Tryptic Soy Broth Research Articles

Porphyromonas gingivalis diffusible signaling molecules enhance Fusobacterium nucleatum biofilm formation via gene expression modulation

ABSTRACT Background Periodontitis is caused by a dysbiotic shift in the dental plaque microbiome. Fusobacterium nucleatum is involved in the colonization of Porphyromonas gingivalis, which plays a key role in dysbiosis, via coaggregation and synergy with this microorganism. Aim We investigated the effect of diffusible signaling molecules from P. gingivalis ATCC 33277 on F. nucleatum TDC 100 to elucidate the synergistic mechanisms involved in dysbiosis. Methods The two species were cocultured separated with an 0.4-µm membrane in tryptic soy broth, and F. nucleatum gene expression profiles in coculture with P. gingivalis were compared with those in monoculture. Results RNA sequencing revealed 139 genes differentially expressed between the coculture and monoculture. The expression of 52 genes was upregulated, including the coaggregation ligand-coding gene. Eighty-seven genes were downregulated. Gene Ontology analysis indicated enrichment for the glycogen synthesis pathway and a decrease in de novo synthesis of purine and pyrimidine. Conclusion These results indicate that diffusible signaling molecules from P. gingivalis induce metabolic changes in F. nucleatum, including an increase in polysaccharide synthesis and reduction in de novo synthesis of purine and pyrimidine. The metabolic changes may accelerate biofilm formation by F. nucleatum with P. gingivalis. Further, the alterations may represent potential therapeutic targets for preventing dysbiosis.

DEAD Box Protein DhR1 Is a Global Regulator Involved in the Bacterial Fitness and Virulence of Riemerella anatipestifer.

DEAD box proteins perform diverse cellular functions in bacteria. Our group previously reported that the transposon Tn4531 insertion in Riean_0395 (designated dhR1), which encodes a putative DEAD box helicase, attenuated the virulence of R. anatipestifer strain YZb1. Here, we show that, compared to the wild-type (WT) R. anatipestifer strain Yb2, the growth or survival of the ΔdhR1 mutant in tryptic soy broth (TSB) was significantly decreased in response to cold, pH, osmotic stress, ethanol, Triton X-100, and oxidative stress, and the dhR1 deletion significantly reduced biofilm formation and the adhesion capacity to Vero cells, whereas the growth of ΔdhR1 was less impaired in iron-limited TSB. Moreover, the virulence of ΔdhR1 in ducklings was attenuated by about 80-fold, compared to the WT. In addition, a transcriptome analysis showed that the dhR1 deletion in the strain Yb2 affected the expression of 58 upregulated genes and 98 downregulated genes that are responsible for various functions. Overall, our work reveals that the deletion of DhR1 results in a broad effect on the bacterial fitness, biofilm formation, iron utilization, and virulence of R. anatipestifer, which makes it a global regulator. IMPORTANCE R. anatipestifer infection has been a continued and serious problem in many duck farms, but little is known about the mechanism underlying the pathogenesis of R. anatipestifer and how R. anatipestifer adapts to the external environment and thereby persists in duck farms. The results of this study demonstrate that the DEAD box protein DhR1 is required for the tolerance of R. anatipestifer to cold, pH, and other stresses, and it is also necessary for biofilm formation, iron utilization, and virulence in ducklings, demonstrating multiple functions of DhR1.

Biofilm formation by the interaction of fungi (Candida tropicalis) with various bacteria

Acinetobacter baumannii) ABSTRACT: Background: Biofilms are composed of more than one species or often called polymicrobial biofilms, so research is needed on the formation of polymicrobial biofilms, especially between fungi and bacteria. This study aimed to analyze the total biomass and metabolic activity of biofilms formed from the interaction of fungi (Candida tropicalis) with bacteria (Staphylococcus aureus, MRSA, Pseudomonas aeruginosa, and Acinetobacter baumannii); Methods: Bacterial and fungal cultures were suspended in Tryptic Soy Broth (TSB) medium, then biofilm was cultivated in two 96-well microplates, then the microplates were incubated at 37 degrees Celsius for 48 hours. Detection of the biofilm will be carried out using the Crystal Violet assay and reduction of the tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), or MTT assay to obtain the Optical Density (OD) value by using ELISA reader; Results: The results of this study obtained significant data from both parameters, the highest average value was found in the C. tropicalis treatment group (2,412 ± 0.825 on the biomass test results; 1,525 ± 0.473 on the metabolic activity test results), while the lowest was seen in the S. aureus treatment group (0.512 ± 0.224 in the biomass test results; 0.978 ± 0.349 in the metabolic activity test results); and Conclusions: In the total biomass parameter, it was obtained that the biofilm biomass could be formed strongly in each treatment, while the results of the metabolic activity showed that each treatment was able to carry out cell proliferation.

COMPARATIVE ANALYSIS OF CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF WATERMELON SEED EXTRACTS (CITRULLUS LANATUS)

Ponte Academic JournalMar 2023, Volume 79, Issue 3 COMPARATIVE ANALYSIS OF CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF WATERMELON SEED EXTRACTS (CITRULLUS LANATUS)Author(s): Smajovic Alisa ,Deumic Sara, Crncevic Neira, Hadzic Selena, Alagic Sabina, Music Elma Nur, Salihovic Mirsada, Pazalja Mirha, Omeragic Elma, Dedic MirzaJ. Ponte - Mar 2023 - Volume 79 - Issue 3 doi: 10.21506/j.ponte.2023.3.4 Abstract:Resistance to already known antibiotics is a burning issue. Today, plant material is increasingly being tested with the aim of detecting pharmacologically active components. Watermelon seeds are most often discarded as waste, and bearing in mind that other parts of the watermelon contain a lot of bioactive components, the logical sequence is also the examination of the seed extract. The aim of this paper is to examine the content of bioactive components, antibiotic activity, minimum inhibitory concentration (MIC) as well as antibiofilm activity extracts of seeds of two species of watermelon, from Croatia and Greece, obtained from two different extraction methods, maceration and Soxhlet extraction. Phytochemical analysis was performed using three different HPLC methods. The antibacterial effect was done using disc method, MIC was determined using broth dilution method and biofilm formation was done using Tryptic Soy Broth (TSB). The results indicate the presence of some flavonoids in all samples, as well as on antimicrobial activity against certain strains of bacteria. The best result iwas shown by the ethanol extract from Greece and overall ethanol extracts show slightly better antimicrobial activity compared to methanol extracts. Depending on the tested concetration, different biofilm categories appear. The results show that as the extract concentration decreases, the biofilm formation capacity increases. Our data show that extracts of the seeds have the potential to act as antimicrobial agents, and further research is needed. Download full text:Check if you have access through your login credentials or your institution Username Password

Different patterns of germination inhibition by carvacrol and thymol in Bacillus subtilis spores.

This study aimed to clarify how the phenolic monoterpene carvacrol and its structural isomer thymol both as essential oil components (EOCs) inhibit the germination of Bacillus subtilis spore. Germination was evaluated by the OD600 reduction rate in a growth medium and phosphate buffer containing either l-alanine (l-Ala) system or l-asparagine, d-glucose, d-fructose plus KCl (AGFK) system. The germination of the wild-type spores in the Trypticase Soy broth (TSB) was found to be greatly inhibited by thymol than by carvacrol. Such a difference in the germination inhibition was confirmed by the dipicolinic acid (DPA) release from germinating spores in the AGFK buffer system, but not in the l-Ala system. Similar to the wild-type spores, no difference in the inhibitory activity between the EOCs was also indicated with the gerB, gerK-deletion mutant spores in the l-Ala buffer system and the above substantial difference was also done with the gerA-deleted mutant spores in the AGFK. Fructose was found to release spores from the EOC inhibition and inversely even stimulated. Increased concentrations of glucose and fructose partially suppressed the germination inhibition by carvacrol. The results obtained should contribute to the elucidation of the control effects of these EOCs on bacterial spores in foods.

INDOLE ACETIC ACID AND EXOPOLYSACCHARIDE PRODUCTION OF NITROGEN FIXING BACTERIA ISOLATED FROM NUTMEG TREE

The constraint of plant cultivation in the tropics is low nitrogen (N) content in soil. The application of nitrogen fixing bacteria (NFB) is not only fulfill the N demand but also provide the phytohormones Indole acetic acid (IAA) and exopolysaccharides (EPS). The objective of the experiment was to was to isolate the NFB from the rhizosphere of nutmeg tree and characterized their IAA and EPS production in a liquid culture. The NFB was isolated by serial dilution plate method on Tryptic Soy Agar. In order to test the ability of various NFB isolates to produce IAA and EPS, all isolates were grown in Tryptic Soy Broth for 72 hours at room temperature. The results showed that at the end of experiment the media acidity was shift from neutral to alkaline. The population of five isolates of NFB was approximately 8-9 log10 (of CFU/mL). The IAA production in liquid culture of all isolates was similar but the EPS production was varied and depend on the isolates, The highest EPS content was in PL1 culture and the lowest was in the culture of PL5. This experiment verified that the NFB isolated from nutmeg rhizosphere enable to produce IAA and EPS.

Development of a lateral flow device for rapid simultaneous multiple detections of some common bacterial causes of bovine mastitis.

This work was conducted for the development of a 5-combi lateral flow immunochromatographic kit (LFK) for rapid and simultaneous identification of the common bacterial causes of bovine mastitis. The following pathogens are the identification targets of this kit: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes in milk samples from suspected bovine mastitis cases. The conventional microbiological identification of these agents is not only time-consuming and requires a fully equipped laboratory but also requires experienced personnel. Rabbit polyclonal antibodies (PAbs) specific to the antigenic components of the selected pathogens were prepared, and the pathogen-specific IgG was separated, purified, and conjugated with nanogold that was laid on the conjugate pad. Guinea pig PAbs specific to the microbial antigens of the selected pathogens were prepared, and their IgG content was separated, purified, and used as a capture antibody in the test (T) line on the nitrocellulose (NC) strips. Goat anti-rabbit IgG antibodies were used to capture the rabbit antibodies in the control (C) line of NC strips. The kit was held in a device comprising five strip-holding channels for the above-mentioned five bacterial species antigens. The developed LFK was evaluated, and its sensitivity and specificity were determined. The developed kits were applied for the examination of bovine milk samples from suspected mastitis cases, and the average sensitivity, specificity, and accuracy of 5-combi LFK for the detection of the five selected bacterial species compared to bacteriological examination (gold standard test) were 93.90%, 80.83%, and 90.53%, respectively. The minimal microbial count that gave positive results using the developed LFK was 103 colony forming unit/ml. Treatment of the milk samples with an application buffer and its pre-incubation in trypticase soy broth for 6 h at 37°C before testing significantly increased the sensitivity of the prepared LFK. The developed kit proved simple and convenient, and the results could be obtained in less than 10 min.

Inactivation trends of Bacillus cereus and Listeria monocytogenes grown in tryptic soy broth to the early stationary phase and subjected to ultraviolet irradiation

In the present study, the inactivation trends of Bacillus cereus and Listeria monocytogenes, grown in tryptic soy broth to the early stationary phase and subjected to ultraviolet (UV)-C irradiation, were identified and analyzed using mathematical models. The inactivation curves of B. cereus showed a tailing effect and were best fitted with the double Weibull model. In contrast, the inactivation curves of L. monocytogenes showed both shoulder and tailing characteristics and were best fitted with the biphasic shoulder model. The survival of highly resistant pathogens along with non-uniform bactericidal treatment was suggested as the reason for the tailing effect; the multi-hit nature of UV irradiation along with the DNA recovery ability of the pathogens was suggested as the reason for the shoulder effect. Moreover, the pathogens grown to the early stationary phase were more resistant than those grown to the mid-stationary phase. Therefore, a wider range of studies is needed regarding pathogens growing to the early stationary phase.

Screening of the bio-decolorization ability of synthetic dyes and the degradation of hydrocarbon bacteria Serratia marcescens MBC1

Humans are inseparable from using hydrocarbons, including fossil fuels, dyes, and oleochemical products. This has frequently resulted in a high possibility of waste spills polluting the environment. A biological approach using microbes was used to facilitate many of these countermeasures. This study aimed to determine the ability of the bacterium Serratia marcescens MBC 1 isolate as a biodegradation agent for various hydrocarbons and a bio-decolorizing agent for synthetic dyes. The bacterial growth media used were solid and tryptic soy broth. Methylene blue, congo red, methyl red, methyl orange, and crystal violet were synthetic dyes at a concentration per million. Within 7 days, several synthetic dyes tested showed decolorization. Methylene blue has the fastest decolorization time, taking just 24 h. The lipase index method was used to assess the propensity of hydrocarbons to degrade qualitatively. Kerosene had the highest lipolytic index at 6.31, followed by used cooking oil at 5.48 index, used lubricant at 5.37 and diesel at 3.63 index. Quantitative and comprehensive in-depth testing of the potential achievements of this initial test will be used in solving environmental problems that may occur further, especially related to the impact of the use of synthetic dyes.

Staphylococcal Enterotoxins Enhance Biofilm Formation by Staphylococcus aureus in Platelet Concentrates.

Biofilm formation and slow growth by Staphylococcus aureus in platelet concentrates (PCs) cause missed detection of this bacterium during routine PC screening with automated culture systems. This heightens the chances of false-negative screening transfusions and pre-disposes transfusion patients to an elevated risk of sepsis due to secretion of staphylococcal enterotoxins (SEs) in PCs. A hybrid approach of comparative RNAseq analyses and CRISPR mutagenesis of SE genes was employed to investigate the effect of SEs in S. aureus growth and biofilm formation in PCs. RNAseq data showed no differential expression for key biofilm genes, whereas SE genes were upregulated (>0.5- to 3.6-fold change) in PCs compared to trypticase soy broth (TSB). Remarkably, growth and biofilm formation assays revealed increased growth for the S. aureus SE mutants, while their ability to form biofilms was significantly impaired (-6.8- to -2.4-fold change) in comparison to the wild type strain, in both PCs and TSB. Through the well-established superantigen mechanism of SEs, we propose three roles for SEs during biofilm development in PCs: (1) provide a scaffold for biofilm matrix, (2) mediate cell-to-cell aggregation, and (3) guarantee biofilm survival. Furthermore, SE contribution to both growth and biofilm development seems to be centrally regulated by agr via quorum sensing and by saeSR and sigB. This study reveals new roles for SEs, which enforce their relevance in ensuring PC safety for transfusion patients. It further deciphers the underlying reasons for failed S. aureus detection in PCs during screening with automated culture systems.

500. Impact of an iron chelator on in vitro and in vivo growth of A. baumannii

Abstract Background Acinetobacter baumannii (AB) has become multidrug-resistant in recent years and currently there is no consensus on the optimal treatment for these infections. To combat this urgent threat, novel treatment options are needed to minimize spread and lower mortality rates. Nutrient metals (e.g., iron) are essential to the metabolic functions of AB. If the bioavailability of essential nutrients is restricted, this can cause a bacterial stress response and alter their ability to multiply. Methods The objective of the study was to examine the impact of iron chelation on the growth of AB in vitro and in vivo. MDR AB bloodstream isolates (n=4) were recovered from unique patients between 2011 and 2018. Clonal diversity was ascertained by Fourier Transform Infrared Spectroscopy. In vitro bacterial densities were measured longitudinally over 20-hours to determine the growth profile. Isolates were started at a baseline concentration of approximately 5.5 log CFU/mL in tryptic soy broth. Variable amounts of an iron chelating agent (deferiprone, 0.2-2mM) were added to create a concentration gradient. Galleria mellonella larvae (100-150mg) were inoculated with approximately 5 log CFU of an isolate, with and without deferiprone (10mM). Quantitative culture was used to ascertain the bacterial burden of aggregate larvae (n=10) immediately and 4h post infection. Additional infected larvae (n=20 in each group) were incubated at 35°C and monitored hourly for 24h. Results Time to mortality was compared using Kaplan-Meier survival analysis and log-rank test. Increasing concentrations of the chelating agent caused a transient and concentration-dependent hindrance of in vitro bacterial growth, compared to the no-treatment group. Bacterial burden immediately post-infection of the control and treatment group were comparable at 6.4 log CFU/g. After 4 hours the bacterial burden was much higher in the control group than the treatment group, 8.7 and 6.7 log CFU/g respectively. A trend of delayed mortality was shown in the iron chelator group (median survival 6.5 vs 10 hours, p=0.15). Conclusion These results support that micro-nutrient limitation has the potential to be a novel approach for treating high-risk infections due to MDR AB. Future studies are warranted to further define its optimal place in therapy. Disclosures All Authors: No reported disclosures.

Transcriptomic and phenotypic changes of Cronobacter sakazakii ATCC 29544 grown under desiccation stress

In the present study, the effect of desiccation stress on the transcriptomic and phenotypic changes of Cronobacter sakazakii ATCC 29544 was identified. Glucose or lactose was added to the commonly used tryptic soy broth (TSB) to induce desiccation stress (aw = 0.975). From transcriptomic analysis, 126 and 64 upregulated differentially expressed genes (DEGs) were identified for glucose added TSB and lactose added TSB, respectively, and validated by qRT–PCR. Formate metabolism-related genes were highly overexpressed by desiccation stress among the DEGs. When phenotypic changes were investigated, the addition of glucose increased the ratio of unsaturated fatty acids in the membrane lipids of C. sakazakii, which in turn decreased membrane rigidity and made it more susceptible to lipid oxidation. The opposite trend was observed for the pathogen grown in lactose-supplemented TSB. In this regard, the heat resistance of C. sakazakii grown in TSB was lower than that grown in lactose-supplemented TSB, while the opposite trend was observed for the pathogen grown in glucose-supplemented TSB. These results indicated that the transcriptome and phenotype of C. sakazakii would be changed by the materials used to nutrify the pathogen.

Phenotyping and Genotyping of Salmonella enterica as Biofilm Producer Isolated from Diarrheal Animal and Human

This study aimed to find out how different stress conditions (like temperature and pH) affect Salmonella enterica biofilm formation. This was done by looking at the phenotypic and genotypic features of isolates. 12 Salmonella enterica Isolate from animals, and 13 Salmonella enterica Isolate from people were used. S. enterica isolates were grown in tryptic soy broth (TSB) at (37°C, 25°C, and 42°C), and at pH levels (7, 5, and 9). The results revealed that the percentage was 52% in the standard conditions (temperature 37ºC and pH 7) while, in another condition, observed in the same temperature (37 °C) but with pH differences (pH 5, pH 9). S. enterica, did not produce biofilm. As for the stability pH in the, pH 7 with a change in, the temperature at 25°C percentage, biofilm produce (44%) while in 42 °C (64%). The detection rates of genes, biofilm-related PCR was used to find BapA and CsgD, were 100%. Although the biofilm formation of the phenotype did not give 100% results, the genotype gave 100%, which indicates that the gene is present but not expressed. Based on the findings in this study provided valuable information on the biofilm formation of Salmonella isolated from animals and humans.

Growth Media of Escherichia coli Does Not Affect Its Survival in Soil under Static Conditions

Many studies have examined the survival of Escherichia coli and foodborne pathogens in agricultural soils. The results of these studies can be influenced by various growth conditions and growth media used when preparing cultures for an experiment. The objectives of this study were to (i) determine the growth curves of rifampin (R)-resistant E. coli in three types of growth media containing R: tryptic soy agar (TSA-R); tryptic soy broth (TSB-R); and poultry pellet extract (PPE-R) and (ii) evaluate the influence of growth media on the survival of E. coli in agricultural soil. Poultry pellet extract (PPE) was prepared by filter sterilizing a 1:10 suspension of heat-treated poultry pellets in sterile water. Generic E. coli (TVS 353) acclimated to 80 μg/mL of R was grown in TSA-R, TSB-R, and PPE-R at 3.0 to 3.5 log CFU/mL and incubated at 37°C. Growth curves were determined by quantifying E. coli populations at 0, 4, 8, 16, 24, and 32 h. Soil microcosms were inoculated with E. coli (6.0 log CFU/g) previously cultured in one of the three media types and stored at 25°C, and soil samples were quantified for E. coli on days 0, 1, 3, 7, 14, 28, and 42. Growth curves and survival models were generated by using DMFit and GInaFiT, respectively. E. coli growth rates were 0.88, 0.77, and 0.69 log CFU/mL/h in TSA-R, TSB-R, and PPE-R, respectively. E. coli populations in the stationary phase were greater for cultures grown in TSA-R (9.4 log CFU/mL) and TSB-R (9.1 log CFU/mL) compared with PPE-R (7.9 log CFU/mL). The E. coli populations in the soil remained stable up to 3 days before declining. An approximate 2 log CFU/g decline of E. coli in soil was observed for each culture type between days 3 and 7, after which E. coli populations declined more slowly from days 7 to 42. A biphasic shoulder model was used to evaluate E. coli survival in soils on the basis of growth media. Using standardized culture growth preparation may aid in determining the complex interactions of enteric pathogen survival in soils.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

Variability in Adaptive Resistance of Salmonella Typhimurium to Sublethal Levels of Antibiotics.

This study was designed to evaluate the adaptive resistance of Salmonella Typhimurium under continuous sublethal selective pressure. Salmonella Typhimurium ATCC 19585 (STATCC) and S. Typhimurium CCARM 8009 (STCCARM) were sequentially cultured for 3 days at 37 °C in trypticase soy broth containing 1/2 × MICs of cefotaxime (CEF1/2), chloramphenicol (CHL1/2), gentamicin (GEN1/2), and polymyxin B (POL1/2). The STATCC and STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were evaluated using antibiotic susceptibility, cross-resistance, and relative fitness. The susceptibilities of STATCC exposed to GEN1/2 and POL1/2 were increased by a 2-fold (gentamicin) and 8-fold (polymyxin B) increase in minimum inhibitory concentration (MIC) values, respectively. The MIC values of STCCARM exposed to CEF1/2, CHL1/2, GEN1/2, and POL1/2 were increased by 4-fold (cefotaxime), 2-fold (chloramphenicol), 2-fold (gentamicin), and 8-fold (polymyxin B). The highest heterogeneous fractions were observed for the STATCC exposed to CEF1/2 (38%) and POL1/2 (82%). The STCCARM exposed to GEN1/2 was cross-resistant to cefotaxime (p < 0.05), chloramphenicol (p < 0.01), and polymyxin B (p < 0.05). The highest relative fitness levels were 0.92 and 0.96, respectively, in STATCC exposed to CEF1/2 and STCCARM exposed to POL1/2. This study provides new insight into the fate of persistent cells and also guidance for antibiotic use.

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 μg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 μg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 μg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

Shelf-life prediction and storage stability of Aeromonas bacteriophage vB_AsM_ZHF

Phage therapy is a potential alternative to antibiotics for the treatment of bacterial infections. Due to the good antibacterial and therapeutic effects of phages, phage therapy has received attention and the demand for clinical applications has gradually increased. Phage storage stability and shelf life are key aspects of biopharmaceutical development and registration. In this study, Aeromonas salmonicida phage was stored at different temperatures for 12 months. We found that 4 °C was the optimal storage temperature. In the case of cryopreservation, 10% dimethyl sulfoxide (DMSO) was more effective at protecting the phage at -20 °C and -80 °C than 30% glycerin. Indeed, the phage titer decreased by only one order of magnitude within one year when DMSO was added. Hydroxyapatite (HAP) reduced the inactivation of phages by six orders of magnitude during storage at 28ºC for 1 year, significantly lower than that of in SM buffer. In addition, for excipients in lyophilization, tryptic soy broth (TSB) and tryptone or skim milk powder (SMP) in combination with trehalose alleviated phage inactivation during lyophilization and subsequent storage at 28 °C. Furthermore, a model for predicting the phage shelf-life was established with the Accelerated Stability Assessment Program (ASAP) based on the Arrhenius equation. The error of the model was less than 15% by comparing the predicted value with the actual value at 28 °C, indicating high accuracy. The study demonstrated the storage stability and shelf-life model of phage for the first time, which provided a theoretical basis for the development and application of phage products.

Influence of Different Stainless Steel Finishes on Biofilm Formation by Listeria monocytogenes

Biofilm formation of Listeria monocytogenes on stainless steel, a widely used abiotic surface in the food processing industry, was investigated by focusing on the attachment tendency and behavior of L. monocytogenes 08-5578 on eight different stainless steel surfaces: glass bead blasted (rough and fine), deburred (Timesaver), drum deburred, pickled, pickled and drum polished, electrolytic polished, and cold rolled (untreated control). The aim was to see whether there are finishes with significantly lower bacterial attachment. Surface roughness data (measured via four roughness parameters), determined by interferometry, was also compared with the number of adhering cells to detect possible correlations. Cultivation of L. monocytogenes biofilms was carried out using a CDC biofilm reactor with 1% tryptic soy broth set at 20°C for 4, 8, and 24 h. In addition, a cultivation trial was run with continuous nutrient flow (1% tryptic soy broth, 6.2 mL/min) for 24 h. Eight-hour results showed a significant difference (P < 0.05) in biofilm cell counts in biofilms between the glass bead-blasted surfaces (3.23 and 3.26 log CFU/cm2 for the fine and rough, respectively) and deburred (Timesaver) surface (2.57 log CFU/cm2), between drum deburred and deburred (Timesaver) surface (3.41 versus 2.57 log CFU/cm2), and between drum deburred and pickled surface (3.41 versus 2.77 log CFU/cm2). Data gained after 4-h, 24-h, and 24-h plus an additional 24-h continuous flow cultivation showed no significant difference in attachment among surfaces. No correlation between roughness data and attachment was found after all four incubation times, suggesting that roughness values, at these ranges, are insufficient in determining the surfaces' affinity to bacteria. Overall, this study suggests that roughness values cannot be used to predict the degree of L. monocytogenes attachment to a specific stainless steel surface.

Sunlight Parameters Influence the Survival and Decline of Salmonella and Escherichia coli in Water

The effect of variations in temperature, UV radiation, and sunlight intensity on Escherichia coli, E. coli O157:H7, Salmonella Newport, and antibiotic resistant (ABR) variants of E. coli O157:H7 and Salmonella Newport exposed to sunlight was evaluated. Bacterial strains suspended in sterile deionized water at a concentration of 8 log CFU/mL were exposed to sunlight on three different days for 180 min; control treatments were stored in the dark. The mean temperature of 30.08 and 26.57°C on day 1 and day 3, respectively, was significantly different (P < 0.05). The UV intensity was significantly different on all 3 days, and sunlight intensity significantly differed on day 3 (P < 0.05). Bacterial population decline positively correlated with temperature, sunlight, and UV intensity. Differences in bacterial population declines differed among species, ABR profile, and day of exposure (P < 0.05). On day 1 and day 2, the populations of E. coli dropped below the limit of detection (1 log CFU/mL), whereas the percentage of live cells was 67 and 6.6%, respectively. The artificial neural network model developed to predict bacterial survival under different environmental conditions suggested that Salmonella cells were more resistant than E. coli cells. The ABR strains had significantly higher numbers of viable cells after sunlight exposure (P < 0.05). Sunlight-exposed cells resuscitated in tryptic soy broth varied in maximum population density and maximum specific growth rate based on bacterial species and presence of ABR. Morphological changes such as viable but nonculturable state transition and filament formation were detected in subpopulations of sunlight-exposed bacteria. Daily fluctuations in UV and sunlight intensity can result in significant variations in bacterial decline and recovery.