Isolation of salmonellas by immunomagnetic separation.

Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10(7] were generally incubated with 10(4) Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.0 +/- 7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9 +/- 12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salm. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Isolation of salmonellas by immunomagnetic separation

A.E.M. VERMUNT, A.A.J.M. FRANKEN AND R.R. BEUMER. 1992. Magnetisable particles, coated with anti-salmonella serum, were used to isolate Salmonella livingstone from pure cultures, mixed cultures and food samples. Beads (10⁷) were generally incubated with 10⁴ Salm. livingstone cells/ml for 60 min at room temperature. The incubation and washing medium (0.01 mol/l phosphate-buffered saline; PBS) contained 0.1% bovine serum albumin (BSA) and 0.1% Tween 20, respectively. This method gave a recovery for Salm. livingstone of 51.9±7.8%. However, other micro-organisms such as Aeromonas hydrophila interfered with this test because of non-specific reactions (recovery 50.9±12.7%). These non-specific reactions could be decreased by using 4% skim milk instead of 0.1% BSA in the incubation medium. The ratio of the recovery of Salim. livingstone relative to the recovery of Aer. hydrophila changed from 0.9 when PBS with 0.1% BSA was used, to 13.4 when PBS with 4% skim milk was used. Immunomagnetic separation of Salmonella spp. from food samples offers good prospects for concentrating salmonella cells from heterogeneous bacterial suspensions, such as enrichment broths.     

Development and optimization of a novel immunomagnetic separation- bacteriophage assay for detection of Salmonella enterica serovar enteritidis in broth

Salmonella is the second-leading cause of food-borne illness in most developed countries, causing diarrhea, cramps, vomiting, and often fever. Many rapid methods are available for detection of Salmonella in foods, but these methods are often insensitive or expensive or require a high degree of technical ability to perform. In this paper we describe development and characterization of a novel assay that utilizes the normal infection cycle of bacteriophage SJ2 for detection of Salmonella enterica serovar Enteritidis in broth. The assay consists of four main stages: (i) capture and concentration of target cells by using immunomagnetic separation (IMS); (ii) infection of the target bacterium with phage; (iii) amplification and recovery of progeny phage; and (iv) assay of progeny phage on the basis of their effect on a healthy population of host cells (signal-amplifying cells). The end point of the assay can be determined by using either fluorescence or optical density measurements. The detection limit of the assay in broth is less than 10(4) CFU/ml, and the assay can be performed in 4 to 5 h. The results of this study demonstrate that the IMS-bacteriophage assay is a rapid, simple, and sensitive technique for detection of Salmonella serovar Enteritidis in broth cultures which can be applied to preenriched food samples.     

Rapid separation and concentration of food-borne pathogens in food samples prior to quantification by viable-cell counting and real-time PCR

Buoyant density gradient centrifugation has been used to separate bacteria from complex food matrices, as well as to remove compounds that inhibit rapid detection methods, such as PCR, and to prevent false-positive results due to DNA originating from dead cells. Applying a principle of buoyant density gradient centrifugation, we developed a method for rapid separation and concentration following filtration and low- and high-speed centrifugation, as well as flotation and sedimentation buoyant density centrifugation, for 12 food-borne pathogens (Salmonella enterica, Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Vibrio cholerae O139, Vibrio parahaemolyticus O3K6, Vibrio vulnificus, Providencia alcalifaciens, Aeromonas hydrophila, Bacillus cereus, Staphylococcus aureus, and Clostridium perfringens) in 13 different food homogenates. This method can be used prior to real-time quantitative PCR (RTi-qPCR) and viable-cell counting. Using this combined method, the target organisms in the food samples theoretically could be concentrated 250-fold and detected at cell concentrations as low as 10(1) to 10(3) CFU/g using the RTi-qPCR assay, and amounts as small as 10(0) to 10(1) CFU/g could be isolated using plate counting. The combined separation and concentration methods and RTi-qPCR confirmed within 3 h the presence of 10(1) to 10(2) CFU/g of Salmonella and C. jejuni directly in naturally contaminated chicken and the presence of S. aureus directly in remaining food items in a poisoning outbreak. These results illustrated the feasibility of using these assays for rapid inspection of bacterial food contamination during a real-world outbreak.     

Identification of a protein bound to the termini of bacteriophage PRD1 DNA.

Lipid-containing bacteriophage PRD1 has a double-stranded DNA genome of about 14,500 nucleotide base pairs. The phage can infect Escherichia coli and Salmonella typhimurium as well as other gram-negative bacteria harboring an appropriate plasmid. [35S]methionine label is incorporated into the DNA band early in infection. The label remains associated with DNA through phenol extraction and boiling with sodium dodecyl sulfate. Nuclease treatment of the genome released a protein which migrated as an early phage-specific protein (P8). This protein is also necessary for phage DNA replication. By restriction enzyme analysis it was shown that protein was associated with the terminal restriction fragments. Extracts of infected cells catalyzed the labeling of protein P8 with [alpha-32P]dGTP.     

Use of bioluminescent Salmonella for assessing the efficiency of constructed phage-based biosorbent

A bacteriophage-based biosorbent for Salmonella enteritidis was constructed, and bacterial bioluminescence was used for assessment of the efficiency of cell capture. A strain of S. enteritidis with bioluminescent phenotype was constructed by transformation with plasmid pT7 carrying the entire lux operon from Photorhabdus luminescens. The relation between relative light output (RLU) and colony-forming units (CFU/ml) of the bioluminescent strain was established. The bacteriophage specific to S. enteritidis was biotinylated, and the biotinylation procedure was optimized based on the maximum retention of phage infectivity. The biotinylated phages were then coated onto streptavidin-labeled magnetic beads, and were used to capture the bioluminescent S. enteritidis cells. Our preliminary results showed that the number of cells captured by constructed biosorbent was five times higher than that of the control, magnetic beads coated with nonbiotinylated phage, indicating the capture is specific. Journal of Industrial Microbiology & Biotechnology (2001) 27, 126–128. Received 06 June 2000/ Accepted in revised form 30 August 2000     

Use of bioluminescent Salmonella for assessing the efficiency of constructed phage-based biosorbent

A bacteriophage-based biosorbent for Salmonella enteritidis was constructed, and bacterial bioluminescence was used for assessment of the efficiency of cell capture. A strain of S. enteritidis with bioluminescent phenotype was constructed by transformation with plasmid pT7 carrying the entire lux operon from Photorhabdus luminescens. The relation between relative light output (RLU) and colony-forming units (CFU/ml) of the bioluminescent strain was established. The bacteriophage specific to S. enteritidis was biotinylated, and the biotinylation procedure was optimized based on the maximum retention of phage infectivity. The biotinylated phages were then coated onto streptavidin-labeled magnetic beads, and were used to capture the bioluminescent S. enteritidis cells. Our preliminary results showed that the number of cells captured by constructed biosorbent was five times higher than that of the control, magnetic beads coated with nonbiotinylated phage, indicating the capture is specific. Journal of Industrial Microbiology & Biotechnology (2000) 25, 273–275. Received 06 June 2000/ Accepted in revised form 30 August 2000     

Salmonella interactions with host cells: in vitro to in vivo

Salmonellosis (diseases caused by Salmonella species) have several clinical manifestations, ranging from gastroenteritis (food poisoning) to typhoid (enteric) fever and bacteraemia. Salmonella species (especially Salmonella typhimurium) also represent organisms that can be readily used to investigate the complex interplay that occurs between a pathogen and its host, both in vitro and in vivo. The ease with which S. typhimurium can be cultivated and genetically manipulated, in combination with the availability of tissue culture models and animal models, has made S. typhimurium a desirable organism for such studies. In this review, we focus on Salmonella interactions with its host cells, both in tissue culture (in vitro) and in relevant animal models (in vivo), and compare results obtained using these different models. The recent advent of sophisticated imaging and molecular genetic tools has facilitated studying the events that occur in disease, thereby confirming tissue culture results, yet identifying new questions that need to be addressed in relevant disease settings.     

Complete genome sequence of Salmonella enterica serovar Typhimurium bacteriophage SPN3UB

Salmonella is one of the major pathogenic bacteria that cause food poisoning. To elucidate the host infection mechanism of Salmonella enterica serovar Typhimurium-targeting phages, the bacteriophage SPN3UB was isolated from a chicken fecal sample. This phage belongs morphologically to the Siphoviridae family and infects the host via the O antigen of lipopolysaccharide (LPS). To further understand its infection mechanism, we completely sequenced and analyzed the genome. Here, we announce its complete genome sequence and report major findings from the genomic analysis results.     

Affinity-selected filamentous bacteriophage as a probe for acoustic wave biodetectors of Salmonella typhimurium

Proof-in-concept biosensors were prepared for the rapid detection of Salmonella typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers. Quantitative deposition studies indicated that approximately 3 x 10(10)phage particles/cm(2) could be irreversibly adsorbed for 1 h at room temperature to prepare working biosensors. The quality of phage deposition was monitored by fluorescent microscopy. Specific-bacterial binding resulted in resonance frequency changes of prepared sensors, which were evaluated using linear regression analysis. Sensors possessed a rapid response time of <180 s, had a low-detection limit of 10(2)cells/ml and were linear over a range of 10(1)-10(7)cells/ml with a sensitivity of 10.9 Hz per order of magnitude of S. typhimurium concentration. Viscosity effects due to increasing bacterial concentration and non-specific binding were not significant to the piezoelectric platform as confirmed by dose-response analysis. Phage-bacterial binding was confirmed by fluorescence and scanning electron microscopy. Overall, phage may constitute effective bioreceptors for use with analytical platforms for detecting and monitoring bacterial agents, including use in food products and possibly biological warfare applications.     

Characterization of the ELPhiS prophage from Salmonella enterica serovar Enteritidis strain LK5

Phages are a primary driving force behind the evolution of bacterial pathogens by transferring a variety of virulence genes into their hosts. Similar to other bacterial genomes, the Salmonella enterica serovar Enteritidis LK5 genome contains several regions that are homologous to phages. Although genomic analysis demonstrated the presence of prophages, it was unable to confirm which phage elements within the genome were viable. Genetic markers were used to tag one of the prophages in the genome to allow monitoring of phage induction. Commonly used laboratory strains of Salmonella were resistant to phage infection, and therefore a rapid screen was developed to identify susceptible hosts. This approach showed that a genetically tagged prophage, ELPhiS (Enteritidis lysogenic phage S), was capable of infecting Salmonella serovars that are diverse in host range and virulence and has the potential to laterally transfer genes between these serovars via lysogenic conversion. The rapid screen approach is adaptable to any system with a large collection of isolates and may be used to test the viability of prophages found by sequencing the genomes of various bacterial pathogens.     

Public health investigations of Salmonella Enteritidis in catering raw shell eggs, 2002-2004

AIMS: In response to a dramatic change in the epidemiology of Salmonella Enteritidis in England and Wales thought to be associated with raw shell eggs, the Health Protection Agency initiated public health investigations to establish the incidence of Salmonella contamination and origin of eggs used by catering premises implicated in outbreaks of Salm. Enteritidis. METHODS AND RESULTS: Between October 2002 and November 2004, 16 971 eggs were sampled and Salmonella were recovered from 3.4%. Salmonella was isolated from 5.5% and 6.3% of Spanish and eggs of unknown origin, respectively, used in catering premises linked to outbreaks, a level significantly higher than that (1.1%) found in nonLion Quality UK eggs sampled. The small sample of UK Lion Quality eggs tested (reflecting their lack of use in premises visited) did not contain Salmonella. Several phage types of Salm. Enteritidis other than phage type 4 (PT 4) were identified with nonUK eggs. CONCLUSIONS: Eggs from Spain were implicated as a major source of infection. Eggs were contaminated more frequently with Salmonella when shells were dirty and/or cracked, and stored at above 8 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Spanish eggs by the catering sector has been identified as a consistent significant factor in many of the outbreaks caused by Salm. Enteritidis nonPT4 in England and Wales during 2002-2004. Advice to caterers and hospitals that raw shell eggs should not be used in food that will either not be cooked or only lightly cooked should be reinforced.     

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoVivatrade mark optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.     

Bacteriocin nanoconjugates: boon to medical and food industry

Resistance to antibiotics is an ongoing problem in the biomedical industry. Developing active, alternative drug therapies would reduce our reliance on antibiotics that induce resistance in micro-organisms. To date, bacteriocins and antimicrobial peptides have shown a positive outcome as antibiotic substitutes and synergists apart from phage therapy, antibodies and probiotics. Bacteriocins are proteinaceous antimicrobial peptides synthesized by lactic acid bacteria extensively used as bio-preservatives and alternative to traditional antibiotics to overcome the problem of drug-resistant pathogens. Nonetheless, the use of bacteriocins has several limitations such as limited antimicrobial spectrum, requiring high dose, sensitivity to proteolytic enzymes, etc. Nanoparticles are one of the promising area of research explored to improve antimicrobial spectrum of bacteriocins. This review therefore highlights the recent developments and research pertaining to use of nanoparticles and bacteriocin conjugates to tackle the resistance crisis as well as its applications in food industry.     

Phage type 193 of Salmonella typhimurium contains different chromosomal genotypes and multiple IS 200 profiles

Abstract The common phage type 193 of Salmonella typhimurium was analyzed with respect to molecular markers of chromosomal genotype. Three profiles of the 16S rRNA genes and seven profiles of the DNA insertion element of IS2 00 were found among tne representative strains of DT193. The IS 200 profiles found within this single phage type were highly diverse, confirming that DT193 is a composite phage type containing several distinct clones and hybrid lines. IS2 00 profiling is thus appropriate both for primary strain discrimination, and for subdivision within certain phage types of S. typhimurium , such as DT193. This rapid molecular definition of clonality will be useful for the epidemiological investigation of food poisoning outbreaks.     

Biosorption of Food Green 3 by a novel green generation composite biosorbent from aqueous environment

A green type composite biosorbent composed of pine, oak, hornbeam, and fir sawdust biomasses modified with cetyltrimethylammonium bromide (CTAB) was first used for biosorption of an unsafe synthetic food dye, Food Green 3 from liquid medium in this study. Batch studies were carried by observing the effects of pH, dye concentration, biosorbent amount, and contact time. The equilibrium data were analyzed using Freundlich, Langmuir, and Dubinin–Radushkevich equations. Freundlich model gave a better conformity than other equations. The maximum dye removal potential of biosorbent was found to be 36.6 mg/g based on Langmuir isotherm. The pseudo-first-order, pseudo-second-order, Elovich, and intra-particle diffusion models were applied to clarify the process kinetics of biosorption. The mechanism studies suggested the biosorption process obeying Elovich kinetics and involving pore diffusion. The estimated values of biosorption free energy from Dubinin–Radushkevich isotherm (E value <8 kJ/mol) and thermodynamic studies (0 < ΔG° < ?20 kJ/mol) implied a spontaneous, feasible, and physical process. Hence, this investigation suggested that the CTAB modified mix sawdust biomass could be a promising biosorbent for biosorption of such problematic dyes from impacted media.     

Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay.

A combination of immunomagnetic separation and polymerase chain reaction (IMS-PCR) was used to detect Salmonella in food samples. Pre-enrichment of samples was combined with filtration through a membrane for the removal of food debris. The IMS-PCR assay combines selective extraction of bacteria by specific antibodies with primer specific PCR amplification that enables to detect Salmonella in non-fatty food samples in 24 h. In comparison with conventional cultural methods, the IMS-PCR is a rapid and specific method. Combined with filtration bags, it partially reduces the negative effects of the food matrix and allows the quick detection of Salmonella cells. The shortened protocols for Salmonella spp. detection described here can improve considerably current methodologies.     

Phage types, plasmid profiles and chromosomal restriction profiles of Salmonella enterica subsp. enterica ser. enteritidis (S. enteritidis) isolated in Poland in 1999-2000]

Salmonella Enteritidis strains are the most often isolated Salmonella serovars in Poland. In the present study, phage typing, plasmid profile analysis, and PFGE have been applied to characterize 140 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 140 S. Enteritidis strains coming from Poland. All 140 strains were typable and six different phage types were observed. A total of 125 (89%) of 140 isolates examined belonged to PT 4. The others PTs were represented by small amount of strains (PT1-2, PT6-6, PT7-1, PT8-4 and PT21-2 strains). Among all tested isolates six different plasmid profiles were observed. Of the 140 examined strains, 128 (91.4%) contained the 57 kb plasmid alone. After XbaI digestion four distinct pulse field chromosomal restriction profiles among studied S. Enteritidis were observed. XbaI and SpeI chromosomal restriction profiles of S. Enteritidis PT4 were identical with reference strain profiles. Our findings confirmed earlier suggestions that the increase of human salmonellosis cases in Poland was caused by S. Enteritidis PT4 and was due to consumption of contaminated food. This study confirmed the importance of using PFGE in combination with phage typing, plasmid typing and antibiotic resistance testing for studying the epidemiology of S. Enteritidis.     

Analysis of Salmonella enteritidis strains isolated from food-poisoning cases in Taiwan by pulsed field gel electrophoresis, plasmid profile and phage typing

AIMS: To establish the molecular typing data for Salmonella enteritidis due to its increasing role in Salmonella infections in Taiwan. METHODS AND RESULTS: Sixty-three Salm. enteritidis strains isolated from related and unrelated patients suffering from food-borne poisoning during 1991-97 were collected and subjected to pulsed field gel electrophoresis (PFGE), plasmid analysis and phage typing. For PFGE, XbaI, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 63 Salmonella strains, 10 PFGE pattern combinations were found. Of these, pattern X3 S3 N3 was the major subtype, since 46 strains isolated from different locations at different times during 1991-97 showed this PFGE pattern. Plasmid analysis showed only three plasmid profiles and phage typing showed that most of the Salmonella strains were of the phage type PT4. CONCLUSION: Most of the Salm. enteritidis strains circulating in Taiwan are of very similar genetic types or are highly related and that strains of PFGE pattern X3 S3 N3 are the prevalent and recirculating strains of Salm. enteritidis which caused food-poisoning cases in Taiwan in 1991-97. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that in Salmonella infection, certain subtypes of Salm. enteritidis should be scrutinized.