Salmonella Enteritidis bacteriophage candidates for phage therapy of poultry

Aims: Salmonella is a worldwide foodborne pathogen causing acute enteric infections in humans. In the recent years, the use of bacteriophages has been suggested as a possible tool to combat this zoonotic pathogen in poultry farms. This work aims to isolate and perform comparative studies of a group of phages active against a collection of specific Salmonella Enteritidis strains from Portugal and England. Also, suitable phage candidates for therapy of poultry will be selected. Methods and Results: The Salm. Enteritidis strains studied were shown to have a significantly high occurrence of defective (cryptic) prophages; however, no live phages were found in the strains. Bacteriophages isolated from different environments lysed all except one of the tested Salm. Enteritidis strains. The bacteriophages studied were divided into different groups according to their genetic homology, RFLP profiles and phenotypic features, and most of them showed no DNA homology with the bacterial hosts. The bacteriophage lytic efficacy proved to be highly dependent on the propagation host strain. Conclusions: Despite the evidences shown in this work that the Salm. Enteritidis strains used did not produce viable phages, we have confirmed that some phages, when grown on particular hosts, behaved as complexes of phages. This is most likely because of the presence of inactive phage‐related genomes (or their parts) in the bacterial strains which are capable of being reactivated or which can recombine with lytic phages. Furthermore, changes of the bacterial hosts used for maintenance of phages must be avoided as these can drastically modify the parameters of the phage preparations, including host range and lytic activity. Significance and Impact of the Study: This work shows that the optimal host and growth conditions must be carefully studied and selected for the production of each bacteriophage candidate for animal therapy.     

The effect of long‐term propionate adaptation on the stress resistance of Salmonella Enteritidis

Aims: Adaptive phenotypes of enteric bacterial pathogens in response to in vivo‐mimicking stress conditions are important because of their potentiality to enhance stress resistance and ameliorate measures intended to control transmission and infectivity. Salmonella enterica serotype Enteritidis (S. Enteritidis) encounters a variety of such environments throughout its infection cycle, including high concentrations of the short‐chain fatty acid, Propionate (PA), during food processing and within the gut of infected hosts. With this study, we aimed to elucidate the significance of PA adaptation on stress resistance in S. Enteritidis. Methods and Results: We have shown (utilizing in vitro stress assays) that S. Enteritidis grown to stationary phase in the presence of PA has a dramatically enhanced resistance to commonly encountered in vivo stressors, including extreme acidity and oxidative/nitrosative stresses when compared to unadapted salmonellae. However, competitive infection between PA adapted and unadapted cells within a murine model showed that adapted cells were at a distinct disadvantage in vivo, resulting in decreased caecal colonization in infected mice. Conclusions: Our results suggest that, while long‐term PA adaptation induces strong resistance to specific stresses in vitro, it also reduces the overall infectivity of the adapted cells by inhibiting the organism’s colonization ability. Significance and Impact of the Study: In S. Enteritidis, PA adaptation is strongly associated with the induction of stress‐resistant phenotypes in vitro. Enhanced stress resistance ex vivo may increase environmental persistence and/or the overall transmissibility of this pathogen, while overall the virulence of the pathogen is likely to be hindered as a result of the decreased colonization ability of PA‐adapted S. Enteritidis.     

Characterization of lysine decarboxylase-negative strains of Salmonella enterica serovar Enteritidis disseminated in Japan

Salmonella enterica serovar Enteritidis is one of the leading causes of food-borne diseases in Japan. Typically, Salmonella spp. test positive for lysine-decarboxylase. However, the number of isolates of serovar Enteritidis without lysine-decarboxylase activity increased in Japan in 2003. Among 109 strains from distinct outbreaks, 10 lacked lysine-decarboxylase activity. Nine of the ten lysine-decarboxylase-negative strains showed quite similar pulsed-field gel electrophoresis profiles. Their lysine-decarboxylase phenotype was recovered by introduction of the cadBA locus from an lysine-decarboxylase-positive strain. Although the cad loci of the lysine-decarboxylase-negative strains seemed to be intact without any insertion sequences, cadC, a positive regulator of cadBA, had a single-base deletion at the same position, the 973rd base (cytosine), in all the nine lysine-decarboxylase-negative strains, whereas the wild-type cadC gene has a 1542 bp coding region (514 amino acids). This deletion was expected to produce a truncated (338 amino acids) form of CadC due to a frameshift. Because CadC senses environmental cues such as external pH and lysine through its putative C-terminal periplasmic domain, it is likely that the truncated CadC is not sensitive enough to external signaling to activate the cadBA operon, resulting in loss of the lysine-decarboxylase activity. Our results suggest that dissemination of these genetically closely related strains of serovar Enteritidis accounts for the unusual increase in the isolation of lysine-decarboxylase-negative strains.     

Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia

AIMS: The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. METHODS AND RESULTS: Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI-pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3.7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. CONCLUSIONS: Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area.     

Reducing colonization of Salmonella Enteritidis in chicken by targeting outer membrane proteins

AIMS: To evaluate the ability of Salmonella enterica ser. Enteritidis outer membrane proteins (OMPs) of 75.6 and 82.3 kDa to inhibit or reduce in vivo colonization of S. Enteritidis on intestinal mucosa in chickens. METHODS AND RESULTS: Nine-week-old specific-pathogen-free chickens were subcutaneously immunized with 75.6 or 82.3 kDa protein, and challenged with a virulent strain of S. Enteritidis. Chickens were killed, and portions of small intestine and caecum were removed at necropsy. The population of S. Enteritidis attached to chicken intestinal mucosa was determined. The population of S. Enteritidis recovered from the small intestine and caecum of chickens immunized with 75.6 or 82.3 kDa protein was significantly (P < 0.05) lower than that recovered from the control birds. CONCLUSIONS: Salmonella Enteritidis OMPs 75.6 kDa and 82.3 kDa were effective in reducing colonization of S. Enteritidis on intestinal mucosa in chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella Enteritidis OMPs 75.6 or 82.3 kDa could be used as potential vaccines to reduce S. Enteritidis colonization in chickens.     

Effect of sub-lethal chemical disinfection on the biofilm forming ability,resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells

Abstract

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

Molecular fingerprinting evidence of the contribution of wildlife vectors in the maintenance of Salmonella Enteritidis infection in layer farms

AIMS: To provide molecular fingerprinting evidence of the contribution of wildlife vectors in the on-farm epidemiology of Salmonella Enteritidis infections. METHODS AND RESULTS: Salmonella Enteritidis strains were isolated from wildlife and from farm environment samples collected in 10 egg layer farms. Isolates were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis and PstI-SphI ribotyping. In all 10 farms we were able to identify the same S. Enteritidis clones in wildlife vectors and farm environment. On several occasions the same clones were found before and after cleansing and disinfecting the farm premises. Also in some instances the same clones were present in mice samples, egg contents and spent hens. CONCLUSIONS: Definitive molecular evidence for the involvement of several wildlife species (mice, rats, flies, litter beetles and foxes) in the maintenance of S. Enteritidis infection on farms has been presented. Failures in biosecurity seriously compromise the control of this pathogen on laying farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper reports on the use of molecular tools for the study of the epidemiology of S. Enteritidis. It gives useful information to be considered in control programmes for this organism on poultry farms.     

Evaluation of steam pasteurization in controlling Salmonella serotype Enteritidis on raw almond surfaces

Aim: To investigate the efficacy of steam pasteurization for reducing Salmonella serotype Enteritidis on raw almond surfaces. Methods and Results: Nonpareil almonds were inoculated to 10^7–8 CFU g^?1 with a Salm. Enteritidis cocktail (Salm. Enteritidis 43353, ME‐13, ME‐14) or Salm. Enteritidis phage type 30, dried overnight and subjected to steam treatments through a pilot‐sized vertical pasteurization machine for 5, 15, 25, 35, 45, 55 and 65 s to investigate the effect of steam on a single layer of almond. Survival of Salm. Enteritidis was evaluated with tryptic soy agar and xylose lysine desoxycholate overlay for total and healthy cells, respectively. No significant differences (P > 0·05) in reduction were observed between the Salm. Enteritidis cocktail and Salm. Enteritidis PT 30 inoculum. Reduction of Salm. Enteritidis increased as a function of treatment time, with 25 s being sufficient to achieve a 5‐log reduction. Discolouration and visible formation of wrinkles were observed following steam pasteurization of more than 35 s. Conclusions: Steam pasteurization of 25 s is sufficient to achieve a 5‐log reduction of Salm. Enteritidis inoculated on raw almonds without visual quality degradation. Significance and Impact of the Study: Steam pasteurization is an effective alternative to reduce or prevent Salm. Enteritidis contamination on raw almonds.     

食源性沙门氏菌耐药性检测及相关质粒

[目的]测定390株沙门氏菌的抗生素药敏性,研究部分多重耐药株中质粒与其宿主耐药表型之间的关系及其在接合过程中对耐药性水平转移的影响.[方法]使用选择性培养基分离到沙门氏菌并通过PCR确认后,按照琼脂稀释法测定分离株对供试抗生素的药敏性,试剂盒提取代表性多重耐药株中的质粒,HindⅢ酶切,DPS软件分析电泳后质粒图谱.通过接合试验研究质粒在抗生素抗性水平转移中的作用.[结果]沙门氏菌分离株对四环素耐药最为普遍(58.2%),其次为链霉素(42.8%)、卡那霉素(39%)和氨苄青霉素(38.2%),对头孢西丁、氯霉素、庆大霉素、头孢曲松、阿莫西林甲氧苄啶、头孢替呋钠和萘啶酮酸的耐药率分别为27.2%、26.9%、21%、19%、18.2%、17.9%、14.6%和12.3%.抗性质粒编码的相同或相似沙门氏菌耐药表型与其中所含的耐药质粒并不呈现出严格的对应关系.质粒携带的抗性基因可通过接合作用转移,接合效率在2.4×10-4到5.6×10-1之间.[结论]食源性沙门氏菌对常用抗生素的多重耐药已经成为普遍现象,抗性质粒的同源性与其宿主耐药表型无直接相关性,其携带的耐药基因可通过接合作用在不同细菌种属之间高频传递.     

Mechanisms of egg contamination by Salmonella Enteritidis

Salmonella Enteritidis (SE) has been the major cause of the food-borne salmonellosis pandemic in humans over the last 20 years, during which contaminated hen's eggs were the most important vehicle of the infection. Eggs can be contaminated on the outer shell surface and internally. Internal contamination can be the result of penetration through the eggshell or by direct contamination of egg contents before oviposition, originating from infection of the reproductive organs. Once inside the egg, the bacteria need to cope with antimicrobial factors in the albumen and vitelline membrane before migration to the yolk can occur. It would seem that serotype Enteritidis has intrinsic characteristics that allow an epidemiological association with hen eggs that are still undefined. There are indications that SE survives the attacks with the help of antimicrobial molecules during the formation of the egg in the hen's oviduct and inside the egg. This appears to require a unique combination of genes encoding for improved cell wall protection and repairing cellular and molecular damage, among others.     

Precise excision and secondary transposition of TnphoA in non-motile mutants of a Salmonella enterica serovar Enteritidis clinical isolate

Mutagenesis with TnphoA has been widely used in many bacteria. Here, we report the excision and secondary transposition of this transposon in three non-motile (fliC, fliF and motB) mutants of Salmonella enterica serovar Enteritidis (S. Enteritidis). Isolation of motile revertants showed that they were kanamycin resistant and conserved a copy of TnphoA in their genome in an insertion site different from the initial one. They also expressed an intact flagella. Characterization of the motile revertant derived from the fliC mutant showed that TnphoA excised precisely from the fliC gene, resulting in an equivalent amount of FliC secreted protein in the revertant compared to that of the wild-type strain. These results show that TnphoA mutants should be used with care and underline the value of using transposon derivatives lacking the transposase gene.     

The use of a bacteriophage cocktail as a biocontrol measure to reduce Salmonella enterica serovar Enteritidis contamination in ground meat and goat cheese

We evaluated the effectiveness of phages on meats and goat cheese contaminated with Salmonella Enteritidis (SE). In meats, reductions of SE were observed during the whole experiment, while in goat cheese a reduction was only observed at day 3. We discuss the relevance of phages as a biocontrol in food.     

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.     

Molecular characterization of antibiotic resistance in clinical Salmonella typhi isolated in Ghana

Fifty-eight clinical Salmonella typhi strains isolated from patients suspected of suffering from typhoid fever were obtained at the Korle-Bu Teaching Hospital and the Noguchi Memorial Institute for Medical Research, both located in Ghana, Africa. Each isolate was examined for susceptibility to ampicillin, chloramphenicol, streptomycin, tetracycline, and trimethoprim/sulfamethoxazole by the disk diffusion assay. Five of the isolates were resistant to all five antibiotics while 10 isolates were resistant to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole, which are considered 'first line' antibiotics in the treatment of typhoid fever. Thirty-four isolates were resistant to at least one of the antibiotics tested and 62% of these isolates possessed conjugable plasmids belonging to incompatibility group IncHI. Ninety percent of the conjugable plasmids conferred a multiple drug-resistant phenotype on the strains harboring them. Additionally, 14 strains contained plasmids that were transformable and six of them encoded multiple drug resistance. Our findings indicate that multiple drug resistance to the 'first line' antibiotics in S. typhi may be more prevalent in Africa than previously thought.     

应用改良庆大霉素保护试验检测肠炎沙门氏菌侵袭力表型

肠炎血清型沙门氏菌(Salmonella enterica serovar Enteritidis,SE)是引起肠炎和全身感染重要的沙门氏菌血清型之一,了解沙门氏菌侵袭力表型对阐明其感染致病机制至关重要。传统庆大霉素保护试验(GPA)存在通量低、重复性差等缺点。文中利用96孔细胞培养和多孔道移液的高通量优势,结合菌落分区微量滴板计数法改良了传统GPA试验方案。应用改良的GPA方法检测了16株SE菌株对非吞噬细胞(HT-29)的入侵表型和43株SE菌株对吞噬细胞(RAW264.7)的胞内复制表型。通过比较分析SE强、弱菌株JL228和LN248对吞噬细胞(RAW264.7)的侵袭力表型发现,改良的GPA得出的数据组内和组间变异系数低、数据重复性强,胞内复制表型也与显微观察结果相符。通过实践应用发现,改良的GPA方法具有通量高、重复性强、结果可靠兼具省时、省力等优点,可作为沙门氏菌菌株侵袭力表型检测的更新方案,为进一步阐明其致病机制提供了更科学有效的方法。