine O-serogroups in all samples positive for Shiga toxin-encoding genes (stx), and to characterize isolates of STEC. The overall prevalence of STEC determined by PCR in 419 selective enrichment broth samples was 35.1% (147/419), and was significantly higher (P = 0.037) in perineum hide swabs (50%) than in 24 h chilled carcass swabs (20%). The maximum total aerobic plate count (TAPC) was 3.8 log10 CFU/100 cm2 for carcass swabs, but was below the South African microbiological standard for meat export at all stages of carcass processing. There was no significant association between TAPC and STEC contamination. Serogroup O113 was the most prevalent serogroup (13.6%; 20/147) detected. Only 33 isolates, all non-O157 STEC, were recovered, amongst which six different genotype combinations were observed. Additionally, the clinically important serogroups O117, O8, and O2 were isolated. Multivariable logistic regression revealed that the odds of STEC contamination was lower in post-wash (OR = 0.42; 95% CI: 0.18–0.98; P = 0.045) and 24 h chilled (OR = 0.33; 95% CI: 0.12–0.91; P = 0.033) carcass swabs compared to pre- and post-evisceration swabs. It was concluded that non-O157 STEC serogroups more frequently colonize beef cattle slaughtered at abattoirs in our study area than O157 STEC, and therefore have a higher potential to enter the food chain during carcass processing, with food safety implications.

Group B Streptococcus in Indian Pregnant Women: Its Prevalence and Risk Factors

AbstractPurpose: To study the prevalence of group B streptococci (GBS) in Indian pregnant women and associated risk factors. Methods: Four hundred and fifty pregnant women attending antenatal outpatient/inpatient department between 35-38 weeks of gestation were enrolled in the study. All enrolled subjects were assessed by a predefined proforma for their demographics, socio-economic characteristics, education, toilet habits, obstetric history and clinical outcome. Two cotton swabs each from lower vagina and rectum were collected and plated on selective solid media CHROM agar Strept B (CHROMagarTM) and selective Enrichment Broth LIM RambaQUICK StreptB broth (CHROMagarTM). Presumptive identification of GBS was growth of 1-3 mm grayish white β-hemolytic colonies on BAP or mauve coloured colonies on CHROM agar Strept B. All presumptively identified GBS were confirmed as group B streptococcus by automated identification system-Vitek MS (Bio Merieux). Results: The recto vaginal colonization rate of GBS in this study was observed as 3.3% (n=15). GBS colonization was significantly associated with nulliparous women (p= 0.026) and use of western style toilet (p=0.017). GBS urinary tract infections was also seen more commonly in women with GBS rectovaginal colonization (p=0.002). Conclusion: Due to the low GBS prevalence and no significant association with major risk factors, we recommend to institute universal screening of GBS in pregnant women, instead of risk based screening. Since this was a single centric study with low prevalence of GBS, its applicability may be limited, therefore further larger multi-centric prospective studies are required to understand the true GBS prevalence in Indian society.

A comparison of GenomEra\xae GBS PCR and GeneXpert \xae GBS PCR assays with culture of GBS performed with and without broth pre-enrichment

This study was designed to compare the performance of GeneXpert® and GenomEra® group B streptococcus (GBS) PCR assays, held up against standard culture of GBS performed with and without broth pre-enrichment. In Denmark, the strategy for preventing early onset GBS infection (EOGBS) is risk factor based. Three hundred and sixty six women fulfilling one or more of the criteria for presence of risk factors for EOGBS were prospectively included. Rectovaginal swab samples were taken intrapartum and tested bed-site by the GenomEra® and the GeneXpert® GBS PCR assays and cultured at the microbiology laboratory using Granada agar plates with and without prior growth of sampling material in selective enrichment broth. Among 366 participants tested intrapartum, 99 were GBS-positive by culture, 95 by GenomEra, and 95 by GeneXpert. Compared with culture, the GenomEra and the GeneXpert performed with a sensitivity of 91.8% and 91.7% and a specificity of 98.1% and 97.3%, respectively. A combined reference standard was established by defining true positives as either culture-positive samples or culture-negative samples where both the GeneXpert and the GenomEra GBS PCR assays were positive. Using this, the sensitivity increased to 92.2% and the specificity to 99.6% for GenomEra and to 92.0% and 96.8% for GeneXpert. The use of selective broth enrichment found only three additional GBS culture-positive samples. The performance of the two PCR methods examined was very similar and close to the findings by culture, and both PCR assays are thus applicable as rapid intrapartum bed-site tests.

Improvement of a new selective enrichment broth for culturing Salmonella in ready‐to‐eat fruits and vegetables

AbstractFruits and vegetables are consumed raw, which is a potential health hazard toward consumers. Consequently, a novel selective enrichment broth (selective Luria‐Bertani broth, SLB) combined with a loop‐mediated isothermal amplification (LAMP) kit was developed for inspection of Salmonella in ready‐to‐eat fruits and vegetables. SLB was formulated by improved Luria‐Bertani (LB), the recovery of acid‐injured Salmonella in SLB was superior to Selenite Cystine Broth (SC) (p < 0.05). In addition, the growth speed of Salmonella in SLB was exceeding among LB and SC, and growth inhibition of non‐target bacteria without compromising Salmonella. Fruits and vegetables, inoculated with Salmonella, were inspected using a LAMP kit and by spreading on selective agar plates after incubation in SLB (10 hr) and Buffered Peptone Water (BPW)‐SC (34 hr). The LAMP kit had a high detection rate (100%) for both the culture methods, whereas detection rates after incubation in BPW‐SC were lower than those in SLB using the coating detection method. In general, SLB had suitable solubility and needed less incubation times, and the combination of SLB and LAMP method could enhance the accuracy of test results.

Evaluation of MALDI–TOF MS as a tool for detection of Listeria spp. directly from selective enrichment broth from food and stool samples

We evaluated the detection of Listeria spp. using MALDI-TOF MS directly in enrichment broths, without isolated colonies, with naturally contaminated food and stool samples. The success rate was 77%. Considering the reduced time for diagnosis and the success rate, this is a promising screening tool, but more tests are needed to determine its viability.

Salmonella Contamination of Seafood in Landing Centers and Retail Markets of Mumbai, India.

Seafood contamination with Salmonella enterica is not only a public health concern, but can also lead to economic losses due to import rejections. Continuous monitoring of seafood for Salmonella is necessary for risk assessment and to establish suitable control measures. The objective of this study was to determine the extent of Salmonella contamination of seafood in fish landing centers and retails markets. In this study, we analyzed seafood samples from three fish landing centers and seven retail markets of Mumbai, India for S. enterica contamination. Salmonella was isolated using multiple selective enrichment broths and selective agars, and identified by conventional biochemical tests followed by polymerase chain reaction (PCR). Of 82 samples of seafood, comprising both finfish and shellfish, Salmonella was detected in 17 (20.7%) finfish samples. None of the shellfish samples (shrimps, clams, and cephalopods) were found to be contaminated with Salmonella. Samples from both landing centers and fish markets, as well as from pelagic and demersal sources, harbored Salmonella. Source-wise analysis showed that 11 of 57 (19.3%) samples from retail fish markets and 6 of 25 (24%) samples from fish landing centers were contaminated with Salmonella. The study emphasizes the need to implement measures to prevent anthropogenic contamination of coastal waters and improve the hygiene of retail fish markets in Mumbai. Seafood from landing centers and retail markets, as well as from pelagic and demersalhabitats were similarly contaminated with Salmonella. Fish species widely consumed in the region of this study were found highly contaminated with Salmonella.

Selenite Cystine Agar for Enumeration of Inoculated Salmonella Serovars Recovered from Stressful Conditions During Antimicrobial Validation Studies.

Process validation studies often require the inoculation of select foodborne pathogens into targeted foods to determine the lethality of the process or antimicrobial ingredients, and quantitative recovery of surviving inoculum bacteria helps to make those assessments. Such processes introduce various stressors on the inoculated challenge microorganisms whereby traditional selective media are too harsh to enumerate the remaining viable and injured population quantitatively. Innate antibiotic resistance of challenge organisms has often been used to establish simple selective media (i.e., Tryptic Soy Agar/TSA + antibiotics) for recovering inoculated strains, but sometimes antibiotic resistant background microorganisms are higher than desired. Salmonella Thompson 120, Salmonella Heidelberg F5038BG1, Salmonella Hadar MF60404, Salmonella Enteritidis H3527, and Salmonella Typhimurium H3380 were characterized for antibiotic resistance and acid adaptation in Tryptic Soy Broth containing 0%, 0.25%, or 1.0% glucose. Sodium pyruvate was evaluated for recovery after stress but no enhancing effect was observed, possibly because the strains were acid-adapted. Selenite Cystine Broth, traditionally used as a selective enrichment broth, was used as the basis for Selenite Cystine Agar (SCA) in combination with three antibiotics to which our Salmonella are resistant. Serovars of Salmonella, both individually and in mixtures, were enumerated on TSA, SCA, Xylose Lysine Desoxycholate (XLD), and Hektoen Enteric (HE) selective agars (all containing the same antibiotics) after conditions of nutrient starvation, desiccation, acid stress, and thermal stress. The data show that quantitative enumeration of our Salmonella serovars on SCA was not significantly different (p > 0.05) than those achieved on TSA for all tested stress categories. Levels of Salmonella enumerated on XLD and/or HE were significantly different (p < 0.05) than on TSA and SCA and often more than 1–2-log lower, consistent with the inhibition of injured cells. These data confirm that SCA (+ antibiotics) is a suitable selective medium for enumeration of these acid-adapted Salmonella serovars as challenge organisms recovered from various conditions of stress.

Detection of multiple naturally occurring Salmonella serotypes from commercial broiler carcasses with conventional methods

AbstractMany laboratories sampling foods for Salmonella are interested only in presence or absence of Salmonella, so only one colony may be selected. The objectives of this study were to use two selective enrichment broths and two selective agar plating media for Salmonella recovery from naturally contaminated broiler carcass rinsates and evaluate these media combinations on Salmonella serotypes recovered from each carcass. Broiler carcasses (n = 52) from a commercial processing plant prior to chilling were rinsed with buffered peptone water and after incubation subcultured to gram‐negative and tetrathionate, and after inoculation to Rappaport Vassiliadis broth which was incubated and then streaked onto plates of Brilliant Green Sulfa and Xylose‐Lysine‐Tergitol‐4 agar. On 11/49 positive carcasses, both plating media yielded the same serotypes; for the other 38 positive samples different serotypes were found on the different plating media. Enrichment and plating media combinations influence the serotypes recovered and demonstrates bias even in a limited study such as this.

Optimal detection of extended-spectrum β-lactamase producers, carbapenemase producers, polymyxin-resistant Enterobacterales, and vancomycin-resistant enterococci from stools

This study compared the impact of different approaches, namely, nonenrichment, nonselective enrichment, and selective (antibiotic-containing) enrichment steps, for detecting extended-spectrum β-lactamase producing Enterobacterales (ESBL-E), carbapenemase-producing Enterobacterales (CPE), polymyxin-resistant Enterobacterales (PMR-E), and vancomycin-resistant enterococci (VRE) from spiked stools. The use of a nonselective 18-h enrichment broth culture significantly improved the recovery rate of all types of resistant bacteria after their plating onto selective media. In addition, the detection of ESBL-E, CPE, PMR-E, and VRE was further improved when using an enrichment step using antibiotic-supplemented broths respectively supplemented with cefotaxime (0.1 μg/mL), ertapenem (0.1 μg/mL), colistin (0.5 μg/mL), and vancomycin (1 μg/mL). Therefore, we showed here that a screening strategy based on a selective broth enrichment step significantly contributes to an increased rate of detection of multidrug-resistant bacteria, which may be crucial in term of improvement of infection control.

High prevalence of Clostridium difficile in soil, mulch and lawn samples from the grounds of Western Australian hospitals

Despite being considered a major hospital-associated pathogen for many years, Clostridium difficile has been isolated increasingly from people without hospital contact. In this study, we investigated the prevalence of C. difficile in the immediate outdoor environment of several hospitals in Perth, Western Australia, to provide further insight into potential sources of community-acquired C. difficile infection. Over 6 months, a total of 159 samples consisting of soil, mulch, lawn and sand were collected from outdoor surroundings of four different old (age>50 years) and new (age<10 years) hospitals. Samples were cultured in a C. difficile selective enrichment broth. Toxin gene profiling using PCR, and PCR ribotyping, was performed on all C. difficile recovered. C. difficile was isolated from 96 of the 159 samples (60.4%). Of the 112 isolates, 33 (29.5%) were toxigenic and 49 (43.8%) were identified as novel strains. Ribotypes (RTs) 014/020 (14.3%) and 010 (13.4%) constituted the highest proportion of isolates. Interestingly, RT 017, a strain endemic to the Asia-Pacific region (but not Australia), was found in a newly laid lawn. This study adds to existing knowledge of potential sources of C. difficile in Western Australia. More research is required to determine the route of transmission of C. difficile from community sources into the hospital.

Detection of Foodborne Pathogens Using Proteomics and Metabolomics-Based Approaches.

Considering the short shelf-life of certain food products such as red meat, there is a need for rapid and cost-effective methods for pathogen detection. Routine pathogen testing in food laboratories mostly relies on conventional microbiological methods which involve the use of multiple selective culture media and long incubation periods, often taking up to 7 days for confirmed identifications. The current study investigated the application of omics-based approaches, proteomics using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF MS) and metabolomics using gas chromatography-mass spectrometry (GC-MS), for detection of three red meat pathogens – Listeria monocytogenes, Salmonella enterica and Escherichia coli O157:H7. Species-level identification was achieved within 18 h for S. enterica and E. coli O157:H7 and 30 h for L. monocytogenes using MALDI-ToF MS analysis. For the metabolomics approach, metabolites were extracted directly from selective enrichment broth samples containing spiked meat samples (obviating the need for culturing on solid media) and data obtained using GC-MS were analyzed using chemometric methods. Putative biomarkers relating to L. monocytogenes, S. enterica and E. coli O157:H7 were observed within 24, 18, and 12 h, respectively, of inoculating meat samples. Many of the identified metabolites were sugars, fatty acids, amino acids, nucleosides and organic acids. Secondary metabolites such as cadaverine, hydroxymelatonin and 3,4-dihydroxymadelic acid were also observed. The results obtained in this study will assist in the future development of rapid diagnostic tests for these important foodborne pathogens.

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

Listeria monocytogenes is one of the most important foodborne pathogens due to the high hospitalization and mortality rates associated to an outbreak. Several new molecular methods that accelerate the identification of L. monocytogenes have been developed, however conventional culture-based methods still remain the gold standard. In this work we developed a novel Peptide Nucleic Acid Fluorescence in situ Hybridization (PNA-FISH) method for the specific detection of L. monocytogenes. The method was based on an already existing PNA probe, LmPNA1253, coupled with a novel blocker probe in a 1:2 ratio. The method was optimized for the detection of L. monocytogenes in food samples through an evaluation of several rich and selective enrichment broths. The best outcome was achieved using One Broth Listeria in a two-step enrichment of 24 h plus 18 h. For validation in food samples, ground beef, ground pork, milk, lettuce and cooked shrimp were artificially contaminated with two ranges of inoculum: a low level (0.2–2 CFU/25 g or mL) and a high level (2–10 CFU/25 g or mL). The PNA-FISH method performed well in all types of food matrices, presenting an overall accuracy of ≈99% and a detection limit of 0.5 CFU/25 g or mL of food sample.

Análisis microbiológico del tracto genital materno y de la sangre del cordón umbilical en relación con el daño neonatal

The etiology leading to neonatal damage is multifactorial, being genital infections one of the causes. The objective of the study was to identify microorganisms of the maternal genital tract that are associated with neonatal damage, in order to prevent future perinatal complications. Seven hundred and eleven pregnant patients attended their prenatal control during the period January 2010-July 2013. Ureaplasma urealyticum and Mycoplasma hominis presence was investigated in umbilical cord blood by metabolic substrates (Micofast-Biomerieux) and that of T.vaginalis, by PCR using specific primers. The microbiological study of the vaginal contents of 288 pregnant patients at weeks 35 to 37 was performed by conventional methods, adding the modified thioglycolate culture for T.vaginalis. GroupB streptococcus (GBS) was investigated in anorectal and vaginal introitus swabs, using selective broth enrichment and subsequent isolation in chromogenic medium. The χ2 Yates test and Fisher's test were used for independent samples. A p value <0.05 was considered statistically significant. The pathogens significantly related to neonatal damage were M.hominis (p=0.03), T.vaginalis (p=0.03), and BV (p=0.02).Main complications were preterm birth, premature rupture of membranes (PRM), low weight and Apgar score ≤7. U.urealyticum (p=0.35), Candidaspp. (p=0.94) and GBS (p=0.18) were not related to neonatal damage. Since different microorganisms of the maternal genital tract were related to neonatal damage, it is very important to perform the microbiological study of vaginal contents during pregnancy to prevent possible maternal and perinatal complications.

Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA

The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0–1.3, 0.5–8.3 × 103, and 0–5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9–5.3 × 102, 6.5 × 102−9.1 × 104, and 2.9–3.7× 103 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.

Evaluation of the Impact of Varied Carvacrol Concentrations on Salmonella Recovery in Oregano and How Corn Oil Can Minimize the Effect of Carvacrol during Preenrichment

Phenolic compounds, like carvacrol, in oregano interfere with the detection of foodborne pathogens such as Salmonella enterica. Carvacrol concentration varies based on plant cultivars and growth region. Six oregano cultivars were used to compare the impact of carvacrol concentration on Salmonella and to evaluate the effectiveness of corn oil to help increase Salmonella survival for detection. The results of Agilent 1200 series high-performance liquid chromatography analysis showed that carvacrol concentration in the six oregano cultivars ranged from 64 to 11,200 ppm. Oregano samples were artificially contaminated with S. enterica and were preenriched in Trypticase soy broth with or without 2% (v/v) corn oil. After 18 to 24 h at 37°C, aliquots were transferred to selective enrichment broths. Salmonella was recovered onto xylose lysine Tergitol 4 agar. Six Salmonella serovars were compared, and recovery varied based on carvacrol concentration and serovar. Samples with higher concentrations of carvacrol showed Salmonella recovery only when they were preenriched with corn oil. Based on metagenomic analysis, the microflora associated with the oregano also varied per cultivar. The results show that, as carvacrol levels increased, Salmonella survival decreased. However, the addition of corn oil to the preenrichment broth can minimize the antimicrobial effects of the phenolic compounds, thus allowing for increased detection of Salmonella from oregano cultivars.

A highly selective enrichment broth combined with real-time PCR for detection of Staphylococcus aureus in food samples

A new selective enrichment broth (OSA) was developed for detection of Staphylococcus aureus combined with real-time PCR. Acriflavine, nalidixic acid, potassium tellurite and sodium chloride were added as selective reagents and mannitol, sodium pyruvate, and yeast extract as promoters. The growth of S. aureus (10, 100, 1000 CFU/mL inoculation level) in OSA was superior to that in TSB with 7.5% NaCl (TNaCl). OSA showed a good inhibition ability against 8 strains including 4 Gram-positive and 4 Gram-negative. Further, OSA gave a good recovery of cold-, acid-, and heat-stressed cells comparable to TSBYE but superior to TNaCl. A real-time PCR targeting the nuc gene was developed and the standard curve was established from 9.1 × 107 to 9.1 × 102 cfu/mL with a high regression coefficient of determination (R2 = 0.999) and a high amplification efficiency of 96.362%. Using artificially contaminated food samples (kimbap, milk, and spam) with a level of ∼10 CFU/25 g or mL, OSA better supported the growth of S. aureus and all samples were readily detected after 12 h enrichment by real-time PCR. In summary, OSA was demonstrated as a promising selective enrichment broth for detection of S. aureus combined with real-time PCR with a total assay time of 15 h.

A new selective broth enrichment automated method for detection of carbapenem-resistant Enterobacteriaceae from rectal swabs

We evaluated the performance of an automated, rapid (TAT 4–8 h), liquid-culture method for carbapenem-resistant Enterobacteriaceae (CRE) detection from rectal swabs, in a setting of KPC-producing Klebsiella pneumoniae endemicity. With 600 samples (22 positive for CRE, 3.7%), the system sensitivity and specificity, at 8 h, were 100% and 99.2%, respectively.

Combination of MALDI-TOF MS and PBP2′ latex agglutination assay for rapid MRSA detection

Sample preparation was optimized for MALDI-TOF MS directly from the selective enrichment broth to detect Staphylococcus aureus. A combination of MALDI-TOF MS and the PBP2′ latex agglutination assay was applied for MRSA screening and evaluated on 255 clinical samples. MRSA colonisation can be reported already 18–24h after sample collection.