Selective Enrichment Broth Research Articles

Evaluation of Simultaneous Growth of Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes in Ground Beef Samples in Different Growth Media.

Several multiplex approaches for the simultaneous detection of pathogens in food have been developed in recent years, but the use of a single enrichment medium remains a problem. In this study, six enrichment broths (five non-selective media, tryptic soy broth (TSB), brain heart infusion broth (BHI), buffered peptone water (BPW), universal pre-enrichment broth (UPB), no. 17 broth, and a selective, Salmonella Escherichia Listeria broth (SEL)), were studied for the simultaneous detection of E. coli O157:H7, Salmonella spp., and L. monocytogenes, to validate the suitable enrichment broth to be used for the detection methods. Different ratios of E. coli O157:H7, Salmonella spp., and L. monocytogenes were used. Almost all non-selective broths evaluated in this study showed similar growth parameters and profiles among each other. The only selective enrichment broth under analysis (SEL) showed distinct growth features compared to the non-selective media, allowing for a slower but balanced growth of the three pathogens, which could be beneficial in preventing the overgrowth of fast-growing bacteria. In addition, when tested in ground beef samples, SEL broth seems to be the most distinctive medium with a balanced growth pattern observed for the three pathogens. Overall, this study is intended to provide the basis for the selection of suitable enrichment broths according to the technology detection to be used, the desired time of enrichment, and the expected balanced concentration of pathogens.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Effect of Selective Enrichment Storage Temperature and Duration Time on the Detection of Salmonella in Food.

For pathogen detection in food, there are occasions where samples cannot be processed immediately after selective enrichment or need to be reexamined days or weeks later for confirmation or retest. This study aimed to investigate the effect of different prolonged period of storage of selective enrichments of food at 4 ± 2°C and room temperature (20-22°C) on the detection and isolation of Salmonella. This study included two experiments involving 34 types of foods to compare the effect of 4 ± 2°C and room temperature storage on the detection of Salmonella in 204 selective enrichments (Rappaport-Vassiliadis [RV] and Tetrathionate [TT] broths) during a 42-day storage (Experiment I); and to monitor the survival of Salmonella in 300 selective enrichments (RV and TT) with different pre-enrichment broths (Lactose broth [LB] or Buffered peptone water broth [BPW]), stored at 4°C for 60 days (Experiment II). All the samples were subjected to Salmonella analysis following the FDA BAM method. During multiple samplings, the positive detection rate for Salmonella remained consistent through Day 42 after selective enrichment, irrespective of Salmonella serotype, storage temperature, pre-enrichment broth, or selective enrichment broth in both Experiment I and II. However, on Day 60 sampling in Experiment II, seven previously positive results turned to negatives. These data indicated that storage of RV and TT enrichments at 4 ± 2°C or room temperature for up to 42 days after selective enrichment did not compromise the detection of Salmonella in the tested food categories, regardless of Salmonella serotypes and the broths used for pre-enrichment and selective enrichment. At least for the food types studied in this experiment, the recovery of Salmonella from selective enrichments could be postponed for a limited period of time (e.g., <42 days) if needed without adversely affecting the test results. However, the delayed analysis of TT and RV enrichments does pose a risk of reduced detection sensitivity, as evidenced by the seven negative results on Day 60 compared to previous positives. We do not recommend or endorse delaying the analysis of TT and RV enrichments. In the food matrixes investigated in this experiment, the plating and isolation of Salmonella from selective TT and RV enrichments stored at 4 ± 2°C or room temperature could be deferred for a period (up to 42 days) without any negative effect on the test results, if necessary.

Cefixime-tellurite-deoxycholate tryptic soy broth (CTD-TSB), a selective enrichment medium, for enhancing isolation of Escherichia albertii from wild raccoon fecal samples.

The aim of this study was to develop a selective enrichment broth for efficient isolation of Escherichia albertii. A total of 412 raccoon rectal swabs suspended in PBS (phosphate-buffered saline) were tested by a real-time PCR to quantify the number of E. albertii followed by its isolation. The number of E. albertii in the PBS suspension strongly affected the isolation rate (1.2%-89%), which notably dropped (≤33%) when the number was<4 log10 CFU ml-1. However, enrichment of PBS suspension containing raccoon feces in tryptic soy broth containing cefixime, tellurite, and deoxycholate (CTD-TSB), the selective medium developed in this study, remarkably improved the isolation efficiency (up to 48%) of E. albertii. CTD-TSB is a useful enrichment culture medium for E. albertii and contributes to increase its isolation rate.

Analysis of molecular characteristics of CAMP-negative Streptococcus agalactiae strains.

Streptococcus agalactiae can produce CAMP factor, which can promote the β-hemolysin activity of Staphylococcus aureus, forming an arrow-shaped hemolysis enhancement zone at the intersection of the two bacterial species on a blood agar plate. This characteristic feature of Streptococcus agalactiae has led to the widespread use of the CAMP test as an identification method. Vaginal/rectal swabs, collected from women at 35-37 weeks of pregnancy, were first inoculated into a selective enrichment broth media, then subcultured onto GBS chromogenic agar and 5% sheep blood agar sequentially. The VITEK-2 automatic identification system and MALDI-TOF MS were initially employed for identification, followed by the CAMP test. CAMP-negative strains underwent 16S rDNA and cfb gene sequence analysis, as well as bacterial multilocus sequence typing. A total of 190 strains were isolated, with 15 identified as CAMP-negative. Further 16S rDNA gene sequence analysis confirmed that all 15 strains were Streptococcus agalactiae. The MLST typing assay revealed that these 15 strains were of the ST862 type. The cfb gene was amplified and electrophoresed, but no specific fragments were found, indicating that these strains lack the CAMP factor due to cfb gene deletion. Antibiotic susceptibility tests demonstrated no resistance to penicillin, ampicillin, vancomycin and linezolid among the GBS strains. However, there are significant differences in resistance rates to tetracycline. This study found that 7.9% of GBS strains isolated from the vagina/rectum of pregnant women were CAMP-negative, suggesting that the CAMP test method or primers targeting the cfb gene should not be used as the sole presumptive test for GBS identification.

Does pre-incubation in selective-enrichment media improve the detection of diarrheagenic Escherichia coli using the RIDA®GENE PCR?

We aimed to investigate whether a selective pre-PCR enrichment step improves test performance of RIDA®GENE EHEC/EPEC to detect diarrheagenic Escherichia coli from stool samples. Each of the 250 stool samples was analyzed for the presence of stx1/2 and eae both with and without pre-PCR enrichment in selective broth. In comparison to a reference method, sensitivities for stx1/2 and eae with and without pre-PCR enrichment were 84% (95%CI 70–93) and 89% (stx1/2, 95%CI 76–96), and 71% (95%CI 58–81) and 72% (eae, 95%CI 60–82), respectively. Specificity exceeded 97% for both methods and target genes. In summary, pre-PCR broth enrichment did not improve test performance.

Description of three novel species of Scandinavium: Scandinavium hiltneri sp. nov., Scandinavium manionii sp. nov. and Scandinavium tedordense sp. nov., isolated from the oak rhizosphere and bleeding cankers of broadleaf hosts.

While investigating the bacterial populations of environmental samples taken from a mix of healthy and Acute Oak Decline afflicted Quercus robur (pedunculate or English oak) rhizosphere soil samples and swabs of bleeding lesions on Tilia spp. (lime) and Quercus rubra (red oak) trees, several strains belonging to the order Enterobacterales were isolated using selective media and enrichment broth. Seven strains from the Q. robur rhizosphere, three strains from Tilia spp. and one from Q. rubra were investigated, with their taxonomic status determined via a polyphasic taxonomic approach. Initially stains were identified as potential members of the recently described genus Scandinavium, based on the partial sequencing of three housekeeping genes. Further analysis of phenotypic traits, including fatty acid profiles, coupled with 16S rRNA gene and phylogenomic analysis of whole genome sequences were applied to a subset of the strains. Phylogenetic and phylogenomic analysis repeatedly placed the isolates in a monophyletic clade within Scandinavium, with four distinct clusters observed, one of which corresponded to Scandinavium goeteborgense, the type species of the genus. The remaining three clusters could be phenotypically and genotypically differentiated from each other and S. goeteborgense. As such, we describe three novel species of the genus, for which we propose the names Scandinavium hiltneri sp. nov. (type strain H11S7T = LMG 32612T = CCUG 76179T), Scandinavium manionii sp. nov. (type strain H17S15T = LMG 32613T = CCUG 76183T) and Scandinavium tedordense sp. nov. (type strain TWS1aT = LMG 32614T = CCUG 76188T). Additionally, the descriptions of the genus Scandinavium and the type species, S. goeteborgense, are emended.

The Development and Evaluation of a Selective Enrichment for the Detection of Escherichia albertii in Food.

Escherichia albertii is an emerging pathogen causing foodborne infections with diarrhea, abdominal pain, and fever. E. albertii has been isolated from various food sources, such as chicken and pork. Although many foodborne outbreaks of E. albertii have been reported, the causative food has not been identified. It is necessary to develop effective detection methods for E. albertii. Because enrichment procedure as the first step of food test is important for growing pathogens, this study aimed to develop a novel effective enrichment for E. albertii detection in food. In this study, we investigated the optimal concentration and combination of cefixime and tellurite for supplementing modified EC broth (mEC) to effectively isolate E. albertii from chicken meat. The results showed that mEC supplemented with 50 μg/L cefixime and 2.5 mg/L tellurite (CT-mEC) inhibited the growth of competitive bacteria in chicken meat but not that of E. albertii. Therefore, it was indicated that CT-mEC had strong potential to selectively grow E. albertii. In an E. albertii foodborne outbreak, CT-mEC was evaluated. E. albertii was successfully isolated from a food sample, a kind of salad, by enrichment with CT-mEC but not buffered peptone water and mEC. In this study, CT-mEC as a selective enrichment broth has been developed to detect E. albertii in chicken meat. It was demonstrated that the selective enrichment broth was effective for the efficient detection of E. albertii in food.

Understanding the occurrence of polymerase chain reactions-positive and culture-negative for Shiga toxin producing Escherichia coli in samples from beef production chain

Molecular epidemiological methods have been employed to detect pathogens and transmission pathways, for disease surveillance, outbreak investigation, outbreak monitoring and control. Molecular methods such as polymerase chain reactions (PCR) is used to assess the positivity rate of virulent gene(s) with pathogens, but in many cases, cultural isolation of the pathogen may not be possible in PCR positive cases. This dichotomy between the outcome of results may be associated with low number of cells compared with the large population of background microflora, presence of viable but non-culturable cells, loss of virulence gene (s) after subculture, and the high sensitivity of the PCR assay. Shiga-toxigenic Escherichia coli (STEC) was used as a model for investigating this phenomenon. In this study, duplex PCR was used to screen 335 abattoir and 303 beef retail outlets selective broth enriched samples for the presence of stx1 and stx2 genes. Subsequent culture isolation of stx-positive broth samples was carried out. The overall STEC positivity determined by PCR in 335 and 303 abattoir and beef retail outlets in selective enrichment broth samples, respectively was 35.2% (118/335; 95% CI: 30.1 - 40.6) and 12.5% (38/303; 95% CI: 9 - 16.8). Only 24 (20%; 24/118) abattoir and 8 (21%; 8/38) retail outlet stx-positive samples were culturable. Both yielded only 51 isolates; 30 isolates for abattoir samples and 21 isolates for beef retail outlets, respectively. Our results confirm the dichotomy of PCR positive/culture negative samples, and from an epidemiological perspective, it is recommended that the use of only PCR to detect virulence genes in broth cultures should be acceptable where isolation is not achievable. This may be the best method for generating relevant epidemiologic data for disease control.

Escherichia Coli Infections, and Antimicrobial Resistance in Poultry Flocks, in North Central Nigeria

To investigate Eschericiacoli infections in poultry flocks, 291 tissue samples from 237 necropsied carcasses submitted from ninety-nine (99) poultry flocks in north central Nigeria were analysed. These flocks comprised layer chicken, broiler, pullet, cockerel, turkey, quail, guinea fowl and ducks. Tissue samples were pre-enriched in 10 mL buffered peptone water media and aliquots were inoculated into selective enrichment broth, sub-cultured onEosin Methylene Blue (EMB) agar and MacConkey agar (MCA) and colonies of E. coli was examined based on cultural morphological characteristics. Layer poultry and laying quails exhibited reproductive lesions which correlates with history of disruption and reduction in egg production. Antibiotics resistance rate was significant (p &lt;0.01) with macrolide and penicillin classes been the most resistant antibiotics in layers and broiler, while quinolones and aminoglycoside were most significantly susceptible (p &lt;0.01). Multidrug resistance (MDR) was found in 56% of the E. coli isolates, with high prevalence in younger birds.

Comparison Study and Evaluation of Selective Enrichment Broth for Coliforms with Commercial Broth Media

Background: Several bacteriological broths and solid media are available for growth of coliforms. Many are generalized and some of them works as selective. Now a days, in this category with development of chromogenic and flourogenic media, enumeration and detection have work become very easy. Despite the fact most of the countries are using approved methods like IS-5887 (Part-I) 1976 and IS-5401 Part-1 (2012) protocol for monitoring E. coli and coliforms in dairy products respectively. This is due to reliability of these media in giving results. Even though results obtained using these methods need confirmation as there are chances to have other organisms/contaminants in the broth or media which does not actually belong to coliforms groups. This leads to further testing to get presence of coliforms and E. coli. Methods: The present investigation comprised comparison and evaluation of formulated selective coliforms broth (Gawai, 2020) with commercially available broths namely Lactose broth, Coliform broth, Tergitol-7 broth, MacConkey’s broth, Violet red bile broth, Lauryl sulphate tryptose broth and nutrient broth at specified time intervals. Result: The growth pattern of Escherichia coli ATCC 25922, Coliforms cocktail and Salmonella typhimurium ATCC 14028 were studied in developed selective coliform broth along with seven commercial broths. It was observed that E.coli ATCC 25922 showed good growth in all the broth tested and highest growth was reported with Formulated broth i.e. 9.60 log cfu/ml and least growth was in MacConkey broth i.e. 8.60 log cfu/ml after 24 h of incubation. Coliform cocktail culture showed the best result in Formulated selective broth and Tergitol broth after 24 h of incubation i.e. 9.40 log cfu/ml while formulated coliforms broth showed the best reduction of Salmonella typhi ATCC 14028 in comparison with other tested broths.

Improvement of Mueller-Kauffman Tetrathionate-Novobiocin (MKTTn) enrichment medium for the detection of Salmonella enterica by the addition of ex situ-generated tetrathionate

The detection of Salmonella in food is based on the use of a selective enrichment broth such as Muller-Kauffman Tetrathionate-Novobiocin (MKTTn), in which tetrathionate plays a key role by providing Salmonella with a growth advantage. As sodium tetrathionate is unstable, it is generated in situ by the addition of iodine (Lugol's solution) before seeding. This step is cumbersome as the solution is easily spilled, compromising the performance of the medium and hindering the work of technicians.The aim of this study was to optimize MKTTn broth by generating tetrathionate ex situ through an external reaction between iodine and thiosulphate followed by lyophilization. Quality control procedures were performed to compare the modified and original media, testing pure productivity (enrichment with 50–120 CFU of Salmonella Thyphimurium ATCC 14028 and Salmonella Enteritidis ATCC 13076 and plating on Xylose Lysine Deoxycholate agar, XLD), mixed productivity (50–120 CFU of Salmonella strains and Pseudomonas aeruginosa and Escherichia coli at ≥104 CFU and XLD plating) and selectivity (≥104 CFU of P. aeruginosa and Enterococcus faecalis and plating on Tryptone Casein Soy agar, TSA).The modified MKTTn medium (S/L) performed comparably with the original medium in terms of growth of both Salmonella strains (>300 colonies in XLD), alone or with P. aeruginosa and E. coli. Quantitative assays showed no statistically significant differences in the number of colonies grown on XLD after 10−5 dilution (p = 0.7015 with S. Thyphimurium ATCC 14028 and p = 0.2387 with S. Enteritidis ATCC 13076; ANOVA test). MKTTn medium (S/L) was also selective against E. coli (≤100 colonies) and E. faecalis (<10 colonies). These results suggest that adding tetrathionate as a lyophilisate (S/L) is a feasible alternative to the use of Lugol's solution for the preparation of MKTTn enrichment broth and does not affect the properties of the medium.

Development of Selective Enrichment Broth for Coliforms using Response Surface Methodology

Background: Food Safety and Standard Authority of India has adopted conventional IS-5887 (Part-I) 1976 and IS-5401 Part-1 (2012) protocol for monitoring of E. coli and coliforms in dairy products respectively. These methods are time consuming, relying on bacteriological media and sometimes require further isolation and confirmation to finalize the true contaminant. The current investigation was carried out to develop such a broth media which will support growth of coliforms and inhibit other organisms associated within the micro-environment of coliforms and get entry in dairy products due to wrong processing practices and post production contaminant. Methods: The present investigation involved formulation of selective broth for coliforms by optimizing the rate of addition of Sodium lauryl sulphate salt, Gentamicin sulphate+Amoxycillin (in 1:1 ratio) and Cefsulodin by Response Surface Methodology (RSM). Result: Formulation of selective broth for coliforms and E.coli consisting of Sodium lauryl sulphate salt, Gentamicin sulphate+ Amoxycillin and Cefsulodin which was added at the rate of 0.25 g, 10 µl and 312.5 µl respectively per 100 ml of broth. This combination of ingredients along with base composition of broth were able to increase the growth of coliforms as well as able to inhibit population of Salmonella typhi ATCC 14028, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923.

Wild boars as reservoir for Campylobacter and Arcobacter

Campylobacteriosis is a significant public health concern with Campylobacter jejuni and Campylobacter coli as main causative agents. Moreover, there is an increasing recognition of other pathogenic Campylobacter species and Campylobacter-like organisms as Arcobacter. However, current knowledge on presence of Arcobacter species in wild boars (Sus scrofa) is lacking, and knowledge on Campylobacter species is based on methods favoring growth of thermotolerant species. In this study, fecal samples originating from 76 wild boars hunted in Campania region (Italy) were examined for the presence of Campylobacter(-like) organisms by a culture dependent approach. Three isolation protocols were performed in parallel: Arcobacter-selective agar plates, mCCDA plates and isolation by passive filtration onto non-selective blood agar plates were used as quantitative isolation methods. Enrichment broths, i.e. Arcobacter selective enrichment broth, Preston broth and CAT broth were used for qualitative detection of low levels or stressed Campylobacter(-like) organisms. The Arcobacter and Campylobacter isolates were identified at species level using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S ribosomal RNA (rRNA) sequence analysis. Overall, 41 (53.9%) of the animals excreted Arcobacter or Campylobacter while 38 (50.0%) shed Campylobacter and 8 (10.5%) Arcobacter. Campylobacter lanienae predominated and was isolated from 31 (40.8%) animals. No statistical difference between the age groups or gender with regard to the fecal excretion of Campylobacter(-like) organisms was observed. Thirty animals (39.5%) shed Campylobacter spp. exceeding levels of 10 ³ CFU g−1 feces. As samples were obtained from hunted wild boars intended for consumption, a potential contamination of meat with these bacterial pathogens must be considered.

Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry with Time-of-Flight Peak Analysis for Rapid and Accurate Detection of Group B Streptococcus in Pregnant Women.

ABSTRACTSevere infections in neonates caused by Streptococcus agalactiae, Group B Streptococcus (GBS), are often associated with GBS transmission from their mothers during labor or birth. Hence, it is necessary to develop a universal method for screening vaginal–rectal GBS colonization in pregnant women worldwide. A subculture of vaginal–rectal swabs using a selective enrichment broth and an agar plate is conventionally recommended for GBS screening. However, infants born to mothers who are GBS negative on subculture sometimes contract GBS infections. Therefore, we developed another method with high sensitivity for GBS screening. A total of 178 vaginal–rectal swabs from pregnant women were inoculated into the enrichment broth, of which 126 were suspected of containing GBS due to the change in the color of the broth. The subculture results were positive for GBS in 34 (27.0%) swabs. Each broth was then analyzed using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Analysis of the TOF peaks specific to GBS revealed 45 (35.7%) swabs as GBS positive. Of the 11 GBS positive samples on TOF peak analysis but negative on subculture, S. agalactiae gene targets were detected through PCR in 4 samples. MALDI detection with analysis of peaks of TOF (MDAPT) can detect GBS directly from cultured broth with high sensitivity. MDAPT can be an alternative method for GBS screening in pregnant women and contribute to the prevention of severe GBS infectious diseases in neonates.IMPORTANCE As previously reported, 10%–30% of pregnant women carry Streptococcus agalactiae, Group B Streptococcus (GBS), in their vagina or rectum, and approximately 50% of them vertically transmit GBS to their neonates during labor or birth. Moreover, 1%–2% of the GBS-transmitted neonates develop severe GBS infectious diseases, which have a mortality rate of 19.2% in a preterm infant and 2.1% in a full-term infant. Hence, universal screening for GBS colonization in pregnant women is conducted worldwide using the subculture procedure; however, infants born to GBS negative mothers sometimes contract GBS infections. Therefore, other laboratory techniques are required for detecting GBS more accurately. The proposed method “MALDI detection with analysis of peaks of TOF (MDAPT)” detects GBS directly from cultured broth with high sensitivity. Therefore, it can be an alternative method for GBS screening in pregnant women, thereby contributing to the prevention of severe GBS infectious diseases in neonates.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples.

Escherichia albertii is an emerging diarrheagenic pathogen causing food- and water-borne infection in humans. However, no selective enrichment broths for E. albertii have ever been reported. In this study, we tested several basal media, selective supplements and culture conditions which enabled selective enrichment of E. albertii. We developed a selective enrichment broth, novobiocin-cefixime-tellurite supplemented modified tryptic soy broth (NCT-mTSB). NCT-mTSB supported the growth of 22 E. albertii strains, while inhibited growth of other Enterobacteriaceae at 37°C, except for Escherichia coli and Shigella spp. Enrichment of E. albertii was improved further by growth at 44°C, a temperature that suppresses growth of several strains of E. coli/Shigella. Combined use of NCT-mTSB with XR-DH-agar, xylose-rhamnose supplemented deoxycholate hydrogen sulphide agar, enabled isolation of E. albertii when at least 1CFU of the bacterium was present per gram of chicken meat. This level of enrichment was superior to those obtained using buffered peptone water, modified-EC broth, or mTSB (with novobiocin). Novobiocin-cefixime-tellurite supplemented modified tryptic soy broth enabled effective enrichment of E. albertii from poultry samples and was helpful for isolation of this bacterium. To our knowledge, this is the first report of selective enrichment of E. albertii from poultry samples.

Heterogeneity in single-cell outgrowth of Listeria monocytogenes in half Fraser enrichment broth is affected by strain variability and physiological state

The behaviour of pathogens at the single-cell level can be highly variable and can thus affect the detection efficacy of enrichment-based detection methods. The outgrowth of single cells of three Listeria monocytogenes strains was monitored after fluorescence-activated single-cell sorting in non-selective brain heart infusion (BHI) broth and selective half Fraser enrichment broth (HFB) to quantify outgrowth heterogeneity and its effect on the detection probability. Single-cell heterogeneity was higher in HFB compared to non-selective BHI and heterogeneity increased further when cells were heat-stressed. The increase in heterogeneity was also strain-dependent because the fast-recovering strain Scott A showed less outgrowth heterogeneity than the slower-recovering strains EGDe and H7962. Modelling of the outgrowth kinetics during the primary enrichment demonstrated that starting at low cell concentrations could fail detection of L. monocytogenes at least partly due to cell heterogeneity. This highlights that it is important to take single-cell heterogeneity into account when optimizing enrichment formulations and procedures when L. monocytogenes contamination levels are low.

Comparison of selective enrichment and plating media for Salmonella isolation from broiler carcasses

AbstractSalmonella detection and isolation rely on different selective enrichment media, which can influence which serovars are detected. The objective of this study was to compare Salmonella recovery from broiler carcass rinses using three different selective enrichment protocols and three differential plating agars. Eight prechill broiler carcasses were collected at a commercial slaughter facility. Each carcass was subjected to whole carcass rinse procedure in buffered peptone water (BPW). An aliquot of the rinse and whole carcasses in the remaining rinse were incubated as a pre‐enrichment before subculturing in selective enrichment broths (Rappaport Vassiliadis [RV], Tetrathionate Hajna [TT], and TT to RV in series [TT‐to‐RV]). Enriched samples were streaked on the three differential agars (Hektoen Enteric [HE], Brilliant Green Sulfa [BGS], and Xylose‐Lysine‐Tergitol‐4 [XLT‐4]). Salmonella was isolated from all eight carcasses. Considering all sample preparations as independent subsamples, Salmonella was detected in 88% (128/144) of subsamples with a 100% recovery from the TT‐to‐RV enrichment, and 92 and 71% from RV and TT broths, individually. A high concordance in recovery on BGS versus XLT‐4 agar plates was observed compared to HE versus BGS and HE versus XLT‐4 plates. These data suggest that choice of pre‐enrichment method, selective enrichment medium, and differential agar can influence the recovery of Salmonella from poultry samples.

Research on quantitative method and contamination level of Salmonella enterica in raw pork from farmer's markets in Chengdu

To optimize the quantitative detection method for Salmonella enterica and analyze the quantitative contamination level of Salmonella enterica in raw pork samples from farmer's markets in Chengdu. Based on qualitative detection standard method of Salmonella enterica in China (GB 4789.4-2016) and the quantitative detection method of FSIS in the United States (MLG 4.08 and MLG appendix 2.05 MPN), the selective enrichment broth, screening plate, identification method and quantitative dilution ratio in quantitative detection of Salmonella enterica were optimized using 70 samples of raw pork. The optimized method compared by student's t-test was used to detect 40 samples of raw pork collected from farmer's markets in Chengdu from June to October 2020. For isolation of Salmonella from raw pork samples, the coincidence degree of TTB enrichment solution was significantly higher than that of RV enrichment solution (0.93±0.32 vs 0.35±0.62,t=8.324,P=0.001) and the consistency of suspicious colonies screened by XLT4 plate was significantly higher than that of Salmonella chromogenic medium (0.77±0.09 vs 1.00±0.00,t=2.971,P =0.017). The MPN method used 4 successive gradient dilutions, namely 12 tube method, could obtain more accurate quantitative value for Salmonella enterica. The combined use of selective enrichment broth TTB, XLT4 plate, Real-time PCR and MALDI-TOF mass spectrometry could get better results for screening and identifying Salmonella enterica. The detection rate for Salmonella enterica isolated from raw pork in farmer's markets was 92.5% (37/40). The most of the Salmonella positive samples (83.8%, 31/37) were detected with a contamination level ranged from 0.1 to 55 MPN/g. The optimized quantitative detection method for Salmonella enterica in raw pork in this study can effectively screen the target bacteria and obtain more accurate quantitative value.

Determination of Antimicrobial Resistance Patterns in Salmonella from Commercial Poultry as Influenced by Microbiological Culture and Antimicrobial Susceptibility Testing Methods.

Monitoring antimicrobial resistance of foodborne pathogens in poultry is critical for food safety. We aimed to compare antimicrobial resistance phenotypes in Salmonella isolated from poultry samples as influenced by isolation and antimicrobial susceptibility testing methods. Salmonella isolates were cultured from a convenience sample of commercial broiler ceca with and without selective broth enrichment, and resistance phenotypes were determined for 14 antimicrobials using the Sensititre® platform and a qualitative broth breakpoint assay. The broth breakpoint method reported higher resistance to chloramphenicol, sulfisoxazole, and the combination of trimethoprim and sulfamethoxazole, and lower resistance to streptomycin as compared to the Sensititre® assay in trial one. Selective enrichment of samples containing Salmonella in Rappaport-Vassiliadis broth reported lowered detectable resistance to amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, nalidixic acid, and meropenem, and increased resistance to streptomycin and tetracycline than direct-plating samples in trial one. Using matched isolates in trial two, the Sensititre® assay reported higher resistance to chloramphenicol and gentamicin, and lower resistance to nalidixic acid as compared to the broth breakpoint method. These results suggest methodology is a critical consideration in the detection and surveillance of antimicrobial resistance phenotypes in Salmonella isolates from poultry samples and could affect the accuracy of population or industry surveillance insights and intervention strategies.