Tryptic Soy Broth Culture Research Articles

The impact of different acidic conditions and food substrates on Listeria monocytogenes biofilms development and removal using nanoencapsulated carvacrol

Listeria monocytogenes biofilms present a significant challenge in the food industry. This study explores the impact of different acidic conditions of culture media and food matrices on the development and removal of biofilms developed on stainless steel surfaces by wild-type (WT) L. monocytogenes strains as well as in two mutant derivatives, ΔsigB and ΔagrA, that have defects in the general stress response and quorum sensing, respectively. Additionally, the study investigates the efficacy of nanoencapsulated carvacrol as an antimicrobial against L. monocytogenes biofilms developed in Tryptic Soy Broth (TSB) culture media acidified to different pH conditions (3.5, 4.5, 5.5, 6.5), and in food substrates (apple juice, strained yogurt, vegetable soup, semi-skimmed milk) having the same pH levels. No biofilm formation was observed for all L. monocytogenes strains at pH levels of 3.5 and 4.5 in both culture media and food substrates. However, at pH 5.5 and 6.5, increased biofilm levels were observed in both the culture media and food substrates, with the WT strain showing significantly higher biofilm formation (3.04–6.05 log CFU cm−2) than the mutant strains (2.30–5.48 log CFU cm−2). For both applications, the nanoencapsulated carvacrol demonstrated more potent antimicrobial activity against biofilms developed at pH 5.5 with 2.23 to 3.61 log reductions, compared to 1.58–2.95 log reductions at pH 6.5, with mutants being more vulnerable in acidic environments. In food substrates, nanoencapsulated carvacrol induced lower log reductions (1.58–2.90) than the ones in TSB (2.02–3.61). These findings provide valuable insights into the impact of different acidic conditions on the development of L. monocytogenes biofilms on stainless steel surfaces and the potential application of nanoencapsulated carvacrol as a biofilm control agent in food processing environments.

Potential Application of Bacterial Lipase from Bacillus subtilis in the Production of Biodisel

This work aims to evaluate the potential of bacterial lipase of Bacillus subtilis in biodiesel production. Assays were performed to obtain, purify, and immobilize the enzyme and compare chemical and enzymatic transesterification tests. The Tryptic Soy Broth (TSB) culture medium without supplementation showed the highest enzymatic activity and purification yields when using 60% of (NH4)2SO4. Citral was the most efficient agent for lipase immobilization. Even though the free enzymes showed a higher activity rate, the immobilized enzymes are associated with a better-quality product. In conclusion, the lipase enzyme showed positive potential in biodiesel production.

Abstract B043: Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer

Abstract Breast cancer is the most common cause of mortality among women worldwide. Among all subtypes, HER2-positive breast cancers are highly aggressive and have poor prognosis. Surgical resection is choice of treatment. However, surgery may not be possible if disease is widespread. Trastuzumab, a monoclonal antibody, is globally used for immunotherapy. It is a large sized molecule (141kDa) and show slow pharmacokinetics and high liver uptake. We have explored trastuzumab fragment as a radio-theranostic agent. Trastuzumab Fab was generated using Papain enzyme tagged beads, incubation time 18 to 20 h at 850 rpm in phosphate buffer. Fab were collected with ultrafiltration device, cut off 30 kDa, in phosphate buffer pH 8. Quantification of Fab was done at 280 nm. Fab characterization was done using MALDI-TOF and SDS-PAGE. To enable radiolabelling of Fab with Ga-68 and Lu-177, Fab was conjugated with bifunctional chelator, DTPA. Fab-DTPA was separated by micro bio-spin P-6 chromatography columns and the average number of DTPA/Fab were calculated. Affinity of Fab conjugated Fab-DTPA for HER2 receptors was studied using the biolayer interferometry technique (BLI). Radiolabelling of Fab with Ga-68 for PET imaging and Lu-177 for therapy was optimized. Quality control included radiochemical purity using TLC in sodium citrate (pH 5) mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. Receptor affinity and apoptosis were studied with HER2 positive SKBR-3 cells using flow cytometry assay. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a known amount of Lu-177-DTPA-Fab (0- 18.5 MBq). First in human Ga-68 Fab PET/CT and Lu-177-Fab gamma camera images were acquired after obtaining clearance from Institutional Ethics Committee and inform signed consent. Patients were recruited with histopathological proven HER2 positive breast cancer and uptake on F-18 FDG PET/CT. Ga-68-DTPA-Fab (3.5-5 mCi) and 10-15 mCi Lu-177 DTPA-Fab was injected and images were acquired at 2/3 h and up to 7 days respectively. Dominant Fab peak at 45kDa on MALD-TOF, single band on SDS-PAGE (Non-reduced or reduced) have been obtained. Two DTPA/Fab were conjugated and confirmed by MALDI-TOF. BLI data showed Kd value of trastuzumab, Fab, and conjugated DTPA-Fab as 0.2 nM, 0.57 nM, and 20 nM for HER2. High labelling efficiency (≥98%) of Lu-177-DTPA-Fab and Ga-68-DTPA-Fab was achieved. Apoptosis (%) was 1.5%- 42.5% in SKBR-3 cells treated with Lu-177-DTPA-Fab. Both formulations were found sterile and pyrogen-free. Kidney and bladder activity of Ga-68/Lu-177-DTPA-Fab demonstrate renal clearance. Blood pool activity of both tracers were high. However, it was decreased, and lesion uptake was increased with time. All the lesions on Ga-68-DTPA-Fab, same as in F-18 FDG PET/CT, were picked up accept those near heart. Good lesion retention of Lu-177-DTPA-Fab was observed showing suitability for therapy. Fab-DTPA has potential to target HER2 receptor. It could be radiolabelled with Ga-68 for PET imaging and Lu-177 for targeted therapy. Citation Format: Jaya Shukla, Yogesh Rathore, Komalpreet Kaur, Santosh Irrinky, Rajender Kumar, Amanjit Bal, Bhagwant R Mittal. Radiolabelled Trastuzumab Fab as a theranostic agent for HER2 expressing breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B043.

Non-Antibiotic Immunomodulatory Combination Treatment for Staphylococcus aureus Infections: In vitro Preliminary Studies I

Despite advancements in prevention measures, infectious mastitis is a common disease of economic significance in cattle. Clinical mastitis affects 20 to 25% of cows each year. Staphylococcus aureus (S. aureus) causes an estimated 10-11.7% of clinical bovine infectious mastitis cases in the US. Antibiotic therapy tends to fail 50% of the time and nature and vaccine immunity usually fails, due to S. aureus’ evasion mechanisms, leading to significant morbidity, chronic mastitis, and culling of such cows. Specific IgYs have been found to inhibit the growth of S. aureus in vitro, but such IgYs have been produced especially for such experiments, commercially available presentations have not been compared to the IgY produced by naturally exposed chickens. In the present study, we compare the impact of two sources of IgY. IgY was obtained commercially and purified from the eggs of chickens naturally exposed to S. aureus (RIR IgY). Their effect on the in vitro growth of S. aureus was compared and the commercial chicken anti-SpA IgY (IgY). This was done by mixing tryptic soy broth cultures of S. aureus with 5, 25, or 125 µg/mL of IgY or RIR IgY (3 tubes per concentration). The result was evaluated by the plate count method. Next, the treated and control broths were sampled and plated at 4, 8, and 12 h. IgY (commercial) showed a powerful inhibitory effect over S. aureus. This effect was sustained for over 12 h when compared to RIR IgY and two respectively to the control. Though the RIR IgY effect was significant; however, it subsided after 8 h. Based on our findings, IgY may have an effective component against S. aureus growth which is more observed in the IgY commercially obtained than the IgY purified chickens which are naturally exposed to S. aureus (RIR IgY).

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm2) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.

Two Type VI Secretion Systems of Enterobacter cloacae Are Required for Bacterial Competition, Cell Adherence, and Intestinal Colonization.

Enterobacter cloacae has emerged as an opportunistic pathogen in healthcare-associated infections. Analysis of the genomic sequences of several E. cloacae strains revealed the presence of genes that code for expression of at least one type VI secretion system (T6SS). Here, we report that E. cloacae strain ATCC 13047 codes for two functional T6SS named T6SS-1 and T6SS-2. T6SS-1 and T6SS-2 were preferentially expressed in tryptic soy broth and tissue culture medium (DMEM), respectively. Mutants in T6SS-1-associated genes clpV1 and hcp1 significantly affected their ability of inter- and intra-bacterial killing indicating that T6SS-1 is required for bacterial competition. In addition, the Hcp effector protein was detected in supernatants of E. cloacae cultures and a functional T6SS-1 was required for the secretion of this protein. A clpV2 mutant was impaired in both biofilm formation and adherence to epithelial cells, supporting the notion that these phenotypes are T6SS-2 dependent. In vivo data strongly suggest that both T6SSs are required for intestinal colonization because single and double mutants in clpV1 and clpV2 genes were defective in gut colonization in mice. We conclude that the two T6SSs are involved in the pathogenesis scheme of E. cloacae with specialized functions in the interaction with other bacteria and with host cells.

Mesoporous Hydroxyapatite/Chitosan Loaded With Recombinant-Human Amelogenin Could Enhance Antibacterial Effect and Promote Periodontal Regeneration.

The recovery of impaired periodontium is still a challenge to the treatment of periodontitis. This study was the first to apply the mesoporous hydroxyapatites/chitosan (mHA/CS) composite scaffold to periodontal regeneration. The aim of our study is to evaluate the biological effects of mesoporous hydroxyapatite/chitosan (mHA/CS) loaded with recombinant human amelogenin (rhAm) on periodontal regeneration. The physicochemical properties of mHA/CS scaffolds were examined by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and Brunauer–Emmett–Teller (BET) analysis. Then, the biological effects of the mHA/CS loaded with rhAm were evaluated, including antibacterial effect, controlled-release capacity, osteogenic and cementogenic effects in vitro and in vivo. The antibacterial effect was tested on 1.5 mg/mL CS; 3 mg/mL mHA; 2.25 mg/mL mHA/CS; 4.5 mg/mL mHA/CS and 20 μg/mL rhAm. Tryptic Soy Broth culture medium was used as a baseline control. Osteogenic effect of rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm) on human periodontal ligament cells (hPDLCs) was evaluated in osteogenic media. The hPDLCs treated either with osteogenic media or Dulbecco's modified Eagle's medium (DMEM) alone were used as the baseline control. In the animal model, 4-week-old nude mice (BALB/c) (n = 6) implanted with root slices subcutaneously were used to observe the cementogenic effect in vivo. The root slices were treated with rhAm (20 μg/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 μg/mL rhAm). The root slices treated with osteogenic medium alone were used as the baseline control. The analyses showed that the mHA/CS particles were 2 μm in diameter and had a uniform pore size. The mesoporous structure was 7 nm in diameter and its surface area was 33.95 m2/g. The scaffold exhibited antibacterial effects against Fusobacterium nucleatum and Porphyromonas gingivalis. The mHA/CS scaffold sustainably released rhAm. The mHA/CS loaded with 20 μg/mL rhAm upregulated ALP activity, the expression levels of osteogenesis-related genes and proteins in vitro. Additionally, it promoted the formation of cementum-like tissue in vivo. Our findings suggest that mHA/CS loaded with 20 μg/mL rhAm could inhibit the growth of periodontal pathogens and promote the formation of bone and cementum-like tissue.

Riemerella anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence

Riemerella anatipestifer is a bacterial pathogen responsible for major economic losses within the duck industry. Recent studies have revealed that biotin biosynthesis is critical for the bacterium’s survival and virulence. We previously found that R. anatipestifer AS87_RS09170, a putative bioF gene, is important for bacterial virulence. In the present study, we characterized the AS87_RS09170 gene in R. anatipestifer strain Yb2. Sequence analysis indicated that the AS87_RS09170 gene is highly conserved among R. anatipestifer strains; the deduced protein harbored the conserved pyridoxal 5′-phosphate binding pocket of 8-amino-7-oxononanoate synthase. Western blot analysis demonstrated that the biotin-dependent enzyme was present in smaller quantities in the mutant strain Yb2ΔbioF compared to that of the wide-type strain Yb2, suggesting that the biotin biosynthesis was defective. The mutant strain Yb2ΔbioF displayed a decreased growth rate at the exponential phase in tryptic soy broth culture and in BeaverBeads Streptavidin treated tryptic soy broth culture, but recovered when biotin was supplemented. In addition, the mutant strain Yb2ΔbioF showed an enhanced biofilm formation, as well as increased adhesion and invasion capacities to duck embryo fibroblasts. Moreover, the mutant strain Yb2ΔbioF exhibited irregular shapes with budding vegetations and relatively thickened cell walls under scanning and transmission electron microscope observation, as well as a reduced capacity to establish systemic infection in a duck infection model. These results provide the first evidence that the R. anatipestifer AS87_RS09170 gene is responsible for biotin synthesis, bacterial morphology and virulence.

Failure Analysis of Pitted Copper Pipes Used in Underground Water and Preventive Measures

This study performed an experiment on the causes of pitting corrosion in a copper tubing used for a sprinkler system. Corrosion products of a copper tubing that sustained pitting corrosion were collected and cultured in Culture medium [Luria–Bertani, Brain heart infusion, Tryptic soy broth (TSB), R2A]. Four types of bacteria were found through identification: Micrococcus luteus sp (species)., Staphylococcus sp., Sphingomonas sp., and Bacillus sp. The copper toxicity test was performed for each microorganism. Among the four microorganisms, Micrococcus luteus sp. showed good growth in the environment containing copper ions. On the immersion test, changes in pH and Optical density were measured; On the inductively coupled plasma optical emission spectrometry test, the copper concentration of each culture medium was measured. The surface of each copper sample was observed using a scanning electron microscope. The corrosion potential of a copper sample, after 48 h exposure of the TSB medium containing Micrococcus luteus sp., was measured using a potentiodynamic polarization experiment. The next experiment was conducted to prevent microbial corrosion by suppressing the growth of microorganisms. Six 30 ml TSB culture media with controlled pH value of 4, 5, 6, 7, 8, and 9 through HCl and NaOH were manufactured. Then the microorganisms were cultured in 37 °C 133 rpm, of which the growth status was checked every 24 h for 3 days. It was found that microorganisms did not grow on culture media with the pH value of 6 and lower. The same experiment conducted on culture media controlled with acetic acid, nitric acid, and sulfuric acid, also showed no growth of microorganisms on media with pH value of 6 and lower. Six 5 ml TSB culture media each containing 0.5, 0.25, 0.125, 0.0625, 0.0312%, and 0.0156% NaOCl and NaOBr as germicides were manufactured. 0.01 μl of microorganisms were inoculated on the media and cultured in 37 °C for 48 h. It was found that microorganisms did not grow in media with NaOCl and NaOBr concentration of 0.0625% and higher. Therefore, it can be suggested that in environments with pH value of 6 or lower, or NaOCl and NaOBr concentration of higher than 0.0625% suppresses microbial growth, thereby preventing microbial corrosion.

A two-component signal transduction system contributes to the virulence of Riemerella anatipestifer.

Similar to other studies of bacterial pathogens, current studies of the pathogenesis of Riemerella anatipestifer (RA) are focused mainly on in vitro culture conditions. To elucidate further the pathogenesis of RA in vivo, bacterial RNA was extracted from overnight tryptic soy broth cultures (in vitro) and from the blood of infected ducks (in vivo) for comparative RNA sequencing analysis. In total, 682 upregulated genes were identified in vivo. Among the upregulated genes, a signal transduction response regulator (ArsR) and a signal transduction histidine kinase (SthK) were predicted to be located on the same operon. A mutant was constructed by deletion of both of these genes. Duck infection tests showed that genes ArsR and SthK were related to the virulence of the pathogen in vivo. Differentially expressed genes identified by comparison of in vitro and in vivo conditions provided an insight into the physiological process of RA infection and provided an opportunity to identify additional virulence factors.

Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family.

The biofilm characteristics of many endospore-forming bacilli, especially the thermophiles are still unclear. In this study, a detailed identification and description of biofilm production characteristics of totally 145 isolates and reference strains belonging to Bacillaceae family, displaying thermophilic (n = 115), facultative thermophilic (n = 24) and mesophilic (n = 6) growth from genera Anoxybacillus, Geobacillus, Thermolongibacillus, Aeribacillus, Brevibacillus, Paenibacillus and Bacillus were presented. The incubation temperatures were adjusted to 37, 45 and 55-65 °C for mesophiles, facultative thermophiles, and thermophiles, respectively. The bacilli were evaluated based on their colony morphotypes on Congo red (CR) agar, their complex exopolysaccharide production on calcofluor supplemented tryptic soy agar, and as well as their pellicle formation at the liquid-air surface in tryptic soy broth cultures. Their biofilm production capabilities were also tested on abiotic surfaces of both polystyrene and stainless steel by crystal violet binding assay and viable biofilm cell enumerations, respectively. As a result, the biofilm production capacities of Bacillaceae members from genera to species level, the effects of osmolarity, temperature, incubation time and abiotic surfaces on biofilm formation as well as the CR morphotypes associated with the biofilm production were able to reveal in a wide group of bacilli. Besides, general enrichment-inoculation approaches and methodologies were also offered, which allow and facilitate the screening and determining the biofilm producing endospore forming bacilli.

INACTIVATION OF SHIGA-TOXIN PRODUCING Escherichia coli (STEC) O157:H7 IN MILK BY COMBINED TREATMENT WITH HIGH HYDROSTATIC PRESSURE AND AQUEOUS POMEGRANATE EXTRACT

The aim of this work was to evaluate the synergistic effect of combined treatment with high hydrostatic pressure (HHP) and an aqueous extract of Punica granatum (pomegranate) peels on the survival of Shiga toxin-producing Escherichia coli (STEC). Our results showed that HHP (250 MPa, 60 min, 25 ºC) reduced the STEC bacterial load in milk and tryptic soy broth culture medium by about 2.5 and 3.5 logs, respectively. Under these conditions, HHP did not alter the cytotoxicity of Shiga toxins in Vero and human umbilical vein endothelial cells. Treatment with up to 30 mg of pomegranate extract/mL caused negligible inactivation, but a combination of HHP and pomegranate extract (3 mg/mL) produced bacterial inactivation from 109 CFU/mL to undetectable levels of viable bacteria. These findings suggest that a combination of HHP and pomegranate extract may be potentially effective in bacterial inactivation during food processing, particularly in the elimination of important foodborne pathogens such as STEC. a

The Riemerella anatipestifer AS87_01735 Gene Encodes Nicotinamidase PncA, an Important Virulence Factor.

Riemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate. Riemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.

Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 has been recognized as a major foodborne pathogen responsible for frequent gastroenteritis outbreaks. Phages and essential oils can be used as a natural antimicrobial method to reduce bacterial pathogens from the food supply. The objective of this study was to determine the effect of a bacteriophage cocktail, BEC8, alone and in combination with the essential oil trans-cinnameldehyde (TC) on the viability of a mixture of EHEC O157:H7 strains applied on whole baby romaine lettuce and baby spinach leaves. The EHEC O157:H7 strains used were Nal R mutants of EK27, ATCC 43895, and 472. Exponentially growing cells from tryptic soy (TS) broth cultures were spot inoculated on leaves and dried. EHEC cells were placed at low, medium, and high inoculum levels (10 4, 10 5, and 10 6 CFU/mL, respectively). Appropriate controls, BEC8 (approx. 10 6 PFU/leaf), and TC (0.5% v/v) were applied on treated leaves. The leaves were incubated at 4, 8, 23, and 37 °C in Petri dishes with moistened filter papers. EHEC survival was determined using standard plate count on nalidixic acid (50 μg/mL) Sorbitol MacConkey agar. No survivors were detected when both leaves were treated with BEC8 or TC individually at low inoculum levels after 24 h at 23 and 37 °C. When the EHEC inoculum size increased and/or incubation temperature decreased, the efficacy of BEC8 and TC decreased. However, when the two treatments were combined, no survivors were detected after 10 min at all temperatures and inoculum levels on both leafy greens. These results indicated that the BEC8/TC combination was highly effective against EHEC on both leafy greens. This combination could potentially be used as an antimicrobial to inactivate EHEC O157:H7 and reduce their incidence in the food chain.

Systemic Inflammatory Response Syndrome in Home Delivered Neonates: A Prospective Observational Study

To determine the severity of systemic inflammatory response syndrome (SIRS) at admission, bacteriological profile, antibiotic sensitivity of pathogens and factors associated with fatality in home delivered neonates with sepsis. This was a prospective observational study conducted in the referral neonatal unit of a teaching hospital admitting extramural neonates. The subjects comprised of 80 home delivered neonates presenting with systemic inflammatory response syndrome at admission. Skin temperature, oxygen saturation, capillary refill time and blood sugar were recorded in all the neonates at admission. For Blood culture, blood collected by venipuncture was placed in a tryptic soy broth culture bottle. Serum TNF-α was measured by ELISA kit. Early onset sepsis was seen in 27.5%. The commonest clinical feature in the study population was decreased oral acceptance (53.8%). The mean distance traveled to reach the hospital was 19 ± 3 km. At admission, acute physiological derangement in the form of abnormal skin temperature, oxygen saturation, perfusion and blood sugar was present in 53 neonates and 44% had more than one parameter deranged. Only 11% cases had early sepsis while the SIRS was well established in the rest. Klebsiella pneumoniae was the predominant bacteria isolated in 14 cases. Resistance of Klebsiella isolates to Ampicillin was 90% and to Gentamicin 57%. The fatality was higher in presence of advanced stages of SIRS at admission. SIRS was well established in 89% cases at admission. Klebsiella resistant to antibiotics was the predominant etiological organism. Fatality was higher in culture positive sepsis and in those associated with meningitis and pneumonia.

Bacterial leakage along the implant–abutment interface of premachined or cast components

In recent clinical studies, contamination of the inner parts of dental implants through bacterial penetration along the implant components has been observed. The aim of the present in-vitro study was to investigate leakage of Fusobacterium nucleatum through the interface between implants and premachined or cast abutments. Both premachined ( n = 10) and cast ( n = 10) implant–abutment assemblies were inoculated with 3.0 μL of microbial inoculum. The assemblies were completely immersed in 5.0 mL of tryptic soy broth culture medium to observe leakage at the implant–abutment interface after 14 days of anaerobic incubation. Bacterial growth in the medium, indicative of microbial leakage, was found only in 1 out of 9 samples (11.1%) in each group. Both premachined and cast abutments connected to external hexagonal implants provide low percentages of bacterial leakage through the interface in in vitro unloaded conditions if the manufacturer's instructions and casting procedures are properly followed.

Survival, isolation and characterization of a psychrotrophic Bacillus cereus strain from a mayonnaise-based ready-to-eat vegetable salad

Incidence and population levels of Bacillus cereus in American salad, an industrially manufactured, packaged and refrigerated deli salad containing vegetables and mustard, were determined. Of 12 ready-to-eat samples examined, one (8.3%) was positive for B. cereus at less than 5×10 3 cfu g −1. According to the ISO confirmation procedure, a strain was isolated and further characterized and identified as B. cereus EPSO-35AS by API 50CH/20E phenotypic system, combined with additional tests of motility, oxidase activity and anaerobic growth. This strain produced diarrhoeal enterotoxin in tryptic soy broth culture as detected by BCET-RPLA test, hydrolysed starch and had a low D 90-value (2.1 min), with an estimated z-value of 6.79 °C. After a lengthy lag phase (9–12 days of incubation), the strain was able to grow at 8 °C in both nutrient broth and tyndallized carrot broth with specific growth rates from 0.009 to 0.037 h −1, respectively. In the vegetable substrate, lag time was approximately 3 days (66 h) shorter than in laboratory medium. The effect of temperature abuses on the safety of the product during the time of use or consumption is discussed.

Modeling the growth of Listeria monocytogenes based on a time to detect model in culture media and frankfurters

In the current study, temperature dependent growth of Listeria monocytogenes strain H7776, with an initial inoculum level of approximately 0.1 CFU/mL or g, was modeled based on “time to detect” (TTD) using the Arrhenius equation. The activation energies ( E a) obtained from specific growth rate and lag phase duration in tryptic soy broth (TSB) and frankfurters were compared. At 4 °C, the TTD for L. monocytogenes was 217 h in TSB and 48 h in portions of frankfurters. The TTD decreased as temperature increased from 4 to 36 °C in both culture media and frankfurters, and this relationship was modeled using the Arrhenius equation. Based on this model, the E a values for the TTD in TSB and frankfurters averaged 22.7 and 18.7 kcal/mol, respectively, and were not significantly different ( p < 0.05). Linear regression was performed on the exponential part of the growth curve to evaluate specific growth rate constants at each temperature using the Monod model. The E a values were also calculated based on the specific growth rate and the lag phase of L. monocytogenes in TSB and frankfurters incubated in the same range of temperatures. The average E a values for the specific growth rates and the lag phase durations in TSB cultures were 19 and 21 kcal/mol, respectively. In frankfurters, the average E a values were slightly greater for both specific growth rate and lag phase duration (29 and 35 kcal/mol, respectively), but these values were not significantly different from the E a calculated for the TTD in each medium. These results indicate that the TTD concept can be used to develop and validate safety-based shelf life models.

Characterization of nigerlysin ©, hemolysin produced by Aspergillus niger, and effect on mouse neuronal cells in vitro

Aspergillus niger produced a proteinaceous hemolysin, nigerlysin © when incubated on sheep's blood agar (SBA) at both 23 and 37 °C. Nigerlysin was purified from tryptic soy broth (TSB) culture filtrate and found to have a molecular weight of approximately 72 kDa, with an isoelectric point of 3.45. Nigerlysin is heat stable up to 65 °C but unstable at 75 °C when incubated for 10 min. Circular dichroic analysis revealed that nigerlysin has an alpha helical structure. Exposure of mouse primary cortical neuronal cells to 0.1 μg ml −1 of nigerlysin resulted in the rapid loss of their viability, approximately 50% in 24 h. The IC 50 is estimated to be 0.037 μg ml −1, or between 0.034 and 0.041 μg ml −1 at the 95% confidence level.

Utilization of ram horn hydrolysate as a supplement for recovery of heat- and freeze-injured bacteria

Tryptic soy agar (TSA) has been widely and successfully used as a resuscitation medium for injured bacteria. The purpose of this study was to examine the use of ram horn hydrolysate (RHH) as a supplement for recovery of injured bacteria, to compare it to TSA and to evaluate its usefulness as a possible alternative supplement in microbial media. The TSA was enriched with RHH at various concentrations. 2% RHH was found to be optimal for growth of healthy bacteria. The enriched medium (TSA + 20 ml RHH in 1–l) was termed enriched tryptic soy agar (ETSA). TSA and ETSA were tested for the recovery of heat- and freeze-stressed bacteria. Four species of bacteria ( Escherichia coli, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa) were heat-treated at 55 °C in physiological saline by a capillary tube method. For the freeze-injury studies, 5 ml of the tryptic soy broth cultures were placed in a freezer at −25 °C for 7 days. The numbers of uninjured bacteria on TSA were evaluated as control. The number of viable bacteria on TSA and ETSA was determined by the pour plate method. Recovery rates of bacteria on ETSA were higher than that of TSA. The RHH appears to be a valuable supplement for microbial media.