Phage engineering for development of diagnostic tools.

The bacteriophages rely on the host cell to provide energy and resources for their own replication. Antibody-based diagnostic tests rely on the antibody and the biomarker interactions. Since, most of these diagnostic tools employ the use of antibodies; hence, they require intensive storage protocols at cold conditions and incur high time and capital cost due to their production and purification process. Phage-based diagnostics can overcome this limitation. Bacteriophages, have been used as emerging tools for the detection of various pathogens. Rapid phage-mediated detection assays have become commercial diagnostic tools. Conventional method and new cloning approaches have been followed to specifically detect a disease- causing microbial strains. This review discusses use of Phage typing as diagnostic tools, phage-based detection methods, and their usage for signal amplification. Design rules for reporter phage engineering are also discussed followed by different engineering platforms for phage genome editing. We also discuss recent examples of how phage research is influencing the recent advances in the development of phage-based diagnostics for ultra-sensitive detection of various bio-species, outlining the advantages and limitations of detection technology of phage-based assays.

New Bacteriophages with Podoviridal Morphotypes Active against Yersinia pestis: Characterization and Application Potential.

Phages of highly pathogenic bacteria represent an area of growing interest for bacterial detection and identification and subspecies typing, as well as for phage therapy and environmental decontamination. Eight new phages-YpEc56, YpEc56D, YpEc57, YpEe58, YpEc1, YpEc2, YpEc11, and YpYeO9-expressing lytic activity towards Yersinia pestis revealed a virion morphology consistent with the Podoviridae morphotype. These phages lyse all 68 strains from 2 different sets of Y. pestis isolates, thus limiting their potential application for subtyping of Y. pestis strains but making them rather promising in terms of infection control. Two phages-YpYeO9 and YpEc11-were selected for detailed studies based on their source of isolation and lytic cross activity towards other Enterobacteriaceae. The full genome sequencing demonstrated the virulent nature of new phages. Phage YpYeO9 was identified as a member of the Teseptimavirus genus and YpEc11 was identified as a member of the Helsettvirus genus, thereby representing new species. A bacterial challenge assay in liquid microcosm with a YpYeO9/YpEc11 phage mixture showed elimination of Y. pestis EV76 during 4 h at a P/B ratio of 1000:1. These results, in combination with high lysis stability results of phages in liquid culture, the low frequency of formation of phage resistant mutants, and their viability under different physical-chemical factors indicate their potential for their practical use as an antibacterial mean.

Induction of Viable but Non-Culturable State in Clinically Relevant Staphylococci and Their Detection with Bacteriophage K.

Prosthetic joint infections are frequently associated with biofilm formation and the presence of viable but non-culturable (VBNC) bacteria. Conventional sample culturing remains the gold standard for microbiological diagnosis. However, VBNC bacteria lack the ability to grow on routine culture medium, leading to culture-negative results. Bacteriophages are viruses that specifically recognize and infect bacteria. In this study, we wanted to determine if bacteriophages could be used to detect VBNC bacteria. Four staphylococcal strains were cultured for biofilm formation and transferred to low-nutrient media with different gentamycin concentrations for VBNC state induction. VBNC bacteria were confirmed with the BacLightTM viability kit staining. Suspensions of live, dead, and VBNC bacteria were incubated with bacteriophage K and assessed in a qPCR for their detection. The VBNC state was successfully induced 8 to 19 days after incubation under stressful conditions. In total, 6.1 to 23.9% of bacteria were confirmed alive while not growing on conventional culturing media. During the qPCR assay, live bacterial suspensions showed a substantial increase in phage DNA. No detection was observed in dead bacteria or phage non-susceptible E. coli suspensions. However, a reduction in phage DNA in VBNC bacterial suspensions was observed, which confirmed the detection was successful based on the adsorption of phages.

Genetic engineering of bacteriophages: Key concepts, strategies, and applications.

Bacteriophages are the most abundant biological entity in the world and hold a tremendous amount of unexplored genetic information. Since their discovery, phages have drawn a great deal of attention from researchers despite their small size. The development of advanced strategies to modify their genomes and produce engineered phages with desired traits has opened new avenues for their applications. This review presents advanced strategies for developing engineered phages and their potential antibacterial applications in phage therapy, disruption of biofilm, delivery of antimicrobials, use of endolysin as an antibacterial agent, and altering the phage host range. Similarly, engineered phages find applications in eukaryotes as a shuttle for delivering genes and drugs to the targeted cells, and are used in the development of vaccines and facilitating tissue engineering. The use of phage display-based specific peptides for vaccine development, diagnostic tools, and targeted drug delivery is also discussed in this review. The engineered phage-mediated industrial food processing and biocontrol, advanced wastewater treatment, phage-based nano-medicines, and their use as a bio-recognition element for the detection of bacterial pathogens are also part of this review. The genetic engineering approaches hold great potential to accelerate translational phages and research. Overall, this review provides a deep understanding of the ingenious knowledge of phage engineering to move them beyond their innate ability for potential applications.

Characterization of Staphylococcus aureus isolates from pastry samples by rep-PCR and phage typing.

Background and Objectives: Staphylococcus aureus is one of the most common causes of food poisoning. This study aimed to identify S. aureus isolated from pastries, the virulence factors, antimicrobial resistance patterns, biofilm formation, and then classification based on SCCmec types, phage types, and also Rep types. Materials and Methods: In this study, 370 creamy and dried pastry samples have been randomly collected from different confectioneries in Hamadan city. The S. aureus isolates were identified by conventional microbiological methods and nuc gene amplification. The virulence factors and prophage genes were detected. After that, the biofilm production and antibiotic susceptibility assay of S. aureus isolates were examined. Finally, the isolates were classified by rep-PCR typing. Results: Among 370 samples, 97 creamy (34.64%) and 3 dried (3.33%) pastry samples were contaminated with S. aureus. Antibiotic sensitivity results showed the highest resistance to penicillin (90%) but none of them were MRSA. According to biofilm formation assay, 14 strains (45%) were strongly adhesive. The dominant phage among isolates was SGF, especially SGFa subgroup. About half of the isolates carried SCCmec Types I and III. Analysis of the genetic linkage between isolates by rep-PCR showed ≥80% genetic similarity and also different rep-types of S. aureus isolates. Conclusion: The presence of different prophage encoded virulence factors and antibiotic resistance enable S. aureus strains to produce a broad range of diseases. Thus, consumption of creamy pastries increases the risk of infection with S. aureus and it is a serious warning to the health system.

A PolyQ Membrane Protein of Vibrio cholerae Acts as the Receptor for Phage Infection.

Bacteriophage VP1 is a typing phage used for the phage subtyping of Vibrio cholerae O1 biotype El Tor, but the molecular mechanisms of its receptor recognition and the resistance of its host to infection are mostly unknown. In this study, we aimed to identify the host receptor and its role in resistance in natural VP1-resistant strains. Generating spontaneous resistance mutations and genome sequencing mutant strains found the polyQ protein VcpQ, which carries 46 glutamine residues in its Q-rich region, to be responsible for infection by VP1. VcpQ is a membrane protein and possibly forms homotrimers. VP1 adsorbed to V. cholerae through VcpQ. Sequence comparisons showed that 72% of natural VP1-resistant strains have fewer glutamines in the VcpQ Q-rich stretch than VP1-sensitive strains. This difference did not affect the membrane location and oligomer of VcpQ but abrogated VP1 adsorption. These mutant VcpQs did not recover VP1 infection sensitivity in a V. cholerae strain with vcpQ deleted. Our study revealed that the polyQ protein VcpQ is responsible for the binding of VP1 during its infection of V. cholerae and that glutamine residue reduction in VcpQ affects VP1 adsorption to likely be the main cause of VP1 resistance in natural resistant strains. The physiological functions of this polyQ protein in bacteria need further clarification; however, mutations in the polyQ stretch may endow V. cholerae with phage resistance and enhance survival against VP1 or related phages.IMPORTANCE Receptor recognition and binding by bacteriophage are the first step for its infection of bacterial cells. In this study, we found the Vibrio cholerae subtyping phage VP1 uses a polyQ protein named VcpQ (V. cholerae polyQ protein) as the receptor for VP1 infection. Our study reveals the receptor's recognition of phage VP1 during its adsorption and the VP1 resistance mechanism of the wild resistant V. cholerae strains bearing the mutagenesis in the receptor VcpQ. These mutations may confer the survival advantage on these resistant strains in the environment containing VP1 or its similar phages.

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.

Development and Application of a Prophage Integrase Typing Scheme for Group B Streptococcus.

Group B Streptococcus (GBS) is a gram-positive pathogen mainly affecting humans, cattle, and fishes. Mobile genetic elements play an important role in the evolution of GBS, its adaptation to host species and niches, and its pathogenicity. In particular, lysogenic prophages have been associated with a high virulence of certain strains and with their ability to cause invasive infections in humans. It is therefore important to be able to accurately detect and classify prophages in GBS genomes. Several bioinformatic tools for the identification of prophages in bacterial genomes are available on-line. However, genome searches for most of these programs are affected by the composition of their reference database. Lack of databases specific to GBS results in failure to recognize all prophages in the species. Additionally, performance of these programs is affected by genome fragmentation in the case of draft genomes, leading to underestimation of the number of phages. They also prove impractical when dealing with large genome datasets and they do not offer a quick way of classifying bacteriophages. We developed a GBS-specific method to screen genome assemblies for the presence of prophages and to classify them based on a reproducible typing scheme. This was achieved through an extensive search of a vast number of high-quality GBS sequences (n = 572) originating from different host species and countries in order to build a database of phage integrase types, on which the scheme is based. The proposed typing scheme comprises 12 integration sites and sixteen prophage integrase types, including multiple subtypes per integration site and integrase genes that were not site-specific. Two putative phage-inducible chromosomal islands (PICI) and their insertion sites were also identified during the course of these analyses. Phages were common and diverse in all major clonal complexes associated with human disease and detected in isolates from every animal species and continent included in the study. This database will facilitate further work on the prevalence and role of prophages in GBS evolution, and identifies the roles of PICIs in GBS and of prophage in hypervirulent ST283 as areas for further research.

Phage Types of Vibrio cholerae 01 Biotype ElTor Strains Isolated from India during 2012-2017.

BACKGROUND: Cholera is a primordial disease caused by Vibrio cholerae which existed from centuries in different parts of the world and still shows its periodic, endemic and epidemic presence. Thousands of cholera cases are reported from different parts of India and the disease remains endemic throughout the year. At present, we do not have enough knowledge about the phenotypic nature of the circulating V. cholerae strains in this part of the world. OBJECTIVES: This study was carried out over a period of 6 years with the aim defer with the changes in the prevalence and distribution of biotypes, serotypes and phage types of V. cholerae clinical isolates from various endemic regions of the country to determine phenotypic characteristics of the circulating strains and also to predict the attributes of cholera strains responsible for causing significant outbreaks in future. MATERIALS AND METHODS: A total of 1882 V.cholerae O1 isolates from different cholera endemic areas of India were included in this study. V.cholerae strains which were identified as O1 biotype ElTor further analyzed for serotype and phage types using the standard methodologies. Polyvalent O1 and monospecific Inaba and Ogawa antisera were used for serotyping. A panel of five phages of Basu and Mukherjee phage typing scheme and five phages from the new phage typing scheme were used for phage typing analysis following standard methodology. RESULTS: Maximum numbers of strains were isolated from cholera-endemic states like Gujarat and Maharashtra. All the isolates were confirmed as V. cholerae O1 biotype ElTor and majority of them were serotype Ogawa (93.2%). New phage typing scheme resulted in almost 100% typeable V. cholerae O1 strains included in this study and phage type 27 was the predominant type. Although 80% of the strains used in this study were sensitive to all the vibrio phages, S5 phage was found most efficient in lysing cholera strains indicating its broader host range. CONCLUSION: The current study identified phage type 27 as the most dominant type and serotype Ogawa was found continuous in circulation throughout the year which has caused recent cholera outbreaks in India during the past years. Phage sensitivity data propose an alternative cost-effective approach to prevent cholera outbreak by therapeutic uses of typing phages irrespective of origin or clonality of the strains.

Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentations.

Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.

Structures of O-specific polysaccharides of Pseudomonas psyhrotolerans BIM B-1158G.

Strain of Pseudomonas psychrotolerans was cultured on the nutrient agar and in a liquid nutrient broth. Bacterial cells were phage-typed with bacteriophages specific to Pseudomonas. O-antigen was isolated from cells using phenol-water method and mild acid degradation. The following structures of the polysaccharides extracted were established by sugar analysis and 1D, 2D NMR spectroscopy: PSI→3)-α-D-Manp-(1→2)-α-D-Manp-(1→; PSII→3)-α-D-Rhap-(1→2)-ß-D-Rhap-(1→3)-α-D-Rhap-(1→; α-D-Glcp-(1Ë©; 2.

Isolation and Characterization of Novel Broad Host Range Bacteriophages of Vibrio cholerae O1 from Bengal.

OBJECTIVES: We have isolated a total of five newer cholera phages which are novel broad host range to incorporate with the existing phage typing schemes for an extended typing scheme. MATERIALS AND METHODS: These newly isolated phages were well characterized including the electron micrograph. A total of 300 Vibrio cholerae strains were isolated from the different endemic region in India were included in phage typing study. RESULTS: These phages were found different from the existing phages. Electron microscopic results showed that the phages belonged to myophage and podophage group. Characterization of the phages based on pH, temperature, and organic solvent sensitivity showed differences among the phages used in this study. All the strains of Vibrio O1 were typeable (100%) with the five set of cholera phages. Of these, 40% strains were clustered under Type-1. CONCLUSION: The newer Vibrio phages are novel and broad host range and will be useful to incorporate with the existing phage typing system for more precisely discriminate the strains of Vibrio cholerae.

Recurrent outbreaks caused by the same Salmonella enterica serovar Infantis clone in a German rehabilitation oncology clinic from 2002 to 2009.

BACKGROUND: Repeated outbreaks of salmonellosis caused by Salmonella enterica serovar Infantis at a rehabilitation clinic in Germany were investigated microbiologically from August 2002 to August 2009. AIM: To identify the sources of transmission and characterize the S. enterica serovar Infantis isolates. METHODS: Associated with these outbreaks, isolates from 98 patients, two kitchen staff, five food samples, four swabs of kitchen facilities, three samples of chicken faeces and one sample of sewage water were evaluated by phage typing. All S. enterica serovar Infantis isolates investigated (N=113) were related to phage type (PT) 29. Additionally, 44 of the 113 isolates were selected at random for typing by XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE). FINDINGS: Typing of the 44 isolates showed that the recurrent infections were caused by the single clone PT 29/XB27+44 (42/44, 95.5%). The most likely route of transmission was only identified in the last outbreak in 2009 within the present study. It was found to be cross-contamination in the kitchen facilities (emanating from a contaminated wooden panel), in combination with carriers among the kitchen staff. CONCLUSIONS: This study demonstrated important details of hospital-specific epidemiological processes, and alludes to a long-term reservoir of an epidemic clone of S. enterica serovar Infantis either in a backyard flock of poultry or in an inanimate kitchen reservoir.

Phage typing or CRISPR typing for epidemiological surveillance of Salmonella Typhimurium?

OBJECTIVE: Salmonella Typhimurium is the most dominant Salmonella serovar around the world. It is associated with foodborne gastroenteritis outbreaks but has recently been associated with invasive illness and deaths. Characterization of S. Typhimurium is therefore very crucial for epidemiological surveillance. Phage typing has been used for decades for subtyping of S. Typhimurium to determine the epidemiological relation among isolates. Recent studies however have suggested that high throughput clustered regular interspaced short palindromic repeats (CRISPR) typing has the potential to replace phage typing. This study aimed to determine the efficacy of high-throughput CRISPR typing over conventional phage typing in epidemiological surveillance and outbreak investigation of S. Typhimurium. RESULTS: In silico analysis of whole genome sequences (WGS) of well-documented phage types of S. Typhimurium reveals the presence of different CRISPR type among strains belong to the same phage type. Furthermore, different phage types of S. Typhimurium share identical CRISPR type. Interestingly, identical spacers were detected among outbreak and non-outbreak associated DT8 strains of S. Typhimurium. Therefore, CRISPR typing is not useful for the epidemiological surveillance and outbreak investigation of S. Typhimurium and phage typing, until it is replaced by WGS, is still the gold standard method for epidemiological surveillance of S. Typhimurium.

A Retrospective Study of Salmonella Enteritidis Isolated from Commercial Layer Flocks.

Consumption of shell eggs has been associated with Salmonella Enteritidis (SE) infections in humans in the United States. Because of this, the Pennsylvania Egg Quality Assurance Program (PEQAP) was developed and implemented in 1994. The PEQAP involves periodic flock testing and management practices to minimize SE contamination of shell eggs. Subsequently, the U.S. Food and Drug Administration (FDA) introduced a mandatory federal program in 2010 and 2012 for shell egg producers modeled closely after PEQAP to reduce the incidence and prevalence of SE during production, storage, and transport nationwide. In this study, a retrospective epidemiologic analysis was conducted by characterizing SE isolated from commercial layer environment samples and shell eggs submitted to the Animal Diagnostic Laboratory at The Pennsylvania State University using phage typing and pulsed-field gel electrophoresis (PFGE). The objective of this study was to determine the relatedness of SE isolates from hen house environments and shell eggs and to optimize the existing protocols of egg quality assurance programs by identifying the best layer-house environmental sampling time points in order to minimize SE contamination of shell eggs. A total of 94 SE isolates from 65 hen flocks on 35 premises in Pennsylvania recovered during 2007 to 2015 were used in this study. The SE phage type 8 and PFGE fingerprint type JEGX01.0004 most commonly associated with human SE infection was also the predominant type present in layer-house environments and shell eggs. This reconfirms hen house environmental monitoring is an effective method to identify SE-infected flocks. Further, the PEQAP program allowed SE detection of infected flocks earlier than the FDA program as it included an additional environmental test at 29-31 wk of age, enabling the earlier prevention of SE-contaminated shell eggs going to the market. Therefore, it is recommended to refine the sampling time points of the current FDA Egg Rule by adding hen house environmental testing at 29-31 wk of age.

Subtyping of Canadian isolates of Salmonella Enteritidis using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) alone and in combination with Pulsed-Field Gel Electrophoresis (PFGE) and phage typing.

Salmonella enterica subspecies enterica serovar Enteritidis (SE) is one of the most common causes of human salmonellosis and in Canada currently accounts for over 40% of human cases. Reliable subtyping of isolates is required for outbreak detection and source attribution. However, Pulsed-Field Gel Electrophoresis (PFGE), the current standard subtyping method for Salmonella spp., is compromised by the high genetic homogeneity of SE. Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) was introduced to supplement PFGE, although there is a lack of data on the ability of MLVA to subtype Canadian isolates of SE. Three subtyping methods, PFGE, MLVA and phage typing were compared for their discriminatory power when applied to three panels of Canadian SE isolates: Panel 1: 70 isolates representing the diversity of phage types (PTs) and PFGE subtypes within these PTs; Panel 2: 214 apparently unrelated SE isolates of the most common PTs; and Panel 3: 27 isolates from 10 groups of epidemiologically related strains. For Panel 2 isolates, four MLVA subtypes were shared among 74% of unrelated isolates and in Panel 3 isolates, one MLVA subtype accounted for 62% of the isolates. For all panels, combining results from PFGE, MLVA and PT gave the best discrimination, except in Panel 1, where the combination of PT and PFGE was equally as high, due to the selection criteria for this panel. However, none of these methods is sufficiently discriminatory alone for reliable outbreak detection or source attribution, and must be applied together to achieve sufficient discrimination for practical purposes. Even then, some large clusters were not differentiated adequately. More discriminatory methods are required for reliable subtyping of this genetically highly homogeneous serovar. This need will likely be met by whole genome sequence analysis given the recent promising reports and as more laboratories implement this tool for outbreak response and surveillance.

Phenotypic and Genotypic Eligible Methods for Salmonella Typhimurium Source Tracking.

Salmonellosis is one of the most common causes of foodborne infection and a leading cause of human gastroenteritis. Throughout the last decade, Salmonella enterica serotype Typhimurium (ST) has shown an increase report with the simultaneous emergence of multidrug-resistant isolates, as phage type DT104. Therefore, to successfully control this microorganism, it is important to attribute salmonellosis to the exact source. Studies of Salmonella source attribution have been performed to determine the main food/food-production animals involved, toward which, control efforts should be correctly directed. Hence, the election of a ST subtyping method depends on the particular problem that efforts must be directed, the resources and the data available. Generally, before choosing a molecular subtyping, phenotyping approaches such as serotyping, phage typing, and antimicrobial resistance profiling are implemented as a screening of an investigation, and the results are computed using frequency-matching models (i.e., Dutch, Hald and Asymmetric Island models). Actually, due to the advancement of molecular tools as PFGE, MLVA, MLST, CRISPR, and WGS more precise results have been obtained, but even with these technologies, there are still gaps to be elucidated. To address this issue, an important question needs to be answered: what are the currently suitable subtyping methods to source attribute ST. This review presents the most frequently applied subtyping methods used to characterize ST, analyses the major available microbial subtyping attribution models and ponders the use of conventional phenotyping methods, as well as, the most applied genotypic tools in the context of their potential applicability to investigates ST source tracking.

Longitudinal Monitoring of Successive Commercial Layer Flocks for Salmonella enterica Serovar Enteritidis.

The Pennsylvania Egg Quality Assurance Program (EQAP) provided the framework for Salmonella Enteritidis (SE) control programs, including the Food and Drug Administration (FDA) mandated Final Egg Rule, for commercial layer facilities throughout the United States. Although flocks with ≥3000 birds must comply with the FDA Final Egg Rule, smaller flocks are exempted from the rule. As a result, eggs produced by small layer flocks may pose a greater public health risk than those from larger flocks. It is also unknown if the EQAPs developed with large flocks in mind are suitable for small- and medium-sized flocks. Therefore, a study was performed to evaluate the effectiveness of best management practices included in EQAPs in reducing SE contamination of small- and medium-sized flocks by longitudinal monitoring of their environment and eggs. A total of 59 medium-sized (3000 to 50,000 birds) and small-sized (<3000 birds) flocks from two major layer production states of the United States were enrolled and monitored for SE by culturing different types of environmental samples and shell eggs for two consecutive flock cycles. Isolated SE was characterized by phage typing, pulsed-field gel electrophoresis (PFGE), and clustered regularly interspaced short palindromic repeats-multi-virulence-locus sequence typing (CRISPR-MVLST). Fifty-four Salmonella isolates belonging to 17 serovars, 22 of which were SE, were isolated from multiple sample types. Typing revealed that SE isolates belonged to three phage types (PTs), three PFGE fingerprint patterns, and three CRISPR-MVLST SE Sequence Types (ESTs). The PT8 and JEGX01.0004 PFGE pattern, the most predominant SE types associated with foodborne illness in the United States, were represented by a majority (91%) of SE. Of the three ESTs observed, 85% SE were typed as EST4. The proportion of SE-positive hen house environment during flock cycle 2 was significantly less than the flock cycle 1, demonstrating that current EQAP practices were effective in reducing SE contamination of medium and small layer flocks.

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Prevalence and antimicrobial resistance of Salmonella serovars isolated from poultry in Ghana.

Poultry are possible sources of non-typhoidal Salmonella serovars which may cause foodborne human disease. We conducted a cross-sectional study to determine the prevalence of Salmonella serovars in egg-laying hens and broilers at the farm level and their susceptibility to antimicrobials commonly used in the poultry industry in Ghana. Sampling of faeces by a sock method (n = 75), dust (n = 75), feed (n = 10) and drinking water (n = 10) was performed at 75 commercial egg-laying and broiler farms in two regions of Ghana and skin neck (n = 30) at a local slaughterhouse from broilers representing different flocks. Salmonella was detected in 94/200 (47%) samples with an overall flock prevalence of 44·0%. Sixteen different serovars were identified with S. Kentucky (18·1%), S. Nima (12·8%), S. Muenster (10·6%), S. Enteritidis (10·6%) and S. Virchow (9·6 %) the most prevalent types. The predominant phage type of S. Enteritidis was PT1. All strains were susceptible to cefotaxime, ceftazidime and cefoxitin. Fifty-seven (60·6%) strains were resistant to one or more of the remaining nine antimicrobials tested by disk diffusion, of which 23 (40·4%) showed multi-resistance (resistance to ⩾3 classes of antimicrobials). Of the resistant strains (n = 57), the most significant were to nalidixic acid (89·5%), tetracycline (80·7%), ciprofloxacin (64·9%), sulfamethazole (42·1%), trimethoprim (29·8%) and ampicillin (26·3%). All S. Kentucky strains were resistant to more than two antimicrobials and shared common resistance to nalidixic acid or ciprofloxacin and tetracycline, often in combinations with other antimicrobials. PFGE analysis using XbaI of S. Kentucky demonstrated one dominant clone in the country. In conclusion, poultry produced in Ghana has a high prevalence of multi-resistant Salmonella and the common finding of clonal S. Kentucky in the Kumasi area warrants further investigations into the epidemiology of this serovar. There is an urgent need for surveillance and control programmes on Salmonella and use of antimicrobials in the Ghanaian poultry industry to protect the health of consumers.