Characterization of a Coliphage AS1 isolated from sewage effluent in Pakistan / Caracterização de um colifago AS1 isolado de efluente de esgoto no Paquistão

The emergence of multi-drug resistant (MDR) bacterial strains, which are posing a global health threat has developed the interest of scientists to use bacteriophages instead of conventional antibiotics therapy. In light of an increased interest in the use of phage as a bacterial control agent, the study aimed to isolate and characterize lytic phages from sewage effluent. During the current study, bacteriophage AS1 was isolated from sewage effluent against E.coli S2. The lytic activity of phageAS1 was limited to E.coli S2 strain showing monovalent behavior. The calculated phage titer was 3.5×109 pfu/ml. PhageAS1 was stable at a wide range of pH and temperature. The maximum stability was recorded at 37ºC and pH 7.0, while showing its normal lytic activity at temperature 60ºC and from pH 5.0 to 11.0 respectively. At temperature 70ºC, phage activity was somewhat reduced whereas, further increase in temperature and decrease or increase in pH completely inactivated the phage. From the current study, it was concluded that waste water is a best source for finding bacteriophages against multi-drug resistant bacterial strains and can be used as bacterial control agent.

O surgimento de cepas bacterianas multirresistentes (MDR), que representam uma ameaça global à saúde, desenvolveu o interesse dos cientistas em usar bacteriófagos em vez da terapia convencional com antibióticos. Diante do crescente interesse no uso de fago como agente de controle bacteriano, o estudo visou isolar e caracterizar fagos líticos de efluente de esgoto. Durante o estudo atual, o bacteriófago AS1 foi isolado de efluente de esgoto contra E. coli S2. A atividade lítica de phageAS1 foi limitada à cepa E. coli S2, apresentando comportamento monovalente. O título de fago calculado foi de 3,5 x 109 ufp/ml. PhageAS1 foi estável em uma ampla faixa de pH e temperatura. A estabilidade máxima foi registrada a 37ºC e pH 7,0, enquanto mostrou atividade lítica normal em temperatura de 60ºC e pH 5,0 a 11,0, respectivamente. Na temperatura de 70ºC, a atividade do fago foi um pouco reduzida, enquanto o aumento adicional da temperatura e a diminuição ou aumento do pH inativaram completamente o fago. Com base no estudo atual, concluiu-se que a água residual é a melhor fonte para encontrar bacteriófagos contra cepas bacterianas multirresistentes e pode ser usada como agente de controle bacteriano.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Salmonella spp. transmission in a vertically integrated poultry operation: Clustering and diversity analysis using phenotyping (serotyping, phage typing) and genotyping (MLVA).

The transmission of Salmonella enterica within a vertically integrated poultry operation was investigated longitudinally over an 18-month period (2013-2014). Thirty six percent of all samples collected (1503 of 4219) were positive for salmonellae with seven Salmonella enterica subsp. enterica serovars, and one Salmonella enterica subsp. salamae serovar detected. Both Salmonella enterica subsp. enterica serovars Infantis and Typhimurium were detected in all locations sampled. Salmonella Typhimurium was the most frequently detected serovar (63% of serotyped samples) with 8 phage types (PT) and 41 multiple-locus variable-number tandem-repeats analysis (MLVA) profiles identified. The most frequently identified phage types were PT135a and DT135. A total of 62 PT/MLVA combinations were observed. MLVA profiles 03-14-10-09-525 and 03-15-11-11-525 were the most frequently identified and 83% of the isolates shared at least one MLVA profile with an isolate from another phage type. The use of phage typing and MLVA profiling, on their own or in combination, were insufficient to understand the complexity of the epidemiological relationships between locations within this production system. Despite the high level of apparent diversity, cluster analysis was unable to differentiate the transmission pathways of all S. Typhimurium variants detected within the integrated enterprise. Using additional epidemiological information, the parent breeder rearing site was identified as the most likely point of introduction of two S. Typhimurium isolates into the production system with subsequent dissemination to the broiler flocks via the hatchery. This complexity is unable to be resolved in the absence of intensive sampling programs at all generations of the production system.

Re-evaluation of a 2014 multi-country European outbreak of Salmonella Enteritidis phage type 14b using recent epidemiological and molecular data.

A European multi-country outbreak of Salmonella Enteritidis phage type (PT) 14b occurred from March to November 2014 associated with the consumption of eggs. The outbreak involved more than 400 human cases from France, Luxembourg, Austria and the United Kingdom. In 2016-2017, it has been re-evaluated combining recent epidemiological results with latest molecular data. The outbreak was traced back to one large Bavarian egg producer with four distinct premises, three located in Bavaria, one in the Czech Republic. The outbreak isolates of S. Enteritidis PT 14b were grouped into three closely related clades by whole genome sequencing. Two of these clades could be referred to two Bavarian premises of the egg producer on the basis of epidemiological and molecular data, while epidemiological data presumably linked the third clade to another premises of the egg producer. Interestingly and in contrast to the situation in other European countries where several outbreaks were documented, all notified 91 laboratory-confirmed cases of S. Enteritidis PT 14b from Bavaria were sporadic, singular cases not belonging to any epidemiological outbreaks. In conclusion, as demonstrated here, the resolution of food-related outbreaks with such a high discriminatory power is rare in outbreak investigation.

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.

Clinical Antibiotic-resistant Acinetobacter baumannii Strains with Higher Susceptibility to Environmental Phages than Antibiotic-sensitive Strains.

Antibiotic-resistant Acinetobacter baumannii is associated with nosocomial infections worldwide. Here, we used clinically isolated A. baumannii strains as models to demonstrate whether antibiotic resistance is correlated with an increased susceptibility to bacteriophages. In this study, 24 active phages capable of infecting A. baumannii were isolated from various environments, and the susceptibilities of both antibiotic-sensitive and antibiotic-resistant strains of A. baumannii to different phages were compared. In our study, a total of 403 clinically isolated A. baumannii strains were identified. On average, the phage infection percentage of the antibiotic-resistant A. baumannii strains was 84% (from 81-86%), whereas the infection percentage in the antibiotic-sensitive A. baumannii strains was only 56.5% (from 49-64%). In addition, the risk of phage infection for A. baumannii was significantly increased in the strains that were resistant to at least four antibiotics and exhibited a dose-dependent response (p-trend < 0.0001). Among all of the A. baumannii isolates, 75.6% were phage typeable. The results of phage typing might also reveal the antibiotic-resistant profiles of clinical A. baumannii strains. In conclusion, phage susceptibility represents an evolutionary trade-off in A. baumannii strains that show adaptations for antibiotic resistance, particularly in medical environments that have high antibiotic use.

Phage-protease-peptide: a novel trifecta enabling multiplex detection of viable bacterial pathogens.

Bacteriophages represent rapid, readily targeted, and easily produced molecular probes for the detection of bacterial pathogens. Molecular biology techniques have allowed researchers to make significant advances in the bioengineering of bacteriophage to further improve speed and sensitivity of detection. Despite their host specificity, bacteriophages have not been meaningfully leveraged in multiplex detection of bacterial pathogens. We propose a proof-of-principal phage-based scheme to enable multiplex detection. Our scheme involves bioengineering bacteriophage to carry a gene for a specific protease, which is expressed during infection of the target cell. Upon lysis, the protease is released to cleave a reporter peptide, and the signal detected. Here we demonstrate the successful (i) modification of T7 bacteriophage to carry tobacco etch virus (TEV) protease; (ii) expression of TEV protease by Escherichia coli following infection by our modified T7, an average of 2000 units of protease per phage are produced during infection; and (iii) proof-of-principle detection of E. coli in 3 h after a primary enrichment via TEV protease activity using a fluorescent peptide and using a designed target peptide for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (MALDI-TOF MS) analysis. This proof-of-principle can be translated to other phage-protease-peptide combinations to enable multiplex bacterial detection and readily adopted on multiple platforms, like MALDI-TOF MS or fluorescent readers, commonly found in labs.

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

[THE IDENTIFICATION AND DIFFERENTIATION OF BACTERIOPHAGES OF HUMAN PATHOGENIC VIBRIO].

The issue of identification and differentiation of large group of bacteriophages of human pathogenic vibrio is still unresolved. In research and practical applied purposes it is important to consider characteristics of bacteriophages for establishing similarity and differences between them. The actual study was carried out to analyze specimens of DNA-containing bacteriophages of pathogenic vibrio. The overwhelming majority of them characterized by complicated type of symmetry--phages with double-helical DNA and also phages with mono-helical DNA structure discovered recently in vibrio. For the first time, the general framework of identification and differentiation of bacteriophages of pathogenic vibrio was developed. This achievement increases possibility to establish species assignment of phages and to compare with phages registered in the database. "The collection of bacteriophages and test-strains of human pathogenic vibrio" (No2010620549 of 24.09.210).

Comparison between Salmonella enterica Serotype Enteritidis Genotyping Methods and Phage Type.

A quantitative comparison between discriminatory indexes and concordance among multilocus variable-number tandem-repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE), automated ribotyping, and phage typing has been performed, testing 238 Salmonella enterica serotype Enteritidis isolates not epidemiologically correlated. The results show that MLVA is the best choice, but each typing method provides a piece of information for establishing clonal relationships between the isolates.

A multi-country Salmonella Enteritidis phage type 14b outbreak associated with eggs from a German producer: 'near real-time' application of whole genome sequencing and food chain investigations, United Kingdom, May to September 2014.

We report an outbreak of Salmonella Enteritidis phage type 14b (PT14b) in the United Kingdom (UK) between May and September 2014 where Public Health England launched an investigation to identify the source of infection and implement control measures. During the same period, outbreaks caused by a Salmonella Enteritidis strain with a specific multilocus variable-number tandem repeat analysis (MLVA) profile occurred in other European Union Member States. Isolates from a number of persons affected by the UK outbreak, who had initially been tested by MLVA also shared this particular profile. Cases were defined as any person infected with S. Enteritidis PT14b, resident in England or Wales and without history of travel outside of this geographical area during the incubation period, reported from 1 June 2014 onwards, with a MLVA profile of 2­11­9-7­4-3­2-8­9 or a single locus variant thereof. In total, 287 cases met the definition. Food traceback investigations in the UK and other affected European countries linked the outbreaks to chicken eggs from a German company. We undertook whole genome sequencing of isolates from UK and European cases, implicated UK premises, and German eggs: isolates were highly similar. Combined with food traceback information, this confirmed that the UK outbreak was also linked to a German producer.

Bacteriophage strain typing by rapid single molecule analysis.

Rapid characterization of unknown biological samples is under the focus of many current studies. Here we report a method for screening of biological samples by optical mapping of their DNA. We use a novel, one-step chemo-enzymatic reaction to covalently bind fluorophores to DNA at the four-base recognition sites of a DNA methyltransferase. Due to the diffraction limit of light, the dense distribution of labels results in a continuous fluorescent signal along the DNA. The amplitude modulations (AM) of the fluorescence intensity along the stretched DNA molecules exhibit a unique molecular fingerprint that can be used for identification. We show that this labelling scheme is highly informative, allowing accurate genotyping. We demonstrate the method by labelling the genomes of λ and T7 bacteriophages, resulting in a consistent, unique AM profile for each genome. These profiles are also successfully used for identification of the phages from a background phage library. Our method may provide a facile route for screening and typing of various organisms and has potential applications in metagenomics studies of various ecosystems.

Characterization of the emerging Salmonella 4,[5],12:i:- in Danish animal production.

The monophasic Salmonella variant with the antigenic formula Salmonella 4,[5],12:i:- has emerged in the last decade as one of the main serotypes related to human salmonellosis. In the present study, a collection of 94 isolates of the S. 4,12:i:- and S. 4,5,12:i:- coming from Danish farm animals, swine (86), cattle (7), and poultry (1), with well-defined identification was further typed by polymerase chain reaction serotyping, phage typing, and molecular typing (polymerase chain reaction and multilocus variable-number tandem-repeat analysis [MLVA]). Moreover, the determination of antimicrobial resistance pattern of each isolate was tested. In 68 of the isolates the fljB gene was absent (i.e., they were true monophasic strains), whereas in 26 isolates, the gene was present despite the fact that the isolates did not express it. The results clustered the isolates in three main pulse-types. The predominant cluster was compatible with the previously described pattern STYMXB.0131. All the isolates included in this cluster lacked the fljB gene, and all the isolates except one belonged to phage type DT 193 with the AMP-STR-SMX-TET resistance pattern. MLVA analysis divided the clusters in several MLVA profiles previously reported by other studies. Finally, antimicrobial resistance and multiresistance was frequent, although no resistance was detected in critical compounds: fluoroquinolones and cephalosporins. The present study demonstrates the presence of monophasic Salmonella Typhimurium-like strains in Danish food animal production with well-characterized clones that are described by previous studies, demonstrating the emergence and spread of this serotype in Denmark.

Genetic diversity and evolution of Salmonella enterica serovar Enteritidis strains with different phage types.

Phage typing has been used for the epidemiological surveillance of Salmonella enterica serovar Enteritidis for over 2 decades. However, knowledge of the genetic and evolutionary relationships between phage types is very limited, making differences difficult to interpret. Here, single nucleotide polymorphisms (SNPs) identified from whole-genome comparisons were used to determine the relationships between some S. Enteritidis phage types (PTs) commonly associated with food-borne outbreaks in the United States. Emphasis was placed on the predominant phage types PT8, PT13a, and PT13 in North America. With >89,400 bp surveyed across 98 S. Enteritidis isolates representing 14 distinct phage types, 55 informative SNPs were discovered within 23 chromosomally anchored loci. To maximize the discriminatory and evolutionary partitioning of these highly homogeneous strains, sequences comprising informative SNPs were concatenated into a single combined data matrix and subjected to phylogenetic analysis. The resultant phylogeny allocated most S. Enteritidis isolates into two distinct clades (clades I and II) and four subclades. Synapomorphic (shared and derived) sets of SNPs capable of distinguishing individual clades/subclades were identified. However, individual phage types appeared to be evolutionarily disjunct when mapped to this phylogeny, suggesting that phage typing may not be valid for making phylogenetic inferences. Furthermore, the set of SNPs identified here represents useful genetic markers for strain differentiation of more clonal S. Enteritidis strains and provides core genotypic markers for future development of a SNP typing scheme with S. Enteritidis.

Typing of Salmonella enterica serovar Infantis isolates from 51 outbreaks in Germany between 1974 and 2009 by a novel phage-typing scheme.

We developed a new phage-typing method and evaluated its application in combination with XbaI macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) as a useful tool for the long-term epidemiology of Salmonella enterica serovar Infantis. In this study, we investigated 1008 S. Infantis isolates recovered from humans, various animal species and food products from 1973 to 2009. The typing scheme is based on 17 typing phages, defining 61 different patterns within the strain collection. The experiments showed that phage typing is a reliable method for differentiation of outbreaks and sporadic clinical cases as well as for elucidation of chains of transmission. The combined analysis of phage typing and PFGE revealed the existence of epidemic clones with a high stability over time like PT29/XB27 which was identified in nosocomial salmonellosis, community outbreaks as well as in broiler chickens from 2002 to 2009.

Severe infections caused by Salmonella Enteritidis PT8/7 linked to a private barbecue.

A cohort study on a barbecue-associated Salmonella outbreak was conducted to describe the burden of disease and to identify the outbreak vehicle. Dose-response relationships were tested with Fisher's exact and Wilcoxon rank sum tests (alpha = 0·05). S. Enteritidis isolates were cultured and phage-typed. Information was available for 11 out of 14 individuals attending the barbecue; all were healthy young adults (median age 27 years). The attack rate was 100%. Three cases were hospitalized and two developed acute pancreatitis. The exposure common to all cases was a vegetable pasta salad that had been stored unrefrigerated for 23 h. Consuming higher doses was associated with longer median symptom duration (7 days vs. 4 days, P = 0·11). S. Enteritidis was found in the stools of nine barbecue guests. Phage type 8/7 was identified in the stools of the salad preparer and one barbecue guest. This outbreak shows that S. Enteritidis can cause serious infection in young healthy individuals without well-known risk factors.

A new methodology for differentiation and typing of closely related Salmonella enterica serovar Heidelberg isolates.

This study describe the use of a combination of two recently proposed typing approaches, multiple amplification of prophage locus typing (MAPLT) and multiple-locus variable-number tandem-repeat analysis (MLVA) for subdividing within Salmonella enterica serovar Heidelberg (S. Heidelberg). The combined typing method was compared with pulsed-field gel electrophoresis (PFGE) by Simpson's index of diversity (DI). PFGE was shown to have a DI = 0.84 and was poor at differentiation of the predominant PT1 (Phage Type 1) phenotype. In comparison, the combined MAPLT/MLVA method comprising 3 MLVA and 9 MAPLT primer pairs provided a higher differentiating ability DI = 0.92. More importantly, the combined methodology was found to be superior in the differentiation of the predominant PT1 isolates. In conclusion, this study demonstrated the potential of the rapid and simple amalgamated MAPLT/MLVA approach in determining transmission of isolates of clonal phage type groups from various environmental sources to humans.