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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Group B Streptococcus (GBS) causes severe infections in neonates and adults with comorbidities. Prophages have been reported to contribute to GBS evolution and pathogenicity. However, no studies are available to date on the presence and diversity of prophages in GBS isolates from humans in South America. This study provides insights into the prophage content of 365 GBS isolates collected from clinical samples in the context of an Argentinean multicentric study. Using whole-genome sequence data, we implemented two previously proposed methods for prophage typing: a PCR approach (carried out in silico ) coupled with a blastx-based method to classify prophages based on their prophage group and integrase type, respectively. We manually searched the genomes and identified 325 prophages. However, only 80% of prophages could be accurately categorized with the previous approaches. Integration of phylogenetic analysis, prophage group and integrase type allowed for all to be classified into 19 prophage types, which correlated with GBS clonal complex grouping. The revised prophage typing approach was additionally improved by using a blastn search after enriching the database with ten new genes for prophage group classification combined with the existing integrase typing method. This modified and integrated typing system was applied to the analysis of 615 GBS genomes (365 GBS from Argentina and 250 from public databases), which revealed 29 prophage types, including two novel integrase subtypes. Their characterization and comparative analysis revealed major differences in the lysogeny and replication modules. Genes related to bacterial fitness, virulence or adaptation to stressful environments were detected in all prophage types. Considering prophage prevalence, distribution and their association with bacterial virulence, it is important to study their role in GBS epidemiology. In this context, we propose the use of an improved and integrated prophage typing system suitable for rapid phage detection and classification with little computational processing.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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.

The EN ISO 6579/2002 guideline was used for the detection of Salmonella in retail poultry and pork meat in the Czech Republic in 2013 and 2014. The laboratory confirmed isolates were further typed (slide agglutination, phage typing, resistance to antimicrobial agents, PCR for the detection of selected genes encoding plasmid mediated quinolone resistance). Out of 176 poultry and 223 pork meat samples, 24 (13.6%) and 6 (2.7%) were positive for the detection of Salmonella spp., respectively. In Salmonella isolates from poultry, 14 serotypes were differentiated with S. indiana, S. enteritidis and S. 6,7:-:1,5 being the most common serotypes. S. typhimurium and its monophasic variant S. 4,[5],12:i:- were predominant in pork meat. The overall resistance to one antimicrobial agent at least was high in both groups of isolates - 50% (poultry) and 71.4% (pork). No Salmonella isolate was confirmed to carry any of the selected PMQR genes. The study showed a higher prevalence of Salmonella in poultry, but pork meat also poses a risk to consumers.

The present study aimed to isolate and characterize bacteriophages (phages) specific for Edwardsiella ictaluri to be used as a biological control of the infection in fish as a final goal. Twenty-six phages (PEi1 ~ PEi26), lytic to E. ictaluri were isolated from ayu Plecoglossus altivelis and its surrounding environment (water and mud) using enrichment and double agar layer methods. All phages formed clear plaques ranging in size from 0.3 to 8 mm in diameter. Transmission electron microscopy revealed that all the isolated bacteriophages belonged to the family Myoviridae. According to phage morphology, plaques size and DNA restriction patterns with EcoRI, the phages were classified into four groups (I ~ IV). E. ictaluri strains isolated from ayu (n=57) were sensitive to these phages with various degrees, resulting in twenty-five phage types of the bacterium. E. ictaluri strains isolated from catfish in the USA (type strain; JCM1680), Indonesia (n=4) or Vietnam (n=2) exhibited no or very limited susceptibility to the present phages. In addition, E. tarda, the most closely related species to E. ictaluri, as well as other fish pathogens were not susceptible to any of the phages examined. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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)-β-D-Rhap-(1→3)-α-D-Rhap-(1→; α-D-Glcp-(1˩; 2 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study, 97 Escherichia coli strains isolated from urinary tract infections were evaluated in terms of phenotypic diversity, susceptibility to sixteen antibiotics, and extended spectrum of beta lactamase (ESBL) characteristics. The bacteriophage interactions was used to identify the relations of the E.coli isolates. Results showed that 21 of the 76 typable strains revealed 19 different phage types. Phage types of 21 strains could not be determined. Antibiotic susceptibility tests, performed against sixteen antibiotics, showed that although the isolates revealed 44 different types of resistance profile, the strains showed the greatest resistance to cephalothin( 54.6%), followed by tetracycline (53.6%), nalidixic acid(44.3%), and aztreonam and ofloxacin (29.8%). There was no resistance to amikacin and meropenem, and all strains were susceptible to meropenem. ESBL detection conducted by double disc synergy and multiplex polymerase chain reaction methods showed that 13 of 97 (13.4%) strains were ESBL-producing. Of the 13 ESBL-producing isolates, two carried blaCTX-M, six blaTEM, five blaOXA, and one blaTEM and blaSHV at same time. We concluded that lytic phages could not be successful for typing the strains phenotypically. The monitoring of antimicrobial resistance levels and antimicrobial usage in humans should be an integral part of the prevention and control of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Synthetic and molecular biology tools for designing and constructing engineered phages. • Engineered phage as a nano-machine and a magic bullet in the health sector. • High host specificity (i.e., bacteria), efficiency, stability, and human-friendly nature of engineered phages. • Therapeutic applications of engineered phages in combating bacterial superbugs, cancer treatment, and vaccine development. [ABSTRACT FROM AUTHOR]

Today film-forming strains of bacteria play very important role in clinical pathology. Staphylococci are ones of most dangerous of them. This bacteria can determine different pathological processes, for example, complication of airway mucosa. The ability to form a biofilm is one of the main properties of nosocomial strains. These strains should be monitored and their carriers are to be properly treated. To determine the origin of staphylococci strains we used bacteriophages from the International kit. The aim of research was to determine the phage type of staphylococci film-forming strains, that were isolated from naso-pharingial mucosa. Phage typing has been carried out for 16 film-forming strains of S. aureus. To solve this problem, we used the International phage kit by Fisher’s method. As a result, sensitivity to phages from the International kit showed 53.8% of studied strains of S. aureus. 64.3% of sensitivity strains were lysed by one of the phage, 21.4% – were by two of the phages, 14.3% – by three of the phages. Isolates were sensitive to phages: 81 – 42.9%, 75 – 35.7%, 28.6% were sensitive to phages 47 and 53. All cases of detection of sensitivity to phage 47 coincided with the ability to form biofilm. Among non-film-forming strains there was no sensitive strains for this phage. Film-forming strains resist to erythromycin (62.5%), ciprofloxacin (43.8%), gentamicin (56.3%), tetracycline (87.5%), amoxicillin (93.8%), and cefuroxime (37.5%). All cases of sensitivity to phage 47 coincided with resistance to erythromycin, amoxicillin and tetracycline. For two of these strains, we also defined resistance to gentamicin and for one of them – to ciprofloxacin. Results of research allowed to relate the bacterial cultures for determining the type. This may have implications for studying of film-forming ability, because surface structures of bacterial cell take place in this process. Belonging of an isolate to specific phage type may indicate possible differences in the structure of the cell wall, composition of the cytoplasmic membrane, receptors, etc. So, phage typing may be used as one of tests to study film-forming processes. Besides, phage typing can be helpful in finding of the source and modes of transmission of nosocomial strains of bacteria. Given the increased level of antibiotic resistance among film-forming strains of staphylococci, the importance of this monitoring is indisputable, because spreading of these strains may cause the failure of therapy.

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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. [ABSTRACT FROM AUTHOR]

The EN ISO 6579/2002 guideline was used for the detection of Salmonella in retail poultry and pork meat in the Czech Republic in 2013 and 2014. The laboratory confirmed isolates were further typed (slide agglutination, phage typing, resistance to antimicrobial agents, PCR for the detection of selected genes encoding plasmid mediated quinolone resistance). Out of 176 poultry and 223 pork meat samples, 24 (13.6%) and 6 (2.7%) were positive for the detection of Salmonella spp., respectively. In Salmonella isolates from poultry, 14 serotypes were differentiated with S. indiana, S. enteritidis and S. 6,7:-:1,5 being the most common serotypes. S. typhimurium and its monophasic variant S. 4,[5],12:i:- were predominant in pork meat. The overall resistance to one antimicrobial agent at least was high in both groups of isolates - 50% (poultry) and 71.4% (pork). No Salmonella isolate was confirmed to carry any of the selected PMQR genes. The study showed a higher prevalence of Salmonella in poultry, but pork meat also poses a risk to consumers. [ABSTRACT FROM AUTHOR]

Aim: The study aimed to evaluate the utility of various commonly used fluoroquinolones against Staphylococcus aureus isolates. Materials and Methods: A total of 250 isolates of S. aureus were studied from different clinical specimens like blood, pus, wound swabs, sputum, ear swabs, and body fluids between November 2009 and December 2011. All the isolates were tested for their susceptibility to fluoroquinolones and other antimicrobial agents by Kirby-Bauer disc diffusion method using criteria of standard zone of inhibition. Methicillin-resistant S. aureus (MRSA) detection was done by cefoxitin disk diffusion method. The MRSA isolates were tested for minimum inhibitory concentration (MIC) to vancomycin by E-test strips. All the MRSA strains were sent to National Staphylococcal Phage-typing Centre, Maulana Azad Medical College, New Delhi for phage typing. Results: A total of 107 strains of S. aureus (42.8%) were detected as MRSA. Multidrug resistance was observed among the MRSA strains more commonly than among the MSSA stains. Among the fluoroquinolones, maximum resistance in MRSA was seen to ciprofloxacin (92.5%), followed by ofloxacin (80.4%). None of the S. aureus isolates showed resistance to vancomycin and linezolid. The MICs of vancomycin for the MRSA tested ranged from 0.5 to 2 μg/ml. Phage typing pattern of 107 MRSA isolates revealed that 37 (34.6%) MRSA isolates were nontypeable and 70 (65.4%) were typeable. Conclusion: Ciprofloxacin can no longer be used in empirical therapy against MRSA infections. Use of other members of fluoroquinolone should be limited only to those strains that show laboratory confirmation of their susceptibility. Vancomycin remains the drug of choice to treat MRSA infections.

Panton-Valentine leucocidin (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 (S. aureus) have 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.

Background: Pyoderma is common in India and other tropical countries. Staphylococcus aureus is the commonest causative agent ofpyoderma. Aims and Objectives: To know the antibiotic susceptibility and bacteriophage pattern of Staphylococcus aureus isolated from pyoderma infection. Materials and Methods: One hundred clinically diagnosed pyoderma cases were investigated bacteriologically. A total of 59 isolates of S. aureus were subjected to antibioticsusceptibility testing by Kirby Bauers disk diffusion method and phage typing by routine test dilution X 100 bacteriophages. Results: Most of the strains were resistant to penicillin, ampicillin and were susceptible to gentamicin, streptomycin and erythromycin. Multidrug resistance was also high among these strains. Regarding the phage types, Phage type 52 (15 strains), 96 (8 strains) and 71(16strains) were predominant among the typed strains (55.95%) of S. aureus. The most common group was mixed phage group (17%) followed by phage group I (13.55%). Conclusion: Knowledge of antibioticsusceptibility pattern is essential to give proper antibiotic therapy and avoid unnecessary medication with non-effective drugs, which may increase resistance. Gentamicin, streptomycin and erythromycin are the drugs of choice in that order. Association of phage typing and antibiotic sensitivity of S. aureus showed the predominance of phage group III with greater frequency of penicillin resistance.

A newly isolated smooth colony morphology phage-resistant (SPR) strain 8416 isolated from a 45-year-old cattle farm cleaner with clinical features of brucellosis in China was reported. The most unusual phenotype was its resistance to two Brucella phages Tbilisi and Weybridge, but sensitive to Berkeley 2, a pattern similar to that of B. melitensis biovar 1. VITEK 2 biochemical identification system found that both strain 8416 and B. melitensis strains shared positive ILATk, but negative in other B. abortus strains. However, routine biochemical and phenotypic characteristics of strain 8416 were most similar to that of B. abortus biovar 9 except CO2 requirement. In addition, multiple PCR molecular typing assays including AMOS-PCR, B. abortus special PCR (B-ab PCR) and a novel sub-biovar typing PCR, indicated that strain 8416 may belong to either biovar 3b or 9 of B. abortus. Surprisingly, further MLVA typing results showed that strain 8416 was most closely related to B. abortus biovar 3 in the Brucella MLVA database, primarily differing in 4 out of 16 screened loci. Therefore, due to the unusual discrepancy between phenotypic (biochemical reactions and particular phage lysis profile) and molecular typing characteristics, strain 8416 couldn’t be exactly classified to any of the existing B. abortus biovars and might be a new variant of B. abortus biovar 9. The present study also indicates that the present phage typing scheme for Brucella spp. is subject to variation and the routine Brucella biovar typing needs further studies.

Introduction: This paper describes the epidemiological, microbiological and environmental investigations conducted during an outbreak of Salmonella gastroenteritis in Singapore.Methods: A case-control study was undertaken to identify the vehicle of transmission. Microbiological testing was performed on faecal, food and environmental samples. Isolates of Salmonella were further characterized by phage typing and ribotyping.Results: There were 216 gastroenteritis cases reported from 20 November to 4 December 2007. The causative agent was identified as Salmonella enterica subspecies enterica serotype Enteritidis for 14 out of 20 cases tested. The vehicle of transmission was traced to cream cakes produced by a bakery and sold at its retail outlets (P < 0.001, OR = 143.00, 95% Cl = 27.23–759.10). More than two-thirds of the 40 Salmonella strains isolated from hospitalized cases, food samples and asymptomatic food handlers were of phage type 1; the others reacted but did not conform to any phage type. The phage types correlated well with their unique antibiograms. The ribotype patterns of 22 selected isolates tested were highly similar, indicating genetic relatedness. The dendrogram of the strains from the outbreak showed distinct clustering and correlation compared to the non-outbreak strains, confirming a common source of infection.Discussion: The cream cakes were likely contaminated by one of the ingredients used in the icing. Cross-contamination down the production line and subsequent storage of cakes at ambient temperatures for a prolonged period before consumption could have resulted in the outbreak.