Impact of desiccation and heat exposure stress on Salmonella tolerance to acidic conditions

In a recent study, the pH of commonly used Salmonella pre-enrichment media became acidic (pH 4.0 to 5.0) when feed or feed ingredients were incubated for 24 h. Acidic conditions have been reported to injure or kill Salmonella. In this study, cultures of four known feed isolates (S. montevideo, S. senftenberg, S. tennessee, and S. schwarzengrund) and four important processing plant isolates (S. typhimurium, S. enteritidis, S. infantis, and S. heidelberg) were grown on meat and bone meal and later subjected to desiccation and heat exposure to stress the microorganism. The impact of stress on the isolates ability to survive in acidic conditions ranging from pH 4.0 to 7.0 was compared to the non-stressed isolate. Cell injury was determined on xylose lysine tergitol 4 (XLT4) and cell death determined on nutrient agar (NA). When measured by cell death in non-stressed Salmonella, S. typhimurium was the most acid tolerant and S. heidelberg was the most acid sensitive whereas in stressed Salmonella, S. senftenberg was the most acid tolerant and S. tennessee was the most acid sensitive. The pH required to cause cell injury varied among isolates. With some isolates, the pH required for 50% cell death and 50% cell injury was similar. In other isolates, cell injury occurred at a more neutral pH. These findings suggest that the pH of pre-enrichment media may influence the recovery and bias the serotype of Salmonella recovered from feed during pre-enrichment.     

Sodium bisulfate and a sodium bisulfate/tannin mixture decreases pH when added to an in vitro incubated poultry cecal or fecal contents while reducing Salmonella Typhimurium marker strain survival and altering the microbiome

The objective of the present study was to investigate the ability of animal feed-grade sodium bisulfate (SBS) and a mixture of sodium bisulfate/tannin to inhibit the growth of Salmonella using an anerobic in vitro mixed cecal culture to mimic the conditions within the chicken cecum. An initial inoculum of Salmonella Typhimurium was introduced to an anerobic dilution solution containing 1/3000 diluted cecal bacteria and solids consisting of ground chicken feed and different percentages of solid SBS or SBS/tannin, and surviving organisms were enumerated. Two different experimental designs were employed. In the “unadapted” treatment, the S. Typhimurium was added at the beginning of the culture incubation along with cecal bacteria and chicken feed/SBS or chicken feed/SBS/tannin. In the “adapted” treatment, S. Typhimurium was added after a 24 hour pre-incubation of the cecal bacteria with the chicken feed/SBS or chicken feed/SBS/tannin. Adding SBS resulted in reduction of pH in the cultures which paralleled with the reduction of S. Typhimurium. The SBS alone was found to be inhibitory to S. Typhimurium in the adapted treatment at all concentrations tested (0.25, 0.5, and 0.75%), and the degree of inhibition was concentration-dependent. Salmonella Typhimurium was completely killed in the adapted culture with 0.5% SBS after 24 and 48 h. The SBS/tannin mixture was less inhibitory than SBS alone at the same concentrations in side-by-side comparisons. Testing at a 0.5% SBS concentration, chicken age had little or no effect on log reduction of S. Typhimurium relative to age-matched control cultures without SBS, but age did affect the absolute number of S. Typhimurium surviving, with the greatest decreases occurring at 2 and 4 weeks of age (approx. 10³ S. Typhimurium surviving) compared to 6 weeks of age (approx. 10⁵ Salmonella surviving). Microbiome analysis with an Illumina MiSeq platform was conducted to investigate bacterial compositional changes related to the addition of SBS. The relative abundance of Firmicutes (at the phylum level) was decreased, and genera Lactobacillus and Faecalibacterium were increased when SBS was added to the anaerobic mixed culture containing either fecal or cecal material. The antimicrobial action of feed-grade SBS may represent a potential pre-harvest control measure for Salmonella in poultry production.     

Effect of soluble maillard reaction products on cadA expression in Salmonella typhimurium

The presence of Maillard reaction products (MRP) in foods and food components is due to the non-enzymatic reaction between protein and carbohydrate residues triggered by thermal steps during food processing. The objective of this study was to assess the effect of MRPs and increasing lysine concentrations on S. Typhimurium growth and the expression of cadA which may be an indirect determinant of Salmonella virulence response. Variations in lysine concentrations (from 0 to 0.5 mM) did not exert any effect either on the final optical density after 6-hour incubation or the growth rates of S. Typhimurium in media containing MRPs. In contrast to the reduced final absorbancy of the bacterial cultures grown with histidine and arginine MRPs supplementations (0.1%), growth rates, in general, remained unaltered by all MRPs at each lysine concentration when compared to the control (M9 pH 5.8, no MRPs added). The induction levels of cadA in media containing 0.1% MRPs were close to cadA induction in the reference media (M9, pH 5.8 and no MRPs) and did not exceed the corresponding values by more than approximately 30%. Although the observed negligible induction of cadA under these conditions complies with the concept of its potential “anti-virulence” function, additional studies involving various concentrations and more refined MRPs are needed.     

Biodegradation of fatty acylamino acids by fusarium culmorum

Abstract

Biodegradation of the fatty acylamino acids by Fusarium culmorum, measured in terms of the release of radioactive aspartate and lysine, occurred maximally at pH 6.5 and pH 7.0, respectively in 10 day cultures. Thirty‐six percent and twenty‐four percent of the total radioactivity recovered were in released aspartate and lysine, respectivley at 30°C. Twenty degrees (C) was the minimum temperature for biodegradation of these compounds by F. culmorum. Greater degradation was observed at 15°C and 30°C. The data suggest the activity of hydrolytic isoenzymes, with optima at different pH's and temperatures, operating in the biodegradation process.     

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

Foodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.     

The StepOne real-time polymerase chain reaction detection of Salmonella sp., Salmonella enterica ser. typhimurium and enteritidis in milk and meat

The aim of this study was to follow contamination of ready to eat milk and meat products with Salmonella spp. by using the StepOne real-time polymerase chain reaction (PCR). Classical microbiological methods for detection of foodborne bacteria involve the use of pre-enrichment and/or specific enrichment, following isolation of bacteria in solid media and the final confirmation by biochemical and/or serological tests. We used the PrepSEQ Rapid Spin Sample Preparation Kit for isolation of DNA and MicroSEQ® Salmonella spp. Detection Kit for pursuance of the real-time PCR (Applied Biosystems). In samples without incubation we detected strain of Salmonella sp. in 5 out of 25 samples (swabs), as well as in the internal positive control (IPC), which was positive in all samples. This StepOne real-time PCR assay is extremely useful for any laboratory equipped by real-time PCR. It is a fast, reproducible, simple, specific and sensitive way to detect nucleic acids, which could be used in clinical diagnostic tests in the future. Our results indicated that real-time PCR assay developed in this study could sensitively detect Salmonella spp. in ready-to-eat food. This could prevent infection caused by Salmonella, and also could benefit food manufacturing companies by extending their product's shelf-life as well as saving the cost of warehousing their food products while awaiting pathogen testing results.     

Non-molecular characterization of pellicle formation by poultry Salmonella Kentucky strains and other poultry-associated Salmonella serovars in Luria Bertani broth

Abstract

There is limited research concerning the biofilm-forming capabilities of Salmonella Kentucky, a common poultry isolate. The objective was to quantitate pellicle formation of S. Kentucky versus better-characterized Salmonella strains of Enteritidis and Heidelberg. In separate experiments, Salmonella strains and serovars were tested for their biofilm-forming abilities in different Luria-Bertani (LB) broths (1); pellicle formation in different volumes of LB without salt (2); and the potential priming effects on formation after pellicles were transferred three consecutive times (3). Data were analyzed using One-Way ANOVA with means separated using Tukey’s HSD (P?≤?0.05). In the first experiment, there was no significant effect between strain and serovars (P?>?0.05), but media type affected pellicle formation significantly with LB Miller and LB minus NaCl plus 2% glucose resulting in no pellicle formation (P?<?0.001). When grown in 50?mL, Kentucky 38-0085 produced larger pellicles than Kentucky 38-0055, and Heidelberg strain 38-0127 (P?<?0.0001). Serial transfers of pellicles did not significantly affect pellicle formation (P?>?0.05); however, Kentucky 38-0084, 38-0085 and 38-0086 produced larger pellicles than Kentucky 38-0055 and 38-0056 and Heidelberg 38-0126, 38-0127 and 38-0152. The current study demonstrates the consistent biofilm forming capabilities of Kentucky and may explain why Kentucky is frequently isolated in poultry processing facilities.     

Fungicide sensitivity of Trichoderma spp. from Agaricus bisporus farms in Serbia

Trichoderma species, the causal agents of green mould disease, induce great losses in Agaricus bisporus farms. Fungicides are widely used to control mushroom diseases although green mould control is encumbered with difficulties. The aims of this study were, therefore, to research in vitro toxicity of several commercial fungicides to Trichoderma isolates originating from Serbian and Bosnia-Herzegovina farms, and to evaluate the effects of pH and light on their growth. The majority of isolates demonstrated optimal growth at pH 5.0, and the rest at pH 6.0. A few isolates also grew well at pH 7. The weakest mycelial growth was noted at pH 8.0–9.0. Generally, light had an inhibitory effect on the growth of tested isolates. The isolates showed the highest susceptibility to chlorothalonil and carbendazim (ED₅₀ less than 1 mg L^?1), and were less sensitive to iprodione (ED₅₀ ranged 0.84–6.72 mg L^?1), weakly resistant to thiophanate-methyl (ED₅₀ = 3.75–24.13 mg L^?1), and resistant to trifloxystrobin (ED₅₀ = 10.25–178.23 mg L^?1). Considering the toxicity of fungicides to A. bisporus, carbendazim showed the best selective toxicity (0.02), iprodione and chlorothalonil moderate (0.16), and thiophanate-methyl the lowest (1.24), while trifloxystrobin toxicity to A. bisporus was not tested because of its inefficiency against Trichoderma isolates.     

Treatment of cork boiling wastewater using chemical oxidation and biodegradation

Three cultures were enriched from cork boiling wastewater using tannic acid as the selective carbon substrate, at 25 degrees C and pH 7.2, 25 degrees C and pH 4.7 and 50 degrees C and pH 4.7. The enrichment culture obtained at neutral pH was composed of five culturable isolates, whereas from each acidic enrichment two bacterial strains were isolated. Mesophilic isolates were Gram negative bacteria belonging to the genera Klebsiella, Pseudomonas, Stenotrophomonas and Burkholderia. Thermophilic isolates were members of the genus Bacillus. Despite the capability of the enrichment cultures to use tannic acid as single carbon and energy source, those cultures were unable to reduce the total polyphenols or the total organic carbon content of cork boiling wastewater. In order to increase the bioavailability of the organic carbon in cork boiling wastewater, biodegradation was preceded by Fenton oxidation. It was demonstrated that the combined process, using small amounts of Fenton reagents and biodegradative inoculum added almost simultaneously to cork boiling wastewater, leads to TOC reductions of more than 90%.     

Prevalence and antimicrobial resistance of Shiga toxin-producing Escherichia coli in milk and dairy products in Egypt

Abstract

Food contaminated with Shiga toxin-producing Escherichia coli (STEC) represents a hazardous public health problem worldwide. Therefore, the present study was performed to elucidate the virulent and antimicrobial resistance characteristics of STEC isolated from milk and dairy products marketed in Egypt. A total of 125 samples (raw market milk, bulk tank milk, Kareish cheese, white soft cheese, and small scale-produced ice cream, 25 each) were collected for determination the prevalence and antimicrobial resistance profiling of STEC. Thirty-six STEC isolates were recovered from milk and dairy products. Serological analysis illustrated that three isolates were E. coli O157:H7 and 33 isolates belonged to different serotypes. Molecular examination indicated that all isolates harboured stx1 and/or stx2 genes, 14 isolates expressed eaeA gene and 3 isolates possessed rfbE gene. Antimicrobial resistance profiling of the isolates was both phenotypically and genetically examined. Interestingly, 31 out of 36 (86.11%) isolates were multidrug-resistant and harboured the extended-spectrum β-lactamase encoding genes, namely, bla_CTX-M-15, bla_SHV-12 and bla_CTX-M-14. Moreover, 12 isolates (33.33%) harboured plasmid-mediated quinolone resistant gene, qnrS. The overall conclusion of the current investigation indicated insufficient hygienic measures adopted during milking, handling, and processing leading to development of pathogenic and multidrug-resistant STEC.     

In vitro toxicity of fungicides with different modes of action to alfalfa anthracnose fungus,Colletotrichum destructivum

Abstract

Sensitivity of 24 isolates of Colletotrichum destructivum O’Gara, collected from alfalfa plants in Serbia, to eight selected fungicides, was investigated in this study. Molecular identification and pathogenicity test of isolates tested were also performed. Fungicide sensitivity was evaluated in vitro, using mycelial growth assay method. All isolates exhibited significant pathogenicity, causing necrosis at the alfalfa seedling root tips two days after inoculation. Using the primer pair GSF1-SR1 and by comparing the amplified fragments of the tested isolates with the marker (M), the presence of the amplicon of the expected size of about 900?bp was determined for all isolates. The isolates tested in this study showed different sensitivity towards fungicides in vitro. Mycelial growth was highly inhibited by QoI (quinone outside inhibitors) fungicide pyraclostrobin (mean EC₅₀=0.39?µg mL^?1) and by DMI (demethylation-inhibiting) fungicide tebuconazole (mean EC₅₀=0.61?µg mL^?1), followed by azoxystrobin (mean EC₅₀=2.83?µg mL^?1) and flutriafol (mean EC₅₀=2.11?µg mL^?1). Multi-site fungicide chlorothalonil and MBC (methyl benzimidazole carbamate) fungicide thiophanate-methyl evinced moderate inhibition with mean EC₅₀=35.31 and 62.83?µg mL^?1, respectively. Thirteen isolates were sensitive to SDHI (succinate dehydrogenase inhibitors) fungicide boscalid and fluxapyroxad, (mean EC₅₀=0.49 and 0.19?µg mL^?1, respectively), while the rest of isolates were highly resistant.     

Rapid immobilization of viable Bacillus pseudomycoides in polyvinyl alcohol/glutaraldehyde hydrogel for biological treatment of municipal wastewater

A new approach for easy synthesis of Bacillus pseudomycoides immobilized polyvinyl alcohol (PVA)/glutaraldehyde (GA) hydrogel for application in a wastewater treatment system is reported. Optimization studies revealed that GA/PVA mass ratio of 0.03 and acidic pH of 2 were required for hydrogel synthesis and eventually for bacterial cell immobilization. The synthesized crosslinked matrix possessed a pore size suitable for microbial cell entrapment while maintaining cell accessibility to external environment for bioremediation. Possible crosslinking and bacterial cell immobilization in the hydrogel were evidenced by FTIR, XRD, and SEM studies, respectively. Further, the extent of crosslinking of GA with PVA was investigated and confirmed by transmittance and permeability experiments. The viability and proliferation of hydrogel embedded cells (after 25 days) was confirmed by confocal fluorescence microscopy which also indicated that acidic pH of polymer solution did not affect the immobilized live cells. B. pseudomycoides immobilized hydrogel were demonstrated to be effective for treatment of municipal wastewater and reduced biochemical oxygen demand (BOD), chemical oxygen demand (COD), and protein content below the recommended levels. Overall, the results from this bench-scale work show that employing bacteria-embedded PVA/GA hydrogel for the treatment of municipal wastewater yield promising results which should be further explored in pilot/field-scale studies.

     

Cross-reactivity of anti-Salmonella egg-yolk antibodies to Salmonella serovars

The cross-reactivity of egg yolk antibodies specific to antigens of Salmonella Enteritidis and Salmonella Typhimurium to killed bacterial cells of common Salmonella serovars were tested using an indirect Enzyme Linked Immunosorbent Assay (ELISA). Egg yolk antibodies were produced against purified fimbriae, flagella and lipopolysaccharide (LPS) of S. Enteritidis strain ATCC13076 and flagella, LPS and outer membrane proteins (OMP) of S. Typhimurium strain ATCC13311. For immunological specificity of egg yolk antibodies against killed bacterial cells, we found that the titers of the anti-S. Enteritidis egg yolk antibodies were higher than those of the anti-S. Typhimurium antibodies. In the evaluation of cross-reactivity of these egg yolk antibodies to various Salmonella serovars, we observed that the anti-S. Enteritidis antibodies exhibited more specific affinity than those of the anti-S. Typhimurium antibodies. All S. Enteritidis strains reacted specifically with the anti-S. Enteritidis fimbrial and flagellar egg yolk antibody whereas anti-S. Enteritidis LPS and anti-S. Typhimurium LPS, OMP and flagellar antibodies displayed non–specific reactivity to all Salmonella serovars used in this study. This finding suggests that it may be possible to design a anti-fimbrial egg yolk antibody of S. Enteritidis as a diagnostic tool and a cocktail of OMP and LPS antigens of S. Enteritidis and S. Typhimurium could be used for administering broad spectrum passive immunity to protect against the colonization of pathogenic Salmonella strains in food animals.     

Comparison of Spontaneous Antibiotic Resistance Frequency of Salmonella Typhimurium Growth in Glucose Amended Continuous Culture at Slow and Fast Dilution Rates

Abstract

The objective of the study was to determine the frequency of spontaneous acquisition of resistance to select antibiotics by Salmonella Typhimurium (ST) when grown in glucose amended continuous flow culture at slow (D = 0.025 h^{? 1}) or fast (D = 0.27 h^{? 1}) dilution rates. The bacterium was grown in LB minimal medium (pH 6.25) containing no antibiotics. Upon achieving steady state, samples were plated to tryptic soy agar (TSA) alone or supplemented (per ml) with 2 and 16 μg oxytetracycline, 4 and 16 μg tetracycline, 2 and 64 μg kanamycin, and 0.25 and 2 μg enrofloxacin. Regardless of growth rate, CFU of resistant ST from the TSA containing antibiotics was less than 2 × 10¹ except for 2 μg kanamycin and 0.25 μg enrofloxacin treatments (higher than 1 × 10⁹ and 4 × 10⁷ CFU of resistant ST for trials 1 and 2, respectively). Frequency of recovering resistant ST from the TSA containing the higher antibiotic concentrations was less than 1 in 10⁹ for all antibiotics, but was higher on the media containing 2 μg kanamycin and 0.25 μg enrofloxacin at both slow and fast growth rates. In general, minimal susceptibility differences were detected for isolates from slow and fast dilution rates.     

Toxicity of pH,heavy metals and bisulfite to a freshwater green alga

Low pH markedly reduced the growth and photosynthetic activity of an $\text{Ankistrodesmus}$ sp. The alga could not grow at pH 3 and only slight growth occurred at pH 4. The alga grew well above pH 4 with maximum growth occurring at pH 6. The fixation of ¹⁴CO₂ followed a similar pattern with pH. The algal cells were also sensitive to mercury and bisulfite in acidic conditions. Algal growth and photosynthesis were reduced by mercury and bisulfite more at pH 5 than at pH 7. Lead was relatively non-toxic to the algal cells at both pH levels. Bisulfite inhibited the membrane transport of α-amino-¹⁴C-isobutyric acid at pH 5 but not at pH 7. The results suggest that algal growth and activity may be reduced in acidic lakes by low pH and that the toxicity of mercury and bisulfite is enhanced in acidic conditions.     

The Combination of Zinc Compounds and Acidic pH Limits Aerobic Growth of a Salmonella typhimurium Poultry Marker Strain in Rich and Minimal Media

Abstract

The objective of the present study was to examine the combined effects of zinc compounds with different acidic pH levels on the aerobic growth of a S. typhimurium poultry isolate in either rich or minimal media. When overall main effects of pH levels of medium or concentrations of Zn compounds were compared, growth rates of the S. typhimurium poultry isolate were significantly ( p < 0.05) decreased by stepwise increase of pH levels of medium (pH 4, 5, 6, and 7) or concentrations (0.67, 3.35, and 6.03%) of Zn compounds (Zn acetate and Zn sulfate). In general growth rates of S. typhimurium poultry isolate appeared to be more reduced by Zn acetate than by Zn sulfate and more reduced in minimal media compared to rich media.     

Effect of the appetite stimulant cyproheptadine on deoxynivalenol ‐ induced reductions in feed consumption and weight gain in the mouse 1

Abstract

Deoxynivalenol (DON, vomitoxin), a Fusarium mycotoxin, is suspected of inducing its anorectic/feed refusal activity through a serotoninergic (5HT) mechanism, possible via 5HT₂‐receptors. In this study the efficiency of cyproheptadine (CYP), a serotonin antagonist and known appetite stimulant, to attenuate the adverse effect of DON was investigated in mice. CYP was administered in the feed for two days before animals began receiving the DON, which was also added to the feed. Both agents were administered concurrently thereafter for a 12‐day period. Dosing levels included various combinations of the two compounds, ranging from 0–16 ppm DON and 0–20 ppm CYP.

Results showed that CYP could effectively offset the reduction in feed intake caused by dietary DON, but only when dose levels were optimized (CYP appeared to have a narrow effective dose range, which was also dependent on the DON concentration). In fact, optimum CYP doses were able to enhance feed intake in DON‐fed mice above control levels, indicating that more than just a direct block of the toxin's effect was occurring. Conversely though, the effect of CYP on weight gain (WG) was ambiguous. At the lower CYP doses (≤ 5 ppm), alone or in combination with the lowest DON level tested (4 ppm), a modest positive effect was noted, but at the higher DON concentrations (≥ 8 ppm) the overall WG generally remained depressed, and tended to be reduced further by increasing doses of CYP.

These results provide additional support that serotoninergic mechanisms probably play a role in mediating DON‐induced reduction in feed intake. Where and how DON actually initiates its anorectic effect, however, has not been resolved.     

Impact of phosphate additive on organic carbon component degradation during pig manure composting

Phosphate, as an additive to composting, could significantly reduce ammonia emission and nitrogen loss but may also cause adverse effects on the degradation of organic matter. However, there is little information about the influence of pH change, salt content, and phosphate on different organic fraction degradation during composting with the addition of phosphate at a higher level. In this study, the equimolar phosphoric acid (H₃PO₄), sulfuric acid (H₂SO₄), and dipotassium phosphate (K₂HPO₄) were added into pig manure composting with 0.25 mol mass per kilogram of dry matter basis addition amount to evaluate the effect of H⁺, PO₄^3?, and salinity on carbon component transformation and organic matter degradation. The results showed that both H₃PO₄ and K₂HPO₄ additives could lead to shorter duration in the thermophilic phase, lower degradation of lignocellulose, and lesser carbon loss compared to CK, even though had different pH, i.e., acidic and alkaline conditions, respectively. Besides, the addition of H₃PO₄, H₂SO₄, and K₂HPO₄ could increase the degradation of soluble protein and lipid during composting. Redundancy analysis demonstrated that the variation in different organic carbon fractions was significantly correlated with the changes of pH and the presence of PO₄^3?, but not with SO₄^2? and electrical conductivity, suggesting that pH and phosphate were the more predominant factors than salinity for the inhibition of organic matter degradation. Taken together, as acidic phosphate addition produces a true advantage of controlling nitrogen loss and lower inhibition of organics transformation during composting, the expected effects may result in more efficient composting products.