Phage Type Research Articles

Bacteriophage Therapy for Critical and High-Priority Antibiotic-Resistant Bacteria and Phage Cocktail-Antibiotic Formulation Perspective.

Phage therapy is revolving to address the issues mainly dealing with antibiotic resistance in the pathogenic bacteria. Among the drug-resistant microbial populations, the bacterial species have been categorized as high-priority or critical-priority bacteria. This review summarizes the efficiency and development in phage therapy used against these drug-resistant bacteria in the past few years mainly belonging to the critical- and high-priority list. Phage therapy is more than just an alternative to antibiotics as it not only kills the target microbial population directly but also leads to the chemical and physical modifications in bacterial cell structures. These phage-mediated modifications in the bacterial cell may make them antibiotic sensitive. Application of phage therapy in antibiotic-resistant foodborne bacteria and possible modulation in gut microbes has also been explored. Further, the phage cocktail antibiotic formulation, containing more than one type of phage with antibiotics, has also been discussed.

On the potential of microscale electrokinetic cascade devices.

Phages used for phage therapy of multidrug resistant bacteria must be highly purified prior to use. There are limited purification approaches that are broadly applicable to many phage types. Electrokinetics has shown great potential to manipulate phages, but obstructions from the cell debris produced during phage propagation can severely diminish the capacity of an electrokinetic device to concentrate and purify phage samples. A multipart insulator-based electrokinetic device is proposed here to remove the larger, undesirable components of mixtures from phage preparations while transferring the freshly purified and concentrated sample to a second stage for downstream analysis. By combining the large debris prescreen and analysis stages in a streamlined system, this approach simultaneously reduces the impact of clogging and minimizes the sample loss observed during manual transferring of purified samples. Polystyrene particles were used to demonstrate a diminished sample loss of approximately one order of magnitude when using the cascade device as opposed to a manual transfer scheme. The purification and concentration of three different phage samples were demonstrated using the first stage of the cascade device as a prescreen. This design provides a simple method of purifying and concentrating valuable samples from a complex mixture that might impede separation capacity in a single channel.

Comparative assessment of the disinfection effectiveness of thymol and benzalkonium chloride against adapted and non-adapted to thymol biofilm cells of a Salmonella Typhimurium epidemic phage type DT193 strain

Nowadays there is a need to develop cost-effective, safe, and preferably eco-friendly methods to combat the pathogenic and other detrimental robust biofilms. Ιn this work, the disinfection actions of thymol (THY), a phytocompound exhibiting wide antimicrobial action, and benzalkonium chloride (BAC), a synthetic biocide commonly used as surface disinfectant in the food industry and elsewhere, were comparatively evaluated against adapted and non-adapted to THY biofilm cells of a Salmonella enterica ser. Typhimurium epidemic phage type DT193 strain. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each compound were determined against planktonic bacteria, together with their minimum biofilm inhibitory and eradication concentrations (MBICs, MBECs) against biofilms formed on polystyrene (PS). Bacteria were subsequently left to form biofilms on model stainless steel (SS) surfaces incubated in 1/10 diluted tryptone soy broth (dTSB) containing or not a sub-inhibitory THY concentration. Those sessile populations were finally submitted to disinfection (for 15 min) with THY or BAC and the viable biofilm bacteria were quantified by using in parallel agar plating and selective fluorescence staining. The results showed that when the terpenoid was applied at sub-MIC during biofilm formation, it was able to cause significant reductions of the final sessile populations on both surfaces (PS and SS). Disinfection results revealed the significant anti-biofilm action of THY on the non-adapted (control) biofilms (5 log reductions at MBC) and its superiority to that of BAC (<2 log reductions at 2 x MBC). However, the disinfection treatments applied on the THY-adapted biofilms were quite less efficient (achieving 2.6 and 1.3 log reductions, for THY and BAC, respectively). This demonstrates adaptation of Salmonella to THY, conferring co- and cross-disinfection resistance, as for many other antibacterial chemicals. Overall, the results demonstrate that natural compounds extracted from plants may be promising agents in helping to combat biofilms, multicellular microbial structures well-known for their remarkable hardiness against many stresses and antimicrobials.

Genomic Characterization of Salmonella typhimurium DT104 Strains Associated with Cattle and Beef Products

Salmonella enterica subsp. enterica serovar Typhimurium DT104, a multidrug-resistant phage type, has emerged globally as a major cause of foodborne outbreaks particularly associated with contaminated beef products. In this study, we sequenced three S. Typhimurium DT104 strains associated with a 2009 outbreak caused by ground beef, including the outbreak source strain and two clinical strains. The goal of the study was to gain a stronger understanding of the genomics and genomic epidemiology of highly clonal S. typhimurium DT104 strains associated with bovine sources. Our study found no single nucleotide polymorphisms (SNPs) between the ground beef source strain and the clinical isolates from the 2009 outbreak. SNP analysis including twelve other S. typhimurium strains from bovine and clinical sources, including both DT104 and non-DT104, determined DT104 strains averaged 55.0 SNPs between strains compared to 474.5 SNPs among non-DT104 strains. Phylogenetic analysis separated the DT104 strains from the non-DT104 strains, but strains did not cluster together based on source of isolation even within the DT104 phage type. Pangenome analysis of the strains confirmed previous studies showing that DT104 strains are missing the genes for the allantoin utilization pathway, but this study confirmed that the genes were part of a deletion event and not substituted or disrupted by the insertion of another genomic element. Additionally, cgMLST analysis revealed that DT104 strains with cattle as the source of isolation were quite diverse as a group and did not cluster together, even among strains from the same country. Expansion of the analysis to 775 S. typhimurium ST19 strains associated with cattle from North America revealed diversity between strains, not limited to just among DT104 strains, which suggests that the cattle environment is favorable for a diverse group of S. typhimurium strains and not just DT104 strains.

Characterisation of Phage Susceptibility Variation in Salmonellaenterica Serovar Typhimurium DT104 and DT104b.

The surge in mortality and morbidity rates caused by multidrug-resistant (MDR) bacteria prompted a renewal of interest in bacteriophages (phages) as clinical therapeutics and natural biocontrol agents. Nevertheless, bacteria and phages are continually under the pressure of the evolutionary phage–host arms race for survival, which is mediated by co-evolving resistance mechanisms. In Anderson phage typing scheme of Salmonella Typhimurium, the epidemiologically related definitive phage types, DT104 and DT104b, display significantly different phage susceptibility profiles. This study aimed to characterise phage resistance mechanisms and genomic differences that may be responsible for the divergent phage reaction patterns in S. Typhimurium DT104 and DT104b using whole genome sequencing (WGS). The analysis of intact prophages, restriction–modification systems (RMS), plasmids and clustered regularly interspaced short palindromic repeats (CRISPRs), as well as CRISPR-associated proteins, revealed no unique genetic determinants that might explain the variation in phage susceptibility among the two phage types. Moreover, analysis of genes coding for potential phage receptors revealed no differences among DT104 and DT104b strains. However, the findings propose the need for experimental assessment of phage-specific receptors on the bacterial cell surface and analysis of bacterial transcriptome using RNA sequencing which will explain the differences in bacterial susceptibility to phages. Using Anderson phage typing scheme of Salmonella Typhimurium for the study of bacteria-phage interaction will help improving our understanding of host–phage interactions which will ultimately lead to the development of phage-based technologies, enabling effective infection control.

Inactivation of Different Salmonella enteriditis Phage Types and Safety and Efficacy of Inactivated Products in Chicken.

This study was conducted to inactivate Salmonella enteriditis phage types (SE pt) and to determine the safety and efficacy of inactivated SE pt in chickens. SE pt 1, 3A, 6A, 7, and 35 were inactivated and inoculated (0.20 mL) in 124 chickens divided into 6 groups (CV1, CV3A, CV6A, CV7, CV35, and CV0 as a control). Sampling was conducted on day 14 after inoculation (pi). Eight chickens from each group were separated on day 14 pi for oral challenge with 0.20 mL/chicken (1010 cfu/mL) SE pt 6A and designated CV1C, CV3AC, CV6AC, CV7C, CV35C, and CV0C as control chickens. On days 7 and 14 postchallenge (pc), 4 chickens from every group were sacrificed for sampling. There was no significant difference in the body weight between different groups. In challenged groups, there was no significant association between different tissues and isolation of Salmonella on days 7 and 14 pc. There was significance (p < 0.05) in isolation of Salmonella when CV0C group was compared with other challenged groups. Significance was not observed between different tissues with respect to induction of microscopic changes. Significance was not observed between day 7 pc and day 14 pc with respect to scoring of lesions induced. Clinical signs and gross lesions were also recorded. ELISA was applied. Only in CV3AC group, the mean antibody titer was 1359 on day 14 pc. The conclusion was that inactivated SE pt 3A and 6A were safe and efficacious for protection against Salmonella enteriditis infection in chickens.

Fate of Salmonella and Enterococcus faecium during Pilot-Scale Spray Drying of Soy Protein Isolate

Outbreaks and recalls associated with microbial contamination of powdered foods have raised concern for the safety of the spray-drying process and its products. However, little research on the fate of bacteria during the spray-drying process has been done, leaving much unknown about the risks of contamination in spray dryers. Therefore, quantifying the contamination levels of Salmonella and Enterococcus faecium (as a surrogate) in various locations within a pilot-scale spray dryer can help illustrate the distribution of bacterial contamination, including in the final product. A 10% (w/w) dispersion of water and soy protein isolate was mixed with tryptic soy broth containing yeast extract inoculated with Salmonella enterica serovar Enteritidis phage type 30 (PT30) or E. faecium strain NRRL B-2354. This dispersion was spray dried using a pilot-scale tall-form cocurrent spray dryer at an inlet air temperature of 180, 200, or 220°C. After drying, samples of powder from eight locations within the system were collected or surface swabbed, plated, and enumerated. Spray drying achieved 2.40 to 4.15 and 2.33 to 2.83 log reductions in the concentrations of Salmonella and E. faecium, respectively, in the final powder product accumulated in the dryer's collectors. Salmonella and E. faecium were found in various concentrations in all locations within the spray dryer after a complete drying cycle. Differences in inlet air temperature between 180 and 220°C had no significant effect on the inactivation levels. As a surrogate, E. faecium was more resistant to spray drying than Salmonella. Overall, spray drying is capable of significant bacterial reduction in the final powder product, which can be combined with other hurdle technologies. However, adequate cleaning and sanitization procedures must be taken into consideration to prevent cross-contamination.

Analyses of propagation processes of Staphylococcus aureus bacteriophages S13′ and S25-3 in two different taxonomies by definitive screening design

To introduce phage therapy against multidrug-resistant Staphylococcus aureus in Western medicine, the establishment of phage manufacturing, particularly phage propagation, is indispensable. For the propagation of S. aureus phages, knowledge of the effects of phage types, process parameters, and analytical methodologies should be investigated. In this study, S. aureus phage propagations were studied in a flask with a new class of design of experiments, definitive screening design, using S. aureus phages S13′ and S25-3 in different taxonomies. Four process parameters, namely, multiplicity of infection, bacterial density at infection, time of harvest, and temperature, were evaluated with the regression models based on the phage concentration data measured using plaque assay and quantitative polymerase chain reaction. As a result, phage propagations measured using plaque assay and quantitative polymerase chain reaction were overall similar to each other in the case of phage S13′, while they differed in the case of phage S25-3. These results suggest that the propagation processes need to be developed according to phage type, and the choice of methodologies for phage concentration measurements should be carefully considered.

Characterisation of IncI1 plasmids associated with change of phage type in isolates of Salmonella enterica serovar Typhimurium

BackgroundAcquisition of IncI1 plasmids by members of the Enterobacteriaceae sometimes leads to transfer of antimicrobial resistance and colicinogeny as well as change of phage type in Salmonella Typhimurium. Isolates of S. Typhimurium from a 2015 outbreak of food poisoning were found to contain an IncI1 plasmid implicated in change of phage type from PT135a to U307 not previously reported. The origin of the changes of phage type associated with this IncI1 plasmid was investigated. In addition, a comparison of its gene composition with that of IncI1 plasmids found in local isolates of S. Typhimurium typed as U307 from other times was undertaken. This comparison was extended to IncI1 plasmids in isolates of phage types PT6 and PT6 var. 1 which are thought to be associated with acquisition of IncI1 plasmids.ResultsAnalysis of IncI1 plasmids from whole genome sequencing of isolates implicated a gene coding for a 1273 amino acid protein present only in U307 isolates as the likely source of change of phage type. The IncI1 plasmids from PT6 and PT6 var. 1 isolates all had the ibfA gene present in IncI1 plasmid R64. This gene inhibits growth of bacteriophage BF23 and was therefore the possible source of change of phage type. A fuller comparison of the genetic composition of IncI1 plasmids from U307 isolates and PT6 and PT6 var. 1 isolates along with two IncI1 plasmids from S. Typhimurium isolates not showing change of phage type was undertaken. Plasmids were classified as either ‘Delta’ or ‘Col’ IncI1 plasmids according to whether genes between repZ and the rfsF site showed high identity to genes in the same location in R64 or ColIb-P9 plasmids respectively. Comparison of the tra gene sets and the pil gene sets across the range of sequenced plasmids identified Delta and Col plasmids with almost identical sequences for both sets of genes. This indicated a genetic recombination event leading to a switch between Delta and Col gene sets at the rfsF site. Comparisons of other gene sets showing significant variation among the sequenced plasmids are reported. Searches of the NCBI GenBank database using DNA and protein sequences of interest from the sequenced plasmids identified global IncI1 plasmids with extensive regions showing 99 to 100% identity to some of the plasmids sequenced in this study indicating evidence for widespread distribution of these plasmids.ConclusionTwo genes possibly associated with change of phage type were identified in IncI1 plasmids. IncI1 plasmids were classified as either ‘Delta’ or ‘Col’ plasmids and other sequences of significant variation among these plasmids were identified. This study offers a new perspective on the understanding of the gene composition of IncI1 plasmids. The sequences of newly sequenced IncI1 plasmids could be compared against the regions of significant sequence variation identified in this study to understand better their overall gene composition and relatedness to other IncI1 plasmids in the databases.

Efficacy of Individual Bacteriophages Does Not Predict Efficacy of Bacteriophage Cocktails for Control of Escherichia coli O157.

Effectiveness of bacteriophages AKFV33 (Tequintavirus, T5) and AHP24 (Rogunavirus, T1), wV7 (Tequatrovirus, T4), and AHP24S (Vequintavirus, rV5), as well as 11 cocktails of combinations of the four phages, were evaluated in vitro for biocontrol of six common phage types of Escherichia coli O157 (human and bovine origins) at different multiplicities of infection (MOIs; 0.01–1,000), temperatures (37 or 22°C), and exposure times (10–22 h). Phage efficacy against O157 was highest at MOI 1,000 (P < 0.001) and after 14-18 h of exposure at 22°C (P < 0.001). The activity of individual phages against O157 did not predict the activity of a cocktail of these phages even at the same temperature and MOI. Combinations of phages were neutral (no better or worse than the most effective constituent phages acting alone), displayed facilitation (greater efficacy than the most effective constituent phages acting alone), or antagonistic (lower efficacy than the most effective constituent phages acting alone). Across MOIs, temperatures, exposure time, and O157 strains, a cocktail of T1, T4, and rV5 was most effective (P < 0.05) against O157, although T1 and rV5 were less effective (P < 0.001) than other individual phages. T5 was the most effective individual phages (P < 0.05), but was antagonistic to other phages, particularly rV5 and T4 + rV5. Interactions among phages were influenced by phage genera and phage combination, O157 strains, MOIs, incubation temperatures, and times. Based on this study, future development of phage cocktails should, as a minimum, include confirmation of a lack of antagonism among constituent phages and preferably confirmation of facilitation or synergistic effects.

Probiotic Bacilli Inhibit Salmonella Biofilm Formation Without Killing Planktonic Cells.

Salmonellosis is a foodborne infection caused by Salmonella. Domestic poultry species are one of the main reservoirs of Salmonella, which causes the foodborne infection salmonellosis, and are responsible for many cases of animal-to-human transmission. Keeping backyard chickens is now a growing trend, increasing the frequency of direct contact with the flock and, by consequence, the incidence of Salmonella infections. Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 are probiotic bacilli that produce the bacteriocins subtilosin A and subtilin, respectively. The antimicrobial activity of the two strains was determined against the reference strain Micrococcus luteus ATCC 10420. The cell-free supernatant of B. subtilis KATMIRA1933 inhibited biofilm formation by Salmonella enterica subsp. enterica serovar Hadar, Salmonella enterica subsp. enterica serovar Enteritidis phage type 4, and Salmonella enterica subsp. enterica serovar Thompson by 51.1, 48.3, and 56.9%, respectively. The cell-free supernatant of B. amyloliquefaciens B-1895 inhibited the biofilm formation of these Salmonella strains by 30.4, 28.6, and 35.5%, respectively. These findings suggest that the bacillus strains may have the potential to be used as probiotics and antibiotic alternatives for the control of Salmonella in poultry. The number of planktonic cells was unaffected by treatment with the cell-free supernatant. A co-culture of the Salmonella strains with either bacilli showed no signs of growth inhibition, suggesting that it might have been quorum sensing that is affected by the two Bacillus strains.

Aztreonam Lysine Increases the Activity of Phages E79 and phiKZ against Pseudomonas aeruginosa PA01.

Pseudomonas aeruginosa is a pernicious bacterial pathogen that is difficult to treat because of high levels of antibiotic resistance. A promising alternative treatment option for such bacteria is the application of bacteriophages; the correct combination of phages plus antibiotics can produce synergistic inhibitory effects. In this study, we describe morphological changes induced by sub-MIC levels of the antibiotic aztreonam lysine (AzLys) on P. aeruginosa PA01, which may in part explain the observed phage–antibiotic synergy (PAS). One-step growth curves for phage E79 showed increased adsorption rates, decreased infection latency, accelerated time to lysis and a minor reduction in burst size. Phage E79 plus AzLys PAS was also able to significantly reduce P. aeruginosa biofilm growth over 3-fold as compared to phage treatment alone. Sub-inhibitory AzLys-induced filamentation of P. aeruginosa cells resulted in loss of twitching motility and a reduction in swimming motility, likely due to a reduction in the number of polar Type IV pili and flagella, respectively, on the filamented cell surfaces. Phage phiKZ, which uses Type IV pili as a receptor, did not exhibit increased activity with AzLys at lower sub-inhibitory levels, but still produced phage–antibiotic synergistic killing with sub-inhibitory AzLys. A one-step growth curve indicates that phiKZ in the presence of AzLys also exhibits a decreased infection latency and moderately undergoes accelerated time to lysis. In contrast to prior PAS studies demonstrating that phages undergo delayed time to lysis with cell filamentation, these PAS results show that phages undergo accelerated time to lysis, which therefore suggests that PAS is dependent upon multiple factors, including the type of phages and antibiotics used, and the bacterial host being tested.

Synthesis of carbon dots with antiphage activity using caffeic acid.

Recent studies on preservation properties in the field of materials science suggest that a newly synthesized material can retain the biological properties of the raw material. Still, further study is necessary since these features critically influence research at the intersection of materials science and biology, and could provide a unique research direction for the synthesis of new materials. Thus, caffeic acid (CA) is used as a raw material to synthesize novel carbon quantum dots (CA-CDs) using a simple and rapid hydrothermal method. CA-CDs exhibit antiphage and antibacterial activities. Using three types of phages as models, it is confirmed that CA-CDs inhibit phage proliferation. The viral titres decline by 4, 5, or 6 orders of magnitude, and CA-CDs display potential universal antiphage ability; the mechanism suggests that the CA-CDs could change the protein structure of these phages and impact the phage adsorption stage leading to loss of infectivity. Meanwhile, the antibacterial activity is retained in CA-CDs, significantly inhibiting the growth of Gram-positive bacteria; this also reveals the preservation properties of CA-CDs. CA-CDs synthesized from CA, does not only expand the range of antiviral activity but also increases its many unique optical properties as a carbon dot material. This finding is meaningful in order to promote the development of new CDs with remarkable biological activity and acts as a basis for future scientific research on novel and alternative antiviral treatments.

A brief overview of emergencies and dissemination of Shiga-toxin-producing E. coli (STEC) and Salmonella enterica serovar Typhimurium DT104 in humans and food producing animals

Shiga-toxin-producing Escherichia coli (STEC) and Salmonella enterica serovar Typhimurium (STDT104) are foodborne pathogens of public health significance. It is less known that Shiga-toxin-producing Escherichia coli (with cattle being the most probable natural reservoir) can be isolated from pigs, sheep and wildlife as well. The basic information about detection of Shiga-toxin-producing genes in STEC as well as the origin of Salmonella Typhimurium definite phage type 4 (STDT4) the virulence and resistance mechanisms including their distribution in the world is presented. Due to the foodborne transmission mechanisms we emphasize the role of veterinary scientist in Serbia in implementing good management practice on animal farms and in strengthening laboratory diagnostic capacities.

It is unclear how important CRISPR-Cas systems are for protecting natural populations of bacteria against infections by mobile genetic elements

Articles on CRISPR commonly open with some variant of the phrase “these short palindromic repeats and their associated endonucleases (Cas) are an adaptive immune system that exists to protect bacteria and archaea from viruses and infections with other mobile genetic elements.” There is an abundance of genomic data consistent with the hypothesis that CRISPR plays this role in natural populations of bacteria and archaea, and experimental demonstrations with a few species of bacteria and their phage and plasmids show that CRISPR-Cas systems can play this role in vitro. Not at all clear are the ubiquity, magnitude, and nature of the contribution of CRISPR-Cas systems to the ecology and evolution of natural populations of microbes and the strength of selection mediated by different types of phage and plasmids to the evolution and maintenance of CRISPR-Cas systems. In this perspective, with the aid of heuristic mathematical–computer simulation models, we explore the a priori conditions under which exposure to lytic and temperate phage and conjugative plasmids will select for and maintain CRISPR-Cas systems in populations of bacteria and archaea. We review the existing literature addressing these ecological and evolutionary questions and highlight the experimental and other evidence needed to fully understand the conditions responsible for the evolution and maintenance of CRISPR-Cas systems and the contribution of these systems to the ecology and evolution of bacteria, archaea, and the mobile genetic elements that infect them.

Analysis Shiga Toxin-Encoding Bacteriophage in a Rare Strain of Shiga Toxin-Producing Escherichia coli O157:H7 stx2a/stx2c.

In December 2015, six cases of Shiga toxin (Stx)-producing Escherichia coli (STEC) O157:H7 stx2a/stx2c phage type (PT) 24 were identified by the national gastrointestinal disease surveillance system at Public Health England (PHE). Frozen grated coconut imported from India was implicated as the vehicle of infection. Short and long read sequencing data were interrogated for genomic markers to provide evidence that the outbreak strain was from an imported source. The outbreak strain belonged to a sub-lineage (IIa) rare in domestically acquired infection in the United Kingdom, and indicative of an imported strain. Phylogenetic analysis identified the most closely related isolates to the outbreak strain were from cases reporting recent travel not to India, but to Uganda. Phylo-geographical signals based on travel data may be confounded by the failure of local and/or global monitoring systems to capture the full diversity of strains in a given country. This may be due to low prevalence strains circulating in-country under the surveillance radar, or a recent importation event involving the migration of animals and/or people. Comparison of stx2a-encoding prophage harbored by the outbreak strain with publicly available stx2a-encoding prophage sequences revealed that it was most closely related to stx2a-encoding prophage acquired by STEC O157:H7 that caused the first outbreak of STEC-hemolytic uremic syndrome (HUS) in England in 1982–83. Animal and people migration events may facilitate the transfer of stx2a-encoding prophage from indigenous STEC O157:H7 to recently imported strains, or vice versa. Monitoring the global transmission of STEC O157:H7 and tracking the exchange of stx2a-encoding phage between imported and indigenous strains may provide an early warning of emerging threats to public health.

Highly clonal relationship among Salmonella Enteritidis isolates in a commercial chicken production chain, Brazil.

In this study, we described the comparison among pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), ribotyping, and PCR-ribotyping methods for subtyping Salmonella Enteritidis isolated from an industrial chicken production chain. One hundred and eight S. Enteritidis were isolated at all stages of poultry meat processing plant. These isolates were pheno- and genotypically characterized by using antimicrobial susceptibility test, phage typing, RAPD, PFGE, ribotyping, and PCR-ribotyping. The highest antibiotic resistance rates were observed for enrofloxacin (18.5%) followed by furazolidone (15.7%), cefoxitin (1.8%), ciprofloxacin, and ampicillin with 0.9% each one, while seven isolates (6.4%) were pan-susceptible. Most strains belonged to the globally disseminated phage type PT4 (n = 74; 69.2%). Additionally, we identified strains belonging to phage types PT1 (n = 19; 17.8%) and PT7a (n = 14; 13.1%). Moreover, our results showed that these four molecular methods indicate similar results showing high similarity (≥ 90%) among S. Enteritidis strains, suggesting that these isolates appear to be from a common ancestor being spread at all stages of the poultry production chain. In summary, the combined molecular approaches of these methods remain a suitable alternative to efficiently subtyping S. Enteritidis in the absence of high-resolution genotyping methods and these results may serve as a baseline study for development of mitigation strategies.

History of Early Bacteriophage Research and Emergence of Key Concepts in Virology.

The viruses of bacteria - bacteriophages - were discovered 20 years after the discovery of viruses. However, this was mainly the bacteriophage research that, after the first 40 years, yielded the modern concept of the virus and to large extent formed the grounds of the emerging molecular genetics and molecular biology. Many specific aspects of the bacteriophage research history have been addressed in the existing publications. The integral outline of the events that led to the formation of the key concepts of modern virology is presented in this review. This includes the opposition of F. d'Herelle and J. Bordet viewpoints over the nature of the bacteriophage, the history of lysogeny discovery and of determination of the mechanisms of underlying this phenomenon, the work of the Phage group led by M. Delbruck in USA, the development of the genetic analysis of bacteriophages and other research that eventually led to emergence of the concept of the virus (bacteriophage) as a transmissive genetic program. The review also covers a brief history of early applications of the bacteriophages such as phage therapy and phage typing.

Sublethal concentrations of undissociated acetic acid may not always stimulate acid resistance in Salmonella enterica sub. entericaserovar Enteritidis Phage Type 4: Implications of challenge substrate associated factors.

Acid adaptation enhances survival of foodborne pathogens under lethal acid conditions that prevail in several food-related ecosystems. In the present study, the role of undissociated acetic acid in inducing acid resistance of Salmonella Enteritidis Phage Type 4 both in laboratory media and in an acid food matrix was investigated. Several combinations of acetic acid (0, 15, 25, 35 and 45 mM) and pH values (4.0, 4.5, 5.0, 5.5, 6.0) were screened for their ability to activate acid resistance mechanisms of pathogen exposed to pH 2.5 (screening assay). Increased survival was observed when increasing undissociated acetic acid within a range of sublethal concentrations (1.9-5.4 mM), but only at pH 5.5 and 6.0. No effect was observed at lower pH values, regardless of the undissociated acetic acid levels. Three combinations (15mM/pH5.0, 35mM/pH5.5, 45mM/pH6.0) were selected and further used for adaptation prior to inoculation in commercial tarama (fish roe) salad, i.e., an acid spread (pH 4.35 ± 0.02), stored at 5°C. Surprisingly and contrary to the results of the screening assay, none of the acid adaptation treatments enhanced survival of Salmonella Enteritidis in the food matrix, as compared to non-adapted cells (control). Further examination of the food pH value, acidulant and storage (challenge) temperature on the responses of the pathogen adapted to 15mM/pH5.0, 35mM/pH5.5 and 45mM/pH6.0 was performed in culture media. Cells adapted to 35mM/pH5.5 were unable to induce acid resistance when exposed to pH 4.35 (tarama salad pH value) at 37°C and 5°C, whereas incubation under refrigeration (5°C) at pH 4.35 sensitized 45mM/pH6.0 adapted cells against the subsequent acid and cold stress. In conclusion, pre-exposure to undissociated acetic acid affected the adaptive responses of Salmonella Enteritidis Phage Type 4 in a concentration- and pH-dependent manner, with regard to conditions prevailing during acid challenge.

Thermal Resistance of Foodborne Pathogens and Enterococcus faecium NRRL B-2354 on Inoculated Pistachios

Process control validations require knowledge of the resistance of the pathogen(s) of concern to the target treatment and, in some cases, the relative resistance of surrogate organisms. Selected strains of Escherichia coli O157:H7 (five strains), Listeria monocytogenes (five strains), and Salmonella enterica (five strains) as well as Salmonella Enteritidis phage type (PT) 30 and nonpathogenic Enterococcus faecium NRRL B-2354 were inoculated separately (as individual strains) onto inshell pistachios. The thermal tolerance of each strain was compared via treatment of inoculated pistachios to hot oil (121°C) or hot water (80°C) for 1 min. Survivor curves in hot oil or hot water (0.5 to 6 min, n = 6 to 15) were determined for one or two of the most resistant strains of each pathogen, as well as E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30, and the Weibull model was fit to the data. A pilot-scale air-impingement oven was used to compare the thermal tolerance of E. faecium NRRL B-2354 and Salmonella Enteritidis PT 30 on pistachios with or without a brining pretreatment and at either dry (no steam) or 30% humidity (v/v) oven conditions. No significant difference in the time to a 4-log reduction in hot oil or hot water was predicted for any of the strains evaluated, on the basis of the 95% confidence interval. In the pilot-scale oven, E. faecium NRRL B-2354 was more thermally resistant than Salmonella in a broad set of differing treatments, treatment times, and temperatures. Salmonella is a suitable target pathogen of concern in pistachios for thermal processes because no other pathogen tested was more thermally resistant under the conditions evaluated. E. faecium NRRL B-2354 was at least as thermally resistant as Salmonella under all conditions evaluated, making it a good potential surrogate for Salmonella on pistachios.