Tryptic Soy Broth Research Articles

The influence of different culture media on Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus biofilm formation

Microorganisms such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus are frequently isolated in samples of urinary, blood, intestinal, and respiratory infections, among others. These bacteria are also associated with microbial biofilm formation. E. coli, P. aeruginosa, and S. aureus biofilm infections are particularly hard to manage and often associated with nosocomial problems. This study investigated the influence of different culture media on E. coli, P. aeruginosa, and S. aureus biofilm formation. Bacterial performance was evaluated in brain heart infusion broth, Mueller-Hinton broth, or tryptic soy broth, with or without supplementing with different glucose levels (1-5%). The study quantified biofilm biomass and the count of viable biofilm colonies. This is the first study that compares the biofilm formation of E. coli, P. aeruginosa and S. aureus in polystyrene using different culture media and with different glucose concentrations. The most robust growth of E. coli, P. aeruginosa, and S. aureus occurred in brain heart infusion broth supplemented with 5% glucose, Mueller-Hinton broth without glucose, and tryptic soy broth with 2% glucose, respectively. Our data demonstrate that behavioral and morphological characteristics of each bacterium require a specific broth to enhance the growth of these microorganisms. These findings will contribute to future tests for therapeutic alternatives with anti-biofilm potential.

Comparison of Phenotype Nutritional Profiles and Phosphate Metabolism Genes in Four Serovars of Salmonella enterica from Water Sources.

The surveillance of foods for Salmonella is hindered by bias in common enrichment media where serovars implicated in human illness are outgrown by less virulent serovars. We examined four Salmonella serovars, two common in human illness (Enteritidis and Typhimurium) and two that often dominate enrichments (Give and Kentucky), for factors that might influence culture bias. The four serovars had similar growth kinetics in Tryptic Soy Broth and Buffered Peptone Water. Phenotype microarray analysis with 950 chemical substrates to assess nutrient utilization and stress resistance revealed phenotype differences between serovars. Strains of S. Enteritidis had better utilization of plant-derived sugars such as xylose, mannitol, rhamnose, and fructose, while S. Typhimurium strains were able to metabolize tagatose. Strains of S. Kentucky used more compounds as phosphorus sources and grew better with inorganic phosphate as the sole phosphorus source. The sequences of nine genes involved in phosphate metabolism were compared, and there were differences between serovars in the catalytic ATP-binding domain of the histidine kinase phoR. Analysis of the predicted PhoR amino acid sequences from additional Salmonella genomes indicated a conservation of sequences each within the Typhimurium, Give, and Enteritidis serovars. However, three different PhoR versions were observed in S. Kentucky.

Refrigerated multi-dose insulin vials remain sterile through 6 months of use.

To evaluate sterility in refrigerated multi-dose insulin vials through 6 months of routine aspiration. Twelve vials of insulin, six of insulin glargine U100 (Lantus®, 10 mL multi-dose vial, Sanofi, Bridgewater, NJ) containing the preservative metacresol, and six of protamine zinc insulin U40 (ProZinc®, 10 mL multi-dose vial, Boehringer Ingelheim, Duluth, GA) containing the preservative phenol, were refrigerated and aspirated twice daily for 6 months, using a new insulin syringe each time. Three vials of each insulin type were wiped with a single-use alcohol swab before sampling. Three times weekly, aspirated samples were inoculated in Tryptic Soy Broth enrichment media and incubated for evidence of microbial growth. Positive broth was cultured and speciated. Endpoints were microbial vial contamination (defined as three consecutive positive cultures of the same organism) and completion of the six-month study period. Microbial contamination was not identified in any vial throughout the study period. A total of 454 aspirated samples were cultured, one of which exhibited non-repeatable growth of Staphylococcus epidermidis. This vial was prematurely lost to breakage after 59 culture samples (29 after the positive growth). Refrigerated phenol- and metacresol-containing multi-dose insulin products carry minimal risk for iatrogenic infection through 6 months of use, regardless of alcohol swab preparation.

Characteristics of Staphylococcus aureus biofilm matured in tryptic soy broth, low-fat milk, or whole milk samples along with inactivation by 405 nm light combined with folic acid

In the present study, the characteristics of Staphylococcus aureus biofilms matured in tryptic soy broth (TSB), low-fat milk, or whole milk samples were identified along with their resistance to 405 nm light with or without folic acid. Phenotypic properties of carbohydrate and protein contents in extracellular polymeric substance (EPS) of S. aureus biofilms matured in different conditions were identified. The carbohydrate content was higher in the biofilm matured in low-fat milk (1.27) than the samples matured in whole milk (0.58) and TSB (0.10). Protein content in the EPS of biofilm was higher in the sample matured in whole milk (6.59) than the samples matured in low-fat milk (3.24) and TSB (2.08). Moreover, the maturation condition had a significant effect on the membrane lipid composition of the biofilm, producing more unsaturated fatty acids in biofilm matured in milk samples. These changes in biofilm matured in milk samples increased the resistance of S. aureus to 405 nm light in the presence of folic acid (LFA). Additionally, transcriptomic analysis was conducted to identify the response of S. aureus biofilm to LFA treatment. Several genes related to DNA and protein protection from oxidative stress along with biofilm accumulation were overexpressed in the LFA-treated biofilms. These results indicate the maturation of S. aureus biofilm in various samples and the biofilms responses to bactericidal treatments.

Isolation and identification of Bacillus species from soil and assessment of antimicrobial properties

The emergence of multi drug resistant pathogens such as Methicillin resistant Staphylococcus aureus (MRSA) has become a threat to public health. Thus, the development of new antibiotics has become a global concern. Many scientists are attracted towards discovery of new antimicrobial agents from microbial sources. The aim of this study is to isolate potential Bacillus species from soil for the production of antimicrobial substance. During the study, 20 soil samples were collected from various areas of Kathmandu valley. A total of 70 Bacillus isolates were identified by morphological and biochemical characteristics. Altogether 39 isolates were able to produce antibacterial extract when it was grown in Trypticase soya broth (TSB) at 37°C for 48 h. The antimicrobial activity was tested against test organisms like Escherichia coli, Klebsiella pneumoniae, Salmonella, Pseudomonas, Staphylococcus aureus and Candida by Agar well diffusion method. All of the extracts showed zone of inhibition against E. coli except 6 extracts (E39, E43, E36, E25, E45 and E46). The highest zone of inhibition was shown by extract B68 with 11 mm. Only one extract (E62) was able to inhibit the growth of Pseudomonas. The extract from Bacillus isolate (E35) showed highest zone of inhibition against S. aureus (6mm). The protein nature of the extracts was determined by Biuret and Ninhydrin tests. The extracts were relatively stable to heat treatment and unstable to surfactants like 70% methanol, 2-propanol and ethylacetate. The study revealed that soil is a good source for the potential antimicrobial producer bacteria.

First Report of Pectobacterium brasiliense Causing Bacterial Blackleg and Soft Rot of Potato in Pennsylvania.

Potato (Solanum tuberosum) plants showing blackleg and soft rot symptoms were collected at a commercial vegetable farm near Newmanstown, PA in August 2021 (Fig. S1). The incidence of potato blackleg in the unirrigated field was about 5 to 8%, but approximately 30% in the irrigated field. The diseased stems were cut into 5 cm and surface disinfected. The stem segments were placed into a 50-mL tube containing 15 mL of sterile water for 15 min for bacterial release. The bacterial suspension was streaked on crystal violet polypectate (CVP) (Hélias et al. 2012) plates and incubated at 28°C for 48 h. Three single colonies produced pits on CVP were picked and purified. Genomic DNA of all three isolates were extracted using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA). Polymerase chain reaction (PCR) was performed using all three extracted DNAs as a template with the primer pairs gapA 7F/938R (Cigna et al. 2017), recA F/R (Waleron et al. 2001), dnaA F/R (Schneider et al. 2011) and dnaX F/R (Sławiak et al. 2009) targeting the gapA, recA, dnaA and dnaX genes, respectively. Isolate 21PA01 was further studied as a representative isolate. PCR amplicons derived from both forward and reverse primers were sequenced and analyzed using the BLAST algorithm against the NCBI database (https://www.ncbi.nlm.nih.gov). The regions of gapA (GenBank accession No. ON989738), recA (ON989739), dnaA (OP121183), and dnaX (OP121184) had 99.86%, 100%, 98.88%, and 100% identities with Pectobacterium brasiliense strains S1.16.01.3M (MN167062.1), BL-2 (MW721598.1), IPO:4132 (CP059956.1), and BL-2 (MW721603.1), respectively. A phylogenetic maximum-likelihood tree of the concatenated genes with the length of 2551 bp was constructed to visualize the relationship among different species of Dickeya and Pectobacterium. As a result, 21PA01 was in a single monophyletic cluster with other Pectobacterium brasiliense reference strains (Fig. S2 C). To confirm the pathogen, Koch's postulates were performed. Seed pieces of potato 'Lamoka' were planted in potting mix in one-gallon plastic pots in a greenhouse. Three weeks after emergence, the stems of three plants were each injected with 10 µL of bacteria suspension of either 21PA01 at 107 CFU/mL, P. parmentieri ME175 in tryptic soy broth (TSB) at 107 CFU/mL or TSB at 2 cm above the soil line. Seven days after inoculation, stems inoculated with 21PA01 and ME175 showed black and rotten symptoms, whereas the TSB-injected control plants remained symptomless. In addition, 'Lamoka' tubers were inoculated by placing 10 µL 21PA01 and ME175 suspensions at 107 CFU/mL, and TSB in a 1-cm-deep hole poked in a tuber separately and then sealed with petroleum gel, followed by incubation in a moist chamber at 22 °C for 4 d. The 21PA01 and ME175 inoculated tubers showed soft rot symptoms, but the TSB treatment had no symptoms. Bacterial colonies were isolated from the infected stems and confirmed by the DNA sequences as described above. PCR result was negative on control plant samples. Both stem and tuber inoculation trials were repeated two times, and the results were consistent. Thus, 21PA01 was identified as Pectobacterium brasiliense. To our knowledge, this is the first report of P. brasiliense infecting potatoes in Pennsylvania, USA, although it has been reported somewhere else (van der Merwe et al. 2010, Zhao et al. 2018). This could be a new species in Northeastern US.

First identification of Xanthomonas nasturtii as the cause of black rot of watercress in Hawaii.

Watercress (Nasturtium officinale) has been in continuous production in Hawaii for over a century and is part of the local diet. Black rot of watercress was first identified as caused by Xanthomonas nasturtii in Florida (Vicente et al., 2017), but symptoms of this disease have also been regularly observed in Hawaii production in all islands, mostly during the rainy season from December to April in areas with poor air circulation (McHugh & Constantinides, 2004). Initially, this disease was attributed to X. campestris due to similar symptoms to black rot of brassicas. Samples of watercress with symptoms that could be attributed to a bacterial disease including yellow spots and lesions on leaves and stunting and deformation of plants in more advanced stages, were collected from a farm in Aiea in the island of Oahu, Hawaii, in October 2017. Isolations were performed at the University of Warwick. Fluid from macerated leaves was streaked into plates of King's B (KB) medium and Yeast Dextrose Calcium Carbonate Agar (YDC). After 48-72 hrs incubation at 28°C, the plates showed a range of mixed colonies. Single cream-yellow mucoid colonies were sub-cultured several times and pure isolates including WHRI 8984 were stored at -76°C as previously described (Vicente et al., 2017). Colony morphology was observed in KB plates and, in contrast to the type strain from Florida (WHRI 8853 = NCPPB 4600), isolate WHRI 8984 did not cause browning of the medium. Pathogenicity was tested on four-week old watercress and Savoy cabbage cv. Wirosa F1 plants by inoculations on leaves as previously described (Vicente et al., 2017). WHRI 8984 did not produce symptoms when inoculated on cabbage but produced typical symptoms on watercress. A re-isolation from a leaf showing a V-shaped lesion, produced isolates with the same morphology, including isolate WHRI 10007A, that was also shown to be pathogenic to watercress therefore completing the Koch's postulates. Fatty acid profiling was performed on WHRI 8984 and 10007A and controls grown on trypticase soy broth agar (TSBA) plates at 28°C for 48 hrs as described by Weller et al. (2000). Profiles were compared with the RTSBA6 v6.21 library; as the database does not include X. nasturtii, the results were only interpreted at the genus level, and both isolates were shown to be Xanthomonas sp. For molecular analysis, DNA was extracted and the gyrB partial gene was amplified and sequenced as described by Parkinson et al. (2007). Comparisons with sequences available in the National Centre for Biotechnology Information (NCBI) databases using the Basic Local Alignment Search Tool (BLAST) showed that partial gyrB of WHRI 8984 and 10007A were identical to the type strain from Florida therefore confirming that they belong to X. nasturtii. For whole genome sequencing, genomic libraries for WHRI 8984 were prepared using Illumina's Nextera XT v2 kit and sequenced on a HiSeq Rapid Run flowcell. The sequences were processed as previously described (Vicente et al., 2017) and the whole genome assembly has been deposited in GenBank (accession QUZM00000000.1); the phylogenetic tree shows that WHRI 8984 is close, but not identical to the type strain. This is the first identification of X. nasturtii in watercress crops in Hawaii. Control of this disease generally involves the use of copper bactericides and minimizing moisture on leaves by reducing overhead irrigation and increasing air circulation (McHugh & Constantinides, 2004); seed testing might help to select batches that are disease free and, in longer term, breeding for disease resistance might produce cultivars that can be part of management strategies.

Therapeutic efficacy of coriander (Coriandrum sativum) enriched diets in Oreochromis niloticus: effect on hepatic-renal functions, the antioxidant-immune response and resistance to Aeromonas veronii.

In this study, the effects of Coriandrum sativum to control Aeromonas veronii infection in Oreochromis niloticus were determined. Coriandrum sativum extract (CE) was tested in vitro against A. veronii by the disc diffusion assay. In in vivo, 150 O. niloticus (from El-Abbassa, Sharkia, Egypt, weighing 34.95 ± 1.98 g) was distributed in five groups (with three replications) in glass aquariums (80 × 40 × 30 cm). The first group (control) was intraperitoneally injected with 0.2 ml of sterilized tryptic soya broth. Groups 2-5 were intraperitoneally challenged with 0.2 ml of A. veronii (4.3 × 106). The five groups were administered a basal diet until clinical signs appeared, and then therapeutic feeding (15 days) was followed: the first (CONT) and second (AV) groups were administered a normal basal diet. The third (AV+CP) and fourth (AV+CE) groups were administered diets supplemented with C. sativum powder and extract, respectively, each at 30 mg/kg. The fifth group (AV+OT) was administered a diet supplemented with oxytetracycline at 500 mg/kg diet. The results of the in vitro experiment revealed that CE has a zone of inhibition of 43 mm against A. veronii. The in vivo results showed that fish administered a therapeutic diet supplemented with CE showed a significant improvement in hematological, biochemical, and immunological parameters, as well as antioxidant capacity (P < 0.05) and the pathological findings of the liver and kidney tissues. The current findings supported that the administration of a CE-enriched diet (30 mg/kg) is an eco-friendly strategy for controlling A. veronii in O. niloticus.

Immunological response of 35 and 42 days old Asian seabass (Lates calcarifer, Bloch 1790) fry following immersion immunization with Streptococcus iniae heat-killed vaccine.

Early disease prevention by vaccination requires understanding when fry fish develop specific immunity to a given pathogen. In this research, we explored the immune responses of Asian seabass (Lates calcarifer) at the stages of 35- and 42- days post-hatching (dph) to an immersive heat-killed Streptococcus iniae (Si) vaccine to determine whether fish can produce specific antibodies against the pathogen. The vaccinated fish of each stage (V35 and V42) were immersed with the Si vaccine at 107CFU/ml for 3h, whereas the control groups (C35 and C42) were immersed with tryptic soy broth (TSB) in the same manner. Specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) before and post-immunization (i.e., 0, 7, and 14 days post-immunization, dpi). Expression of innate (TNFα and IL-1β) and adaptive (MHCI, MHCII, CD4, CD8, IgM-like, IgT-like, and IgD-like) immune-related genes were evaluated at the same time points with the addition of 1 dpi. The results showed that a subset of immunized fish from both V35 and V42 fry could elicit specific antibodies (IgM) against Si at 14 dpi. All tested innate and adaptive immune genes upregulated at 7 dpi among fish in V35 group. Interestingly, 42 dph fish appeared to respond to the Si vaccine faster than that of 35 dph, as a significant increase in transcripts was observed in CD4, IL-1β, IgM-like, and IgD-like at 1 dpi; and specific antibody titers of some fish, although not all, were higher than a threshold (p=0.05) since 7 dpi. In conclusion, this study reveals that 35-42 dph Asian seabass fry can elicit specific immunity to Si immersion vaccine, suggesting that early vaccination of 35 dph fry Asian seabass is feasible.

Streptococcus angriness isolated from Patients with Acute Infection following Surgical Tooth Extraction

In this study, 250 individuals with acute infection following surgical tooth extraction were treated at a Hilla dental clinic and cultivated at 37°C for 18–24 hours. They cultured on several selective medium at 37°C for 18–24 hours. Identification of Streptococcus anginosus was based on colonial morphology, microscopically, and biochemical tests, and all scientific specimens were successful cultures. Of 250 specimens, only 25(10%) were biochemically identified as Strep. anginosus, while 225(90%) were associated with other microorganisms. Hemolysin-producing Strep. anginosus isolates have been studied. 10(66.66%) isolates produced hemolysin in blood agar that formed a clearance sector around the colonies and a streak on the plate after 24 hours of incubation at 37°C. Strep. anginosus isolates were also tested for siderophores production, 5(33.33%) Strep. anginosus isolates produce siderophores. All bacterial isolates provide protease to hydrolyze protein. After 24 hours of incubation, all Strep. anginosus isolates were able to hydrolyze protein using protease enzyme (100%) when tested using M9 (supported by 20% glucose and 1% csaien). Adding 3ml of 5% Trichloroacetic acid gave the colony a clean hallo. Quantitative biofilm development tests were performed in a microtiter using Trypticase Soy Broth with 1% glucose. To improve accuracy, this assay was done three times. All Strep. anginosus isolates were biofilm former (100%), 12/15(80%) were strong biofilm formers, and 3/15 (20%) were mild biofilm formers. However, the antibiotic disc diffusion test using six clinically important antibiotics showed that most Strep. anginosus isolates were extremely resistant to the antibiotics, especially the ?-lactams, including Amoxicillin 13(86.6%), Cefixime 12(80%), Ceftriaxone 12(80%), Meropenem, 11(73.33%) Cefotaxime 10(66.66%), and Imipenem 9(60%).

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.

Performance and transcriptome analysis of Salmonella enterica serovar Enteritidis PT 30 under persistent desiccation stress: Cultured by lawn and broth methods

Lawn-harvest method uses a solid medium (e.g., tryptic soy agar, TSA) to produce bacterial lawns and is widely accepted for the culture of microorganisms in microbial studies of low-moisture foods (LMFs, foods with water activity less than 0.85). It produces desiccation-tolerant cells with higher D-values in LMFs; however, little is known about the molecular mechanisms underlying bacterial resistance. Salmonella enterica Enteritidis PT 30 (S. Enteritidis), the most pertinent pathogen in LMFs, was cultured in TSA and tryptic soy broth (TSB). Cells were harvested and inoculated on filter papers to assess their performance under a relative humidity of 32 ± 2%. Transcriptome analysis of cultured cells during long-term desiccation (24, 72, and 168 h) was conducted in TruSeq PE Cluster Kit (Illumina) by paired-end methods. Lawn-cultured S. Enteritidis cells have stronger survivability (only decreased by 0.78 ± 0.12 log after 130 d of storage) and heat tolerance (higher D/β value) than those from the broth method. More desiccation genes of lawn-cultured cells were significantly upregulated from growth to long-term desiccation. Differentially expressed genes were the most enriched in the ribosome and sulfur metabolism pathways in the lawn- and broth-cultured groups. This study tracked the transcriptomic differences between two cultured groups in response to long-term desiccation stress and revealed some molecular mechanisms underlying their different suitability in microbial studies of LMFs.

Effect of pH, temperature, and salt concentration on the growth of Bacillus subtilis T9-05 isolated from fish sauce

Biogenic amines (BAs) are organic compounds found in fermented food products, such as sausage, fermented vegetables, wine, and fish sauce. Due to their potential to be harmful to human health and as markers of food quality, BAs content in food can be used as an indicator of food quality and food safety. Some bacteria have the ability to produce or degrade the BAs during fermentation. Environmental conditions can influence the growth of bacteria that produce biogenic amines. The aim of this study was to investigate the growth of Bacillus subtilis T9-05 isolated from fish sauce. Bacillus subtilis T9-05 was grown in tryptic soy broth with the treatment of pH (5, 6, 7, 8, and 9) and salt concentration (0, 1, 3, 6, 9, and 12% (w/v)) and the growing colonies were counted by the total plate count (TPC) method. Bacterial growth was also observed using different temperatures (25°C, 30°C, 35°C, 40°C, and 45°C). Differences in pH conditions and salt concentration significantly affected the growth of Bacillus subtilis T9-05. The highest growth of Bacillus subtilis T9-05 was achieved at pH 7-8 and a salt concentration of 0-1%. Bacillus subtilis T9-05 is classified as halotolerant bacteria. Bacillus subtilis T9-05 could be categorized as mesophilic bacteria based on its optimal growth rate of 35°C.

The growth of mercury-resistant bacterial consortium at different levels of pH, temperature, and C:N ratio

The study was conducted to investigate the growth of mercury-resistant bacterial consortium under the influence of different pHs, temperatures, and C:N ratios, as a part of mercury bioremediation research. The consortium consisted of five bacterial isolates selected due to their resistance to HgCl2 up to 100 ppm and was proven to effectively reduce mercury from the culture media. Consortium suspension was inoculated into two sets of media: 1) Tryptic soy broth (TSB) with pHs of 4.8, 6.1, 8.5, and 9.7; 2) Mineral salt with the addition of glucose and peptone to create C:N ratios of 20:1, 30:1, and 40:1 while the pH was set to 7.4. For comparison, a solution containing mercury-contaminated soil was added to the same set of media and regarded as a microbial community. The growth curves show that the bacterial consortium tended to grow better compared to the microbial community, especially in the TSB medium at incubation temperatures of 20 and 35 °C. In the same medium at a temperature of 45 °C, the growth of both consortium and community under high pHs (8.5 and 9.7) was higher than the one under lower pHs (4.8 and 6.1). Meanwhile, in the mineral salt medium, the influence of the inoculant was not remarkable except under an incubation temperature of 35 °C. Moreover, the C:N ratio did not affect the growth of either the bacterial consortium or the microbial community (p > 0.05). The variance analysis also indicated that the growths were significantly different only when the pH was 4.8.

Studying the metabolic factors that may impact the growth of co-cultured Listeria monocytogenes strains at low temperature

The simultaneous presence of more than one strains of Listeria monocytogenes in the same food product may affect the growth capacity of each strain. The present study evaluated the metabolites composition that may potentially influence the growth of individual L. monocytogenes strains in a dual strain composite. Based on previous studies, L. monocytogenes strains, C5 (4b) and 6179 (1/2a) were selected due to the remarkable interaction, which was observed during their co-culture. The selected strains were inoculated (2.0 - 3.0 log CFU/mL) in Tryptic Soy Broth with 0.6% Yeast Extract (TSB-YE) in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic conditions (AC). Their resistance to different antibiotics enabled the selective enumeration of each strain in the co-culture. After reaching stationary phase, single and dual cultures were centrifuged and filtered. The cell-free spent medium (CFSM) was either characterized by Fourier transform infrared (FTIR-ATR) spectrometry or re-inoculated, after the addition of concentrated TSB-YE (for nutrient replenishment), with single and two-strain cultures for the evaluation of growth under the influence of metabolites produced from the same singly and co-cultured strains in the different combinations of strains and CFSM origin (7 °C/AC) (n = 2x3). By the end of storage, singly-cultured C5 and 6179 had reached 9.1 log CFU/mL, while in dual culture, 6179 was affected by the presence of C5 attaining only 6.4 ± 0.8 log CFU/mL. FTIR-ATR spectra of CFSM produced by singly-cultured 6179 and the co-culture were almost identical. Characteristic peaks in FTIR-ATR spectrum of CFSM of singly-cultured C5 at 1741, 1645 and 1223 cm−1 represent functional groups which were not present in the CFSM of the co-culture. These molecules may be located intracellularly or mounted on bacterial cell surface and removed from the supernatant during cell filtration of the co-culture. Both singly- and co-cultured 6179 managed to grow similarly regardless of CFSM origin. Contrarily, both singly- and co-cultured C5 managed to outgrow 6179 in CFSM which contained high concentration of C5 metabolites, while in CFSM produced by singly-cultured 6179, C5 did not grow, suggesting that the produced metabolites of strain 6179 appears to be harmful to strain C5. However, during co-culture, C5 may produce molecules that counteract the inhibitory effect of 6179. The findings shed more light on the mechanism behind the inter-strain interactions of L. monocytogenes indicating that both contact of cells and extracellular metabolites may influence the behavior of the different co-existing strains.

The biochemical characteristics of viable but nonculturable state Yersinia enterocolitica induced by lactic acid stress and its presence in food systems

The viable but nonculturable (VBNC) state is adopted by many foodborne pathogenic bacteria to survive in adverse conditions. This study found that lactic acid, a widely used food preservative, can induce Yersinia enterocolitica to enter a VBNC state. Y. enterocolitica treated with 2 mg/mL lactic acid completely lost culturability within 20 min, and 10.137 ± 1.693 % of the cells entered a VBNC state. VBNC state cells could be recovered (resuscitated) in tryptic soy broth (TSB), 5 % (v/v) Tween80−TSB, and 2 mg/mL sodium pyruvate-TSB. In the VBNC state of Y. enterocolitica induced by lactic acid, the intracellular adenosine triphosphate (ATP) concentration and various enzyme activities were decreased, and the reactive oxygen species (ROS) level was elevated, compared with uninduced cells. The VBNC state cells were significantly more resistant to heat and simulated gastric fluid than uninduced cells, but their ability to survive in a high−osmotic−pressure environment was significantly less than that of uninduced cells. The VBNC state cells induced by lactic acid changed from long rod-like to short rod-like, with small vacuoles at the cell edges; the genetic material was loosened and the density of cytoplasm was increased. The VBNC state cells had decreased ability to adhere to and invade Caco−2 (human colorectal adenocarcinoma) cells. The transcription levels of genes related to adhesion, invasion, motility, and resistance to adverse environmental stress were downregulated in VBNC state cells relative to uninduced cells. In meat-based broth, all nine tested strains of Y. enterocolitica entered the VBNC state after lactic acid treatment; among these strains, only VBNC state cells of Y. enterocolitica CMCC 52207 and Isolate 36 could not be recovered. Therefore, this study is a wake−up call for food safety problems caused by VBNC state pathogens induced by lactic acid.

Growth comparison and model validation for growth of Shiga toxin-producing Escherichia coli (STEC) in ground beef

The aim of this study was to compare the growth of non-O157 Shiga toxin-producing Escherichia coli (STEC) with E. coli O157:H7 in ground beef and modified Tryptic Soy Broth (TSB). Additionally, the performance of three available models (ComBase, Huang et al. model, and Cepeda et al. model), for predicting STEC growth in ground beef was evaluated. To achieve this, eleven groups of STEC, including E. coli O157:H7, were inoculated into irradiated ground beef (7% or 27% fat) or modified TSB, and then subjected to programmed water baths to follow time-varying sinusoidal temperature profiles for 300 h (low temperature, 5–15 °C) and 25 h (high temperature, 10–40 °C). The growth data of STEC in ground beef were fitted to the three models, and model performance indices such as mean relative percentage error (MRPE), mean absolute relative error (MARE), root mean squared error (RMSE), bias factor (BF) and accuracy factor (AF) were calculated to assess their accuracy in predicting STEC growth. The study found that the growth of non-O157 STEC was similar to that of E. coli O157:H7, indicating that growth models for E. coli O157:H7 can be used to predict the growth of non-O157 STEC. The fat content did not significantly affect STEC growth in ground beef. Among the three models tested, the Huang et al. model underestimated the growth of all STEC in ground beef, with an RMSE of up to 2.14 log CFU/g. The ComBase model also under-predicted STEC growth in ground beef at high temperatures (10–40 °C) and could not predict growth at temperatures below 10 °C, where STEC might grow slowly. In contrast, the Cepeda et al. model accurately predicted STEC growth in ground beef and is considered an conservative growth model for STEC in commercial ground beef products. In summary, this study provides important insights into the growth of non-O157 STEC and E. coli O157:H7 in ground beef and their modeling, which can aid in predicting the safety of commercial ground beef products.

Proof of concept for detection of staphylococcal enterotoxins in platelet concentrates as a novel safety mitigation strategy.

Staphylococcus aureus is a predominant contaminant of platelet concentrates (PCs) that can evade detection during screening with culture methods. Importantly, S. aureus produces staphylococcal enterotoxins (SEs) during PC storage, which are linked to slow growth and enhanced biofilm formation. This study investigated timing of SE production during PC storage and feasibility of SE detection as a PC safety strategy. Genomic and transcriptomic data of transfusion-relevant S. aureus PS/BAC/169/17/W, PS/BAC/317/16/W, CI/BAC/25/13/W and CBS2016-05 were used to determine the presence and differential expression of exotoxin genes in PCs. Trypticase soy broth (TSB) and PCs were inoculated with 1.0E+06 cfu/mL of S. aureus PS/BAC/169/17/W and CBS2016-05. Expression of SEs at different growth phases was confirmed with Western blotting. PCs were inoculated with 30 cfu/unit of the same strains, and SE detection during PC storage was optimized with a sandwich dot-ELISA assay. S. aureus genomes contain multiple exotoxin genes including those encoding for SEs. Transcriptome data revealed significant upregulation (0.5-6.7-fold, p < 0.05) of SE genes in PCs versus TSB. Western blots demonstrated SE production at all growth phases. Notably, dot-ELISA detected clinically relevant concentrations of SEs (~0.2 μg/mL) at 32 h of PC storage when S. aureus PS/BAC/169/17/W and CBS2016-05 counts were 1.8E+04 and 1.4E+04 cfu/mL, respectively. Genomic analyses revealed that staphylococcal exotoxins are widely distributed and highly conserved among transfusion-relevant S. aureus isolates. Furthermore, SEs are significantly upregulated in PCs and detected at 30 h of PC storage. Therefore, bacterial toxin detection could supplement mitigation strategies to enhance PC safety.

Inhibition of Listeria monocytogenes by Broth Cultures of Surface Microbiota of Wooden Boards Used in Cheese Ripening

Listeria monocytogenes is a significant concern in cheese production. It has been assumed by some that wood surfaces pose a greater risk for pathogen contamination during cheese production. The goal of this study was to evaluate the effect of the microbiota obtained from wooden boards used in cheese ripening on the growth of Listeria monocytogenes. Bacterial communities from the surface of wooden boards obtained from cheese-ripening facilities were inoculated into tryptic soy broth (TSB) and incubated at 11 °C for 48 h. These communities (108 CFU/mL stationary phase cells) were co-incubated with 104–105 CFU/mL L. monocytogenes 2203 at 11 °C for up to eight days. At various times, samples were removed, diluted in sterile saline and plated on modified Oxford agar. Bacterial communities from each of the five boards from three different facilities significantly inhibited growth of L. monocytogenes in vitro, compared to growth of L. monocytogenes 2203 alone. Using 16S rRNA analysis, we identified sequences belonging to the genera Carnobacterium, Leuconostoc and Staphylococcus as the most abundant in the communities grown in TSB. Leuconostoc mesenteroides and Staphylococcus equorum isolated from the most inhibitory community significantly inhibited growth of L. monocytogenes in TSB at 11 °C, compared to growth of L. monocytogenes 2203 alone. These findings suggest that some members of the complex microbial communities on wooden boards in cheese aging facilities might inhibit the growth of L. monocytogenes.

Rapid Identification of Carbapenemase-Producing Klebsiella pneumoniae Using Headspace Solid-Phase Microextraction Combined with Gas Chromatography-Mass Spectrometry.

Carbapenemase-producing Klebsiella pneumoniae is an unprecedented threat to public health, and its detection remains challenging. Analysis of microbial volatile organic compounds (VOCs) may offer a rapid way to determine bacterial antibiotic susceptibility. The aim of this study was to explore the VOCs released by carbapenemase-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) using headspace solid-phase microextraction/gas chromatography-mass spectrometry (HS-SPME/GC-MS). Test bacteria were incubated in trypticase soy broth to the end of exponential growth phase, and imipenem was added in the middle time. Headspace VOCs were concentrated and analyzed using HS-SPME/GC-MS. The compound 3-methyl-1-butanol was found to be a biomarker among the 26 bacterial isolates (10 KPC-positive, 10 NDM-positive, 2 IMP-positive, 2 carbapenemase-negative CRKP, and 2 carbapenem-susceptible K. pneumonoiae). This study explored a promising new strategy for the screening of carbapenemase-producing CRKP strains. Further research with larger sample sizes will potentially accelerate the application of biomarkers in routine microbiology.