Strain Variability of Listeria monocytogenes under NaCl Stress Elucidated by a High-Throughput Microbial Growth Data Assembly and Analysis Protocol.

Listeria monocytogenes causes the severe foodborne illness listeriosis and survives in food-associated environments due to its high stress tolerance. A data assembly and analysis protocol for microbial growth experiments was compiled to elucidate the strain variability of L. monocytogenes stress tolerance. The protocol includes measurement of growth ability under stress (step 1), selection of a suitable method for growth parameter calculation (step 2), comparison of growth patterns between strains (step 3), and biological interpretation of the discovered differences (step 4). In step 1, L. monocytogenes strains (n = 388) of various serovars and origins grown on media with 9.0% NaCl were measured using a Bioscreen C microbiology reader. Technical variability of the growth measurements was assessed and eliminated. In step 2, the growth parameters determined by Gompertz, modified-Gompertz, logistic, and Richards models and model-free splines were compared, illustrating differences in the suitability of these methods to describe the experimental data. In step 3, hierarchical clustering was used to describe the NaCl tolerance of L. monocytogenes measured by strain-specific variation in growth ability; tolerant strains had higher growth rates and maximum optical densities and shorter lag phases than susceptible strains. The spline parameter area under the curve best classified "poor," "average," and "good" growers. In step 4, the tested L. monocytogenes lineage I strains (serovars 4b and 1/2b) proved to be significantly more tolerant toward 9.0% NaCl than lineage II strains (serovars 1/2a, 1/2c, and 3a). Our protocol provides systematic tools to gain comparable data for investigating strain-specific variation of bacterial growth under stress.IMPORTANCE The pathogen Listeria monocytogenes causes the foodborne disease listeriosis, which can be fatal in immunocompromised individuals. L. monocytogenes tolerates several environmental stressors and can persist in food-processing environments and grow in foodstuffs despite traditional control measures such as high salt content. Nonetheless, L. monocytogenes strains differ in their ability to withstand stressors. Elucidating the intraspecies strain variability of L. monocytogenes stress tolerance is crucial for the identification of particularly tolerant strains. To enhance reliable identification of variability in bacterial stress tolerance phenotypes, we compiled a large-scale protocol for the entire data assembly and analysis of microbial growth experiments, providing a systematic approach and checklist for experiments on strain-specific growth ability. Our study illustrated the diversity and strain-specific variation of L. monocytogenes stress tolerance with an unprecedented scope and discovered biologically relevant serovar- and lineage-dependent phenotypes of NaCl tolerance.

Octenidine's Efficacy: A Matter of Interpretation or the Influence of Experimental Setups?

With its broad antimicrobial spectrum and non-specific mode of action via membrane disruption, any resistance to octenidine (OCT) seems unlikely and has not been observed in clinical settings so far. In this study, we aimed to investigate the efficacy of OCT against Escherichia coli and mutants lacking specific lipid head groups which, due to altered membrane properties, might be the root cause for resistance development of membrane-active compounds. Furthermore, we aimed to test its efficacy under different experimental conditions including different solvents for OCT, bacterial concentration and methods for analysis. Our primary goal was to estimate how many OCT molecules are needed to kill one bacterium. We performed susceptibility assays by observing bacterial growth behavior, using a Bioscreen in an analogous manner for every condition. The growth curves were recorded for 20 h at 420-580 nm in presence of different OCT concentrations and were used to assess the inhibitory concentrations (IC100%) for OCT. Bacterial concentrations given in cell numbers were determined, followed by Bioscreen measurement by manual colony counting on agar plates and QUANTOMTM cell staining. This indicated a significant variance between both methods, which influenced IC100% of OCT, especially when used at low doses. The binding capacity of OCT to E. coli was investigated by measuring UV-absorbance of OCT exposed to bacteria and a common thermodynamic framework based on Bioscreen measurements. Results showed that OCT's antimicrobial activity in E. coli is not affected by changes at the membrane level but strongly dependent on experimental settings in respect to solvents and applied bacterial counts. More OCT was required when the active was dissolved in phosphate or Hepes buffers instead of water and when higher bacterial concentration was used. Furthermore, binding studies revealed that 107-108 OCT molecules bind to bacteria, which is necessary for the saturation of the bacterial surface to initiate the killing cascade. Our results clearly demonstrate that in vitro data, depending on the applied materials and the methods for determination of IC100%, can easily be misinterpreted as reduced bacterial susceptibility towards OCT.

The use of bacteriophages against saprophytic mesophilic bacteria in minimally processed food.

BACKGROUND: ood producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic. METHODS: , food producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic bac. RESULTS: , food producers strive to meet the changing needs of consumers while maintaining the highest nutritional value of the products they supply. Physicochemical methods, which include modified atmosphere packaging, membrane techniques or ultrasounds, are the most frequently used to preserve food. Alternatively, biological methods can be applied, one of which is the use of bacteriophages (phages) to limit bacterial growth in the food environment. The purpose of our research was to verify the possibility of the use of bacteriophages as an antibacterial agent in minimally processed food environments of vegetable origin. The first stage of the research involved the isolation of phages against the dominant bacterial microflora in the analyzed products: broccoli sprouts, spinach leaves and freshly squeezed carrot-celery juice. Bacteriophages were isolated from municipal waste collected from sewage-treatment plants. Specific bacteriophages were isolated for twenty-nine out of thirty identified bacterial strains. The lytic activity of the phages was tested using a Bioscreen C automatic growth analyzer. Three methods for applying the phage cocktail were tested: direct addition of the cocktail, spraying it on, and placing the food product on a pad soaked with the phage mixture. The food products were packaged in a protective atmosphere and stored at 20°C. The total number of bacteria after adding the phage cocktail to the products was determined during the subsequent hours of incubation using classical microbial culturing. A significant decrease in the total number of bacteria was observed in the products containing phage suspensions. The obtained results suggest that application of the phage cocktail offers the possibility to extend the shelf life of the analyzed minimally processed food products by reducing the total number of saprophytic bacteria.

Resistance of multidrug resistant Escherichia coli to environmental nanoscale TiO2 and ZnO.

Excessive production and utilization of nanoparticles (NPs) at industrial and household levels releases substantial quantities of NPs into the environment. These can be harmful to different types of organisms and cause adverse effects on ecosystems. Purchased TiO2 and ZnO NPs were characterized via XRD, XPS, FESEM, and Zeta potential. This study elucidates how multidrug resistant Escherichia coli LM13, which was recovered from livestock manure, counteracts the antibacterial activities of TiO2 and ZnO NPs to survive in the environment. E. coli ATCC25922, which is susceptible to antibiotics, was used as control. A dose-response experiment showed that the antibacterial activity of TiO2 was lower than that of ZnO NPs and, LM13 was more resistant to NPs than ATCC25922. An AcrAB-TolC efflux pump along with its regulation genes helped LM13 to minimize NP toxicity. Flow cytometry findings also indicated that the intensity of the side-scatter light parameter increased with TiO2 and ZnO NPs in a dose dependent manner, suggesting NP uptake by the both strains. The generation of reactive oxygen species in LM13 was several-fold lower than in ATCC25922, suggesting that reactive oxygen species mainly contribute to the toxicity mechanism. These results illustrate the necessity to evaluate the impacts of NPs on the survival capacity of bacteria and on the resistance genes in bacteria with higher NP resistance than NP susceptible bacteria.

Effect of Zataria multiflora Boiss. essential oil, NaCl, acid, time, and temperature on the growth of Listeria monocytogenes strains in broth and minced rainbow trout.

The small outbreaks of listeriosis as one of the leading causes of food poisoning-associated deaths occur more than previously reported. In current study, the growth ability of Listeria monocytogenes strains isolated from different sources of food and human origin was measured under salt stress (0.5%, 2.5%, 5%, 7.5%, and 10%) and acid environments (pH = 6.64 and 5.77) for 96 hr by using a Bioscreen C microbiology reader at 37°C. In further steps of this study, after analysis of constituents of Zataria multiflora Boiss. essential oil (ZMEO), the sensory evaluation of the treated fish meat with ZMEO was performed. Then, the fish isolate of L. monocytogenes was exposed to sensory acceptable and subminimum inhibitory concentrations (subMICs) of ZMEO in fish broth and minced fish meat during incubation at abuse (12°C), room (22°C), and optimum (37°C) temperatures for 48 hr. The MIC of NaCl against four strains of L. monocytogenes was 10% at 37°C. The maximum optical densities (ODs) and under curve areas (AUC) of growth patterns in higher pH value and lower contents of NaCl followed the order of 21C > 6F > 66C > 22C of L. monocytogenes strains, while the lag time was prolonged in the reverse order. The maximum OD, growth, and lag times of samples treated with higher contents of NaCl and lower pH value were affected in a different order. The organoleptic evaluation showed that the fish meat treated with less than 0.5% of ZMEO was sensory acceptable. The population of L. monocytogenes remained relatively constant at the inoculation level of 107 cfu/ml (or g) at 12°C in broth and minced fish mediums. The inhibitory antilisterial activity of essential oil as an extensive-used plant for food and pharmacological applications is negligible due to possible adverse sensory and toxic effects at relevant high doses.

Assessment of the Effect of Satureja montana and Origanum virens Essential Oils on Aspergillus flavus Growth and Aflatoxin Production at Different Water Activities.

Aflatoxin contamination of foodstuffs poses a serious risk to food security, and it is essential to search for new control methods to prevent these toxins entering the food chain. Several essential oils are able to reduce the growth and mycotoxin biosynthesis of toxigenic species, although their efficiency is strongly influenced by the environmental conditions. In this work, the effectiveness of Satureja montana and Origanum virens essential oils to control Aspergillus flavus growth was evaluated under three water activity levels (0.94, 0.96 and 0.98 aw) using a Bioscreen C, a rapid in vitro spectrophotometric technique. The aflatoxin concentrations at all conditions tested were determined by HPLC-FLD. Aspergillus flavus growth was delayed by both essential oil treatments. However, only S. montana essential oil was able to significantly affect aflatoxin production, although the inhibition percentages widely differed among water activities. The most significant reduction was observed at 0.96 aw, which is coincident with the conditions in which A. flavus reached the highest levels of aflatoxin production. On the contrary, the treatment with S. montana essential oil was not effective in significantly reducing aflatoxin production at 0.94 aw. Therefore, it is important to study the interaction of the new control compounds with environmental factors before their application in food matrices, and in vitro ecophysiological studies are a good option since they provide accurate and rapid results.

Comparative growth of trichoderma strains in different nutritional sources, using bioscreen c automated system.

Trichoderma is one of the fungi genera that produce important metabolites for industry. The growth of these organisms is a consequence of the nutritional sources used as also of the physical conditions employed to cultivate them. In this work, the automated Bioscreen C system was used to evaluate the influence of different nutritional sources on the growth of Trichoderma strains (T. hamatum, T. harzianum, T. viride, and T. longibrachiatum) isolated from the soil in the Juréia-Itatins Ecological Station (JIES), São Paulo State - Brazil. The cultures were grown in liquid culture media containing different carbon- (2%; w/v) and nitrogen (1%; w/v) sources at 28ºC, pH 6.5, and agitated at 150 rpm for 72 h. The results showed, as expected, that glucose is superior to sucrose as a growth-stimulating carbon source in the Trichoderma strains studied, while yeast extract and tryptone were good growth-stimulating nitrogen sources in the cultivation of T. hamatum and T. harzianum.

Screening for Growth-Inhibitory ORFans in Pseudomonas aeruginosa-Infecting Bacteriophages.

Like all viruses, bacteriophages heavily depend on their host's physiology for reproduction. Therefore, phages have evolved numerous proteins that influence the host metabolism to facilitate the infection process. Some of these proteins strongly perturb the host cell, ultimately leading to cell death. These growth-inhibitory phage proteins presumably target key metabolic processes, which may provide a basis for innovative phage-derived antibacterials. Unfortunately, most of these proteins are the so-called ORFans, since they have no known function or sequence homology to any other gene. We here describe a screening method for the identification of growth-inhibitory ORFans of bacteriophages infecting gram-negative bacteria (e.g., Pseudomonas aeruginosa), using the pUC18-mini-Tn7T-Lac vector system, which allows for stable single-copy integration of the phage ORFans in the Pseudomonas genome under the control of an IPTG-inducible promoter. Furthermore, we describe a method to examine the effect of the phage proteins in different hosts, using different vector copy numbers. Finally, we explain how to investigate the effect of ORFan expression on the host morphology using time-lapse microscopy.

Evaluation of automated time-lapse microscopy for assessment of in vitro activity of antibiotics.

This study aimed to evaluate the potential of a new time-lapse microscopy based method (oCelloScope) to efficiently assess the in vitro antibacterial effects of antibiotics. Two E. coli and one P. aeruginosa strain were exposed to ciprofloxacin, colistin, ertapenem and meropenem in 24-h experiments. Background corrected absorption (BCA) derived from the oCelloScope was used to detect bacterial growth. The data obtained with the oCelloScope were compared with those of the automated Bioscreen C method and standard time-kill experiments and a good agreement in results was observed during 6-24h of experiments. Viable counts obtained at 1, 4, 6 and 24h during oCelloScope and Bioscreen C experiments were well correlated with the corresponding BCA and optical density (OD) data. Initial antibacterial effects during the first 6h of experiments were difficult to detect with the automated methods due to their high detection limits (approximately 105CFU/mL for oCelloScope and 107CFU/mL for Bioscreen C), the inability to distinguish between live and dead bacteria and early morphological changes of bacteria during exposure to ciprofloxacin, ertapenem and meropenem. Regrowth was more frequently detected in time-kill experiments, possibly related to the larger working volume with an increased risk of pre-existing or emerging resistance. In comparison with Bioscreen C, the oCelloScope provided additional information on bacterial growth dynamics in the range of 105 to 107CFU/mL and morphological features. In conclusion, the oCelloScope would be suitable for detection of in vitro effects of antibiotics, especially when a large number of regimens need to be tested.

Effect of Neem (Azadirachta indica) on the Survival of Escherichia coli O157:H7 in Dairy Manure.

Escherichia coli O157:H7 (EcO157) shed in cattle manure can survive for extended periods of time and intervention strategies to control this pathogen at the source are critical as produce crops are often grown in proximity to animal raising operations. This study evaluated whether neem (Azadirachta indica), known for its antimicrobial and insecticidal properties, can be used to amend manure to control EcO157. The influence of neem materials (leaf, bark, and oil) on the survival of an apple juice outbreak strain of EcO157 in dairy manure was monitored. Neem leaf and bark supplements eliminated the pathogen in less than 10 d with a D-value (days for 90% elimination) of 1.3 d. In contrast, nearly 4 log CFU EcO157/g remained after 10 d in neem-free manure control. The ethyl acetate extractable fraction of neem leaves was inhibitory to the growth of EcO157 in LB broth. Azadirachtin, a neem product with insect antifeedant properties, failed to inhibit EcO157. Application of inexpensive neem supplements to control pathogens in manure and possibly in produce fields may be an option for controlling the transfer of foodborne pathogens from farm to fork.

Serum susceptibility in clinical isolates of Burkholderia cepacia complex bacteria: development of a growth-based assay for high throughput determination.

Burkholderia cepacia complex (BCC) bacteria can cause devastating chronic infections in people with cystic fibrosis. Of particular concern is "cepacia syndrome," a rapidly progressive and usually fatal decline in health, characterized by a necrotizing bacteremic pneumonia. An important component of defense against bloodstream infections is the bactericidal action of serum. Traditional methods to determine the capacity of bacterial isolates to resist the bactericidal effects of serum are relatively low-throughput viability assays. In this study, we developed a novel growth-based assay for serum susceptibility, which allows for high throughput analysis. We applied this assay to a range of clinical isolates of BCC as well as isolates comprising the BCC experimental strain panel. Our data demonstrate that isolates from all species of BCC examined can possess serum resistant or serum sensitive/intermediate phenotypes. Of particular clinical significance, we also found no direct link between the last saved pulmonary isolate from patients who subsequently developed "cepacia syndrome" and their capacity to resist the inhibitory effects of human serum, suggesting serum resistance cannot be used as a marker of an isolate's capacity to escape from the lung and cause bacteremia.

Screening, identification and classification of an antimitosis marine bacterium from Eastern China Sea / 第二军医大学学报

Objective: To screen, identify and classify an antimitosis marine bacterium strain 32-11-2-2 of Eastern China Sea. Methods: The marine bacteria were isolated with the infinite dilution and plate streak technique and screened for antimitotic active strains by Pyricularia Oryzae model. The active strain 32-11-2-2 was subjected to morphological, physiological and biochemical study, and was classified by the analysis of the 16S rDNA sequence. Results: A total of 421 strains of marine bacteria were isolated from marine water and sediment samples; 54 of them had antimitotic activity. The study of morphological, physiological and biochemical characteristics showed that the strain 32-11-2-2 was a slight halophilic strain; it had the morphological characteristics of a continent Bacillus and could produce amylase, fluidify glutin; and it also showed a positive reaction in the litmus milk test. The 16S rDNA analysis showed the strain 32-11-2-2 had a 98% homology with Bacillus subtilis. Conclusion: The strain 32-11-2-2 has been identified to be a Bacillus subtilis adapted to the oceanic environment.

Comparative growth of trichoderma strains in different nutritional sources, using bioscreen c automated system / Crescimento de linhagens de Trichoderma em diferentes fontes nutricionais, empregando o sistema automatizado Bioscreen C

Trichoderma is one of the fungi genera that produce important metabolites for industry. The growth of these organisms is a consequence of the nutritional sources used as also of the physical conditions employed to cultivate them. In this work, the automated Bioscreen C system was used to evaluate the influence of different nutritional sources on the growth of Trichoderma strains (T. hamatum, T. harzianum, T. viride, andT. longibrachiatum) isolated from the soil in the Juréia-Itatins Ecological Station (JIES), São Paulo State - Brazil. The cultures were grown in liquid culture media containing different carbon- (2%; w/v) and nitrogen (1%; w/v) sources at 28ºC, pH 6.5, and agitated at 150 rpm for 72 h. The results showed, as expected, that glucose is superior to sucrose as a growth-stimulating carbon source in the Trichoderma strains studied, while yeast extract and tryptone were good growth-stimulating nitrogen sources in the cultivation of T. hamatum and T. harzianum.

Trichoderma é um dos gêneros de fungos produtores de metabólitos de interesse industrial. O crescimento destes organismos é conseqüência das fontes nutricionais utilizadas, juntamente com as condições físicas de cultivo. Neste trabalho, o sistema automatizado Bioscreen C foi utilizado para avaliar a influência de diferentes fontes nutricionais sobre o crescimentode linhagens de Trichoderma (T. hamatum, T. harzianum, T. viride e T. longibrachiatum) isoladas do solo da Estação Ecológica da Juréia-Itatins (JIES), São Paulo - Brasil. Os cultivosforam feitos em meios líquidos de cultura contendo diferentes fontes de carbono (2%; w / v) e nitrogênio (1%; w / v) a 28ºC, pH 6,5 e agitados a 150 rpm durante 72 h. Os resultados mostraram, conforme esperado, que a glicose é melhor do que a sacarose como fonte de carbono indutora de crescimento das linhagens de Trichoderma testadas, enquanto que, o extrato de leveduras e a triptona foram boas fontes de nitrogênio indutorasde crescimento para os cultivos de T. hamatum e T. harzianum.