Microbial-resistant Salmonella enteritidis isolated from poultry samples

Background:

Multidrug resistance in Salmonella enteritidis isolates is a public health problem worldwide; the present study, therefore, was designed for antimicrobial-resistance determination in this strain.

Methods:

Salmonella strains isolated from poultry samples by biochemical positive and negative tests were subjected to PCR and identified as Salmonella enteritidis. For detection and identification of Salmonella enteritidis isolates, sdfI gene-specific primers were used.

Results:

We found that 100% of isolates were resistant to ampicillin, 90% were resistant to cephalothin and streptomycin, 70% were resistant to cefotaxime, and 60% were resistant to kanamycin and gentamicin.

Conclusion:

Salmonella enteritidis isolates had antimicrobial resistance to mentioned antibiotics. Key Words: Antibiotic Resistance, PCR, Poultry, Salmonella enteritidis     

Influence of bacteriological media constituents on the reproduction of Salmonella enteritidis bacteriophages

Three different Salmonella enteritidis phages were isolated and purified from raw sewage by agar-layer technique. The sensitivity of the host organisms toward phages was changed when they were grown on different bacteriological media.The effect of single components of the medium on phage reproduction was determined by the omission of that substance from the medium. CaCl₂, MgSO₄, and glycerol each had a pronounced stimulatory effect on the phage reproduction, while bile salts had a profound inhibitory effect. The inhibitory effect of bile salts on phage growth was much greater on one strain of Salmonella enteritidis than on the other.     

In Vitro Protective Efficacy of Clostridium butyricum Against Fish Pathogen Infections

Most pathogens in intestine are opportunist, called “opportunistic pathogens” that usually do not cause disease in a healthy host. Only when the host’s resistance is lowered or the intestinal microecological balance is destroyed, the opportunistic pathogens are capable of causing disease. Here, two opportunistic pathogens, Salmonella enteritidis and Vibrio parahaemolyticus were chosen to test the possible antagonistic effect of the probiotic agent Clostridium butyricum on these pathogens infections in vitro using fish intestinal epithelial cells (FIECs). The C. butyricum and its spent culture supernatants exhibited significant inhibitory activity on S. enteritidis and V. parahaemolyticus growth and adherence to FIECs. The C. butyricum also showed significant inhibitory effects on S. enteritidis and V. parahaemolyticus induced apoptosis, which may due to its growth and adhesion inhibitory effects. These results indicated that the probiotic bacterium C. butyricum has preventive and therapeutic effects on S. enteritidis and V. parahaemolyticus infections in fish.     

Protection against Mesocestoides corti infection in mice treated with zymosan or Salmonella enteritidis 11RX

Toye P. G. and Jenkin C. R. 1982. Protection against Mesocestoides corti infection in mice treated with zymosan or Salmonella enteritidis 11RX. International Journal for Parasitology12: 399–402. Zymosan and Salmonella enteritidis 11RX were found to partially protect mice against infection with the cestode Mesocestoides corti. Thus, mice previously infected with S. enteritidis 11RX contained fewer parasites in the peritoneal cavity compared to normal mice. Mice pretreated with zymosan contained fewer parasites in the peritoneal cavity and in the liver compared to normal mice and this protection was enhanced by the passive transfer of serum from mice chronically infected with M. corti. Examination of mice in the initial stages of infection revealed that the administration of zymosan led to an alteration in parasite location from the peritoneal cavity to the liver.     

Immunoregulation by macrophages: differential secretion of prostaglandin E and interleukin 1 during infection with Salmonella enteritidis

We have investigated the temporal relationship between bacterial clearance in vivo, macrophage bactericidal activity in vitro, and the secretion of immunoregulatory molecules, prostaglandin E (PGE) and Interleukin 1 (IL1) in vitro, during infection with an avirulent strain of Salmonella, Salmonella enteritidis 11RX. The two model systems used were normal mice challenged intraperitoneally with SE11RX (NSE) and previously sensitized mice rechallenged 24 days later with SE11RX (SESE). The increasing nonspecific bactericidal activity of the peritoneal macrophages from NSE and SESE mice after the second day of infection paralleled the clearance of bacteria observed in vivo. Prostaglandin secretion by normal macrophages cultured for 4 hr with LPS correlated inversely with intracellular bacterial numbers but showed a positive correlation when cultured with opsonized SRBC or C3-zymosan complexes. PGE was the major arachidonate metabolite secreted. The cells from sensitized mice secreted tittle prostaglandin with any stimulus, and this secretion showed a positive correlation with bacterial number. The capacity to secrete IL1 in response to LPS increased during infection in both NSE and SESE mice. There was an inverse correlation between IL1 secretion and PGE production by cells from sensitized mice. We propose that changes in the capacity of peritoneal macrophages to secrete IL1 and PGE in response to stimulants in vitro reflect the initiation and regulation of the immune response through the course of infection.     

Effect of pH and Chelating Agents on the Heat Resistance and Viability of Salmonella typhimurium Tm-1 and Salmonella senftenberg 775W in Egg White

Supplementation of egg white at pH 8.9 with 5 mg of disodium ethylenediaminetetraacetic acid (EDTA) per ml resulted in a kill of Salmonella typhimurium Tm-1 of greater than 10⁶ per ml after 28 days at 2 C. While at 28 C, supplementation with 7 mg of EDTA per ml resulted in approximately a 10⁶ kill in less than 24 hr. Kena supplementation at 40 mg/ml of egg white resulted in a kill of S. typhimurium Tm-1 of greater than 10⁶ after approximately 60 hr of storage at 28 C. This is in contrast to no reduction in viable count in unsupplemented egg white stored at 2 C and a 100-fold increase in viable count in that stored at 28 C. Supplementation of egg white with EDTA at 7 mg/ml or with Kena at 10 mg/ml also affected the heat resistant characteristics of the two organisms at 52.5 C, reducing the time required to kill 90% of the population (D value) at any pH by a factor of 2 to 6. There was a synergistic effect between EDTA and lactic acid when lactic acid was used to adjust EDTA-supplemented egg white to an acidic pH (5.3) which greatly decreased the heat resistance of Salmonella senftenberg 775W (from 100D to D).     

Bacterial Resistance to Ultrasonic Waves under Pressure at Nonlethal (Manosonication) and Lethal (Manothermosonication) Temperatures

The decimal reduction times of Streptococcus faecium, Listeria monocytogenes, Salmonella enteritidis, and Aeromonas hydrophila corresponding to heat treatment at 62°C were 7.1, 0.34, 0.024, and 0.0096 min, and those corresponding to manosonication treatment (40°C, 200 kPa, 117 μm) were 4.0, 1.5, 0.86, and 0.90 min, respectively. The manosonication decimal reduction times of the four species investigated decreased sixfold when the amplitude was increased from 62 to 150 μm and fivefold when the relative pressure was raised from 0 to 400 kPa. In L. monocytogenes, S. enteritidis, and A. hydrophila, the lethal effect of manothermosonication was the result of the addition of the lethal effects of heat and manosonication, whereas in S. faecium it was a synergistic effect.     

Non-additive benefit or cost? Disentangling the indirect effects that occur when plants bearing extrafloral nectaries and honeydew-producing insects share exotic ant mutualists

Background and Aims

In complex communities, organisms often form mutualisms with multiple different partners simultaneously. Non-additive effects may emerge among species linked by these positive interactions. Ants commonly participate in mutualisms with both honeydew-producing insects (HPI) and their extrafloral nectary (EFN)-bearing host plants. Consequently, HPI and EFN-bearing plants may experience non-additive benefits or costs when these groups co-occur. The outcomes of these interactions are likely to be influenced by variation in preferences among ants for honeydew vs. nectar. In this study, a test was made for non-additive effects on HPI and EFN-bearing plants resulting from sharing exotic ant guards. Preferences of the dominant exotic ant species for nectar vs. honeydew resources were also examined.

Methods

Ant access, HPI and nectar availability were manipulated on the EFN-bearing shrub, Morinda citrifolia, and ant and HPI abundances, herbivory and plant growth were assessed. Ant-tending behaviours toward HPI across an experimental gradient of nectar availability were also tracked in order to investigate mechanisms underlying ant responses.

Key Results

The dominant ant species, Anoplolepis gracilipes, differed from less invasive ants in response to multiple mutualists, with reductions in plot-wide abundances when nectar was reduced, but no response to HPI reduction. Conversely, at sites where A. gracilipes was absent or rare, abundances of less invasive ants increased when nectar was reduced, but declined when HPI were reduced. Non-additive benefits were found at sites dominated by A. gracilipes, but only for M. citrifolia plants. Responses of HPI at these sites supported predictions of the non-additive cost model. Interestingly, the opposite non-additive patterns emerged at sites dominated by other ants.

Conclusions

It was demonstrated that strong non-additive benefits and costs can both occur when a plant and herbivore share mutualist partners. These findings suggest that broadening the community context of mutualism studies can reveal important non-additive effects and increase understanding of the dynamics of species interactions.     

SEF17 fimbriae are essential for the convoluted colonial morphology of Salmonella enteritidis

Salmonella enteritidis isolated from poultry infections generated a convoluted colonial morphology after 48 h growth on colonisation factor antigen (CFA) agar at 25°C. A mutant S. enteritidis defective for the elaboration of the SEF17 fimbrial antigen, in which the agf gene cluster was inactivated by insertion of an ampicillin resistance gene cassette, and other wild-type S. enteritidis transduced to this genotype failed to produce convoluted colonies. However, growth of SEF17⁻ mutants at 25°C on CFA agar supplemented with 0.001% Congo red resulted in partial recovery of the phenotype. Immunoelectron microscopy demonstrated that copious amounts of the SEF17 fimbrial antigen were present in the extracellular matrix of convoluted colonies of wild-type virulent S. enteritidis isolates. Bacteria were often hyperflagellated also. Immunoelectron microscopy of SEF17⁻ mutants grown on CFA agar+0.001% Congo red demonstrated the elaboration of an as yet undefined fimbrial structure. Isolates of S. enteritidis which were described previously as avirulent and sensitive to environmental stress failed to express SEF17 or produce convoluted colonies. These data indicate an essential role for SEF17, and possibly for another fimbria and flagella, in the generation of the convoluted colonial phenotype. The relationship between virulence and colonial phenotype is discussed.     

Fate of Staphylococci and Enteric Microorganisms Introduced into Slurry of Frozen Pot Pies

A slurry was prepared from six frozen pot pies diluted 1:5 with distilled water, two chicken, two turkey, and two beef pies of different brands. This slurry formed a reference sample and was placed in sterilized jars, frozen, and used as needed throughout the experiments. A second slurry was prepared in a similar manner from a frozen beef pot pie and a chicken pot pie, and was used as a control in only one experiment. The total count of microorganisms and the number of coliforms, Escherichia coli, salmonellae, and coagulase-positive staphylococci per gram were determined. Samples of slurry were inoculated in decimal dilutions with one or more of the following: Salmonella typhimurium, E. coli, and a strain of staphylococcus that causes food poisoning. The natural flora was found to exert an inhibitory effect upon the growth of the added microorganisms after incubation at 35 C for 18 hr. The inhibitory effect on growth was in part due to pH. The predominating organism isolated from the natural flora after incubation was a lactobacillus, which, when added in mixture with the test organisms in sterilized slurry, did not exert the profound inhibitory effect observed in the case of the natural flora. Some factors which may be concerned with the inhibition were investigated.     

Optimization of Sterilization of Salmonella enteritidis in Meat by Surfactin and Iturin Using a Response Surface Method

In this paper, the sterilization of surfactin and iturin to Salmonella enteritidis was observed, and the optimization of the inactivation of surfactin and iturin to S. enteritidis in meat by a response surface methodology was studied. Results showed that surfactin and iturin had high sterilization to S. enteritidis, whose minimal inhibitory concentration was 6.25 and 12.50 μM respectively. The optimization result indicated that S. enteritidis could be inactivated by five orders of magnitude when the temperature was 4.83°C, the action time was 17.20 h, and the concentration (surfactin/iturin molar ratio 1:2) was 0.69 MIC.     

Efficacies of Novel N-Halamine Disinfectants against Salmonella and Pseudomonas Species

Six novel N-halamine compounds of potential importance as disinfectants to the food-processing industry were tested against Salmonella enteritidis, Salmonella gallinarum, Salmonella typhimurium, and Pseudomonas fluorescens in aqueous solution. Inactivation times for 10⁶-fold reductions were determined as a function of water quality at pH 6.5 and 25°C. Phenol coefficients for the efficacies of the compounds against S. enteritidis have been reported also. When both stability and efficacy data are considered, as well as cost of production, two compounds, 1,3-dichloro-2,2,5,5-tetramethylimidazolidin-4-one and 1-chloro-2,2,5,5-tetramethylimidazolidin-4-one, offer the greatest potential as biocides for the food-processing industry.     

Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10⁵ CFU g⁻¹ was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

Experimental Salmonellosis

Mice were infected with smooth or rough strains of Salmonella enteritidis and Salmonella typhimurium and viable bacterial cells found in the liver of the inoculated animals were enumerated by plating homogenates of tissues on nutrient agar plates containing 0.35 M sucrose. Some rough strains of these Salmonella were recovered in the bacteria seen on these plates and appeared able to form colonies only on the sucrose-containing medium but not on an identical medium without added sucrose. This population did not appear in the liver of animals until at least 24 hr after infection. The number of bacteria capable of forming colonies only on the hypertonic medium was found to vary with the time after infection and the strain of bacteria used for infection. From the results of morphological examination of cells of the colonies developing on the hypertonic plates, these bacterial forms were thought to result from unstable L forms in the infected tissues. Possible processes of the formation of these L forms in vivo and their significance in induction of anti-infectious immunity are discussed.     

Growth, Survival and Characterization of cspA in Salmonella enteritidis Following Cold Shock

Salmonella enteritidis is a major foodborne microbial pathogen that can grow and survive at low temperatures for a considerable period of time. Increased survival was evidenced from a frozen S. enteritidis culture when treated at 10°C prior to freezing. Western blot analysis with Escherichia coli CspA antibody and analysis of radiolabeled proteins from S. enteritidis cultures after cold shock at 10°C and 5°C showed increased expression of a 7.4-kDa major cold shock protein, CS7.4, similar in size to that reported for E. coli. Cloning followed by nucleotide sequence analysis of the cspA gene from S. enteritidis showed a 100% nucleotide sequence identity in the promoter elements (−35 and −10) and the amino acid sequence encoded by the open reading frame (ORF) with the E. coli cspA gene. However, the differences in the nucleotide sequences between E. coli and S. enteritidis cspA genes in the putative repressor protein binding domain, the fragment 7, and in various segments throughout the upstream 0.642-kbp DNA may contribute to the expression of CS7.4 at less stringent temperatures in S. enteritidis. As in E. coli, the actual role of CS7.4 in protecting S. enteritidis from the damaging effects of cold or freezing temperatures is not yet understood. Received: 14 March 1997 / Accepted: 10 July 1997     

A dual mechanism involved in membrane and nucleic acid disruption of AvBD103b,a new avian defensin from the king penguin,against Salmonella enteritidis CVCC3377

The food-borne bacterial gastrointestinal infection is a serious public health threat. Defensins are evolutionarily conserved innate immune components with broad-spectrum antibacterial activity that do not easily induce resistance. AvBD103b, an avian defensin with potent activity against Salmonella enteritidis, was isolated from the stomach contents of the king penguin (Aptenodytes patagonicus). To elucidate further the antibacterial mechanism of AvBD103b, its effect on the S. enteritidis CVCC3377 cell membrane and intracellular DNA was researched. The cell surface hydrophobicity and a N-phenyl-1-naphthylamine uptake assay demonstrated that AvBD103b treatment increased the cell surface hydrophobicity and outer membrane permeability. Atomic absorption spectrometry, ultraviolet spectrophotometry, flow cytometry, and transmission electron microscopy (TEM) indicated that AvBD103b treatment can lead to the release of the cellular contents and cell death through damage of the membrane. DNA gel retardation and circular dichroism analysis demonstrated that AvBD103b interacted with DNA and intercalated into the DNA base pairs. A cell cycle assay demonstrated that AvBD103b affected cellular functions, such as DNA synthesis. Our results confirmed that AvBD103b exerts its antibacterial activity by damaging the cell membrane and interfering with intracellular DNA, ultimately causing cell death, and suggested that AvBD103b may be a promising candidate as an alternative to antibiotics against S. enteritidis.     

Soft cheese supplemented with black cumin oil: Impact on food borne pathogens and quality during storage

Black cumin seed oil (BCSO) was tested for its inhibitory effect against some pathogenic bacteria (Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Listeria monocytogenes Scott A and Salmonella enteritidis PT4) in Domiati cheese during cold storage. Physical, chemical and sensorial changes in cheese during storage were recorded. Pasteurized milk was inoculated before renneting with a mixed culture of bacteria at ca. 4 log CFU mL⁻¹. In vitro and in situ supplementation with BCSO showed antimicrobial impact on the growth of S. aureus, E. coli, L. monocytogenes and S. enteritidis inoculated into media and cheese samples. Supplementing of cheese with BCSO (0.1% or 0.2%, w/w) significantly reduced the counts of the inoculated pathogens by ca. 1.3 log and 1.5 log CFU g⁻¹ after 21 days of storage. In addition, BCSO controlled the development of titratable acidity, limited the changes in ripening indices, flavor components and kept considerable physicochemical and sensorial properties of cheese.     

The variant rpoS allele of S. enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::str^r null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::str^r. Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits.     

KH-type splicing regulatory protein is regulated by nuclear factor-κB signaling to mediate innate immunity in Caco-2 cells infected by <Emphasis Type="Italic">Salmonella enteritidis</Emphasis>

Salmonella enteritidis infection occurs in enterogenous diseases, such as gastroenteritis and parenteral focal infection, which often involve inflammation of intestinal epithelial cells. The nuclear factor kappa B (NF-κB) pathway participates in the innate immune response to many gram-negative pathogenic bacteria and initiates inflammation in epithelial cells. KH-type splicing regulatory protein (KSRP) is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for inflammatory response factors. However, it remains unclear whether KSRP is regulated by NF-κB signaling pathway in response to S. enteritidis infection and affects the development of inflammation. Accordingly, in this study, we investigated the role of KSRP in mediating the response to S. enteritidis in Caco-2 cells. The data revealed that S. enteritidis infection decreased KSRP expression, which was suppressed by blocking the NF-κB pathway. Additionally, S. enteritidis infection significantly increased the expression of inducible nitric oxide synthase and cyclooxygenase-2. Overexpression of KSRP reduced the expression levels of inflammatory factors in Caco-2 cells. KSRP was regulated by the NF-κB signaling pathway and participated in mediating the innate immune response to S. enteritidis infection in Caco-2 cells, and KSRP acted as a negative regulator of inflammatory gene expression.     

Preservation by Freeze-Drying and the Stability of Virulence of Salmonella typhimurium

Differences in the ability to withstand freeze-drying were demonstrated among strains of Salmonella typhimurium. On the average, the number of viable cells in freeze-dried cultures stored at 5 C for 12 to 18 months was approximately one half as large as that found 24 hr after freeze-drying. The viability in samples stored at higher temperatures declined rapidly and was correlated with the dryness of the sample. The virulence for mice of three strains of S. typhimurium did not change appreciably when samples were kept for 1 or 2 years as freeze-dried samples stored at 5 C, or as agar cultures stored at 5 C or at room temperature.