Diverse Genotypes of Cronobacter spp. Associated with Dairy Farm Systems in Jiangsu and Shandong Provinces in China.

Cronobacter spp. are the most concerning foodborne pathogen in infant formula milk powder. Currently, there are many reports on the prevalence of Cronobacter spp. in infant formula milk and its processing environment, but there are few studies on the prevalence of Cronobacter spp. on dairy farms. We have, therefore, undertaken this study to investigate and track genomic epidemiology of Cronobacter spp. isolates from Chinese dairy farms in the provinces of Jiangsu and Shandong. In this study, forty Cronobacter spp. strains, consisting of thirty Cronobacter sakazakii, eight Cronobacter malonaticus, and two Cronobacter dublinensis, were obtained from 1115 dairy farm samples (raw milk, silage, bedding, and feces), with a prevalence rate of 3.57%. These isolates were classified into 10 Cronobacter serotypes and 31 sequence types (STs), including three novel STs which were isolated for the first time. Notably, pathogenic Cronobacter STs 7, 8, 17, 60, and 64, which are associated with clinical infections, were observed. Antimicrobial susceptibility testing showed that all the Cronobacter spp. were highly resistant to cephalothin and fosfomycin, which was consistent with the antimicrobial genotype. All isolates carried core virulence genes related to adherence, invasion, endotoxin, immune evasion, secretion system, and regulation. Approximately half the isolates were also able to produce a strong biofilm. Twenty-one prophages and eight plasmids were detected, with the most common prophage being Cronobacter_ENT47670 and the most common plasmid being IncFIB (pCTU1). In addition, two isolates harbored the transmissible locus of stress tolerance (tLST) which confers high environmental persistence. Phylogenetic analysis showed strong clustering by species level and sequence types. Isolates from different sources or regions with a similar genomic background suggests the cross-contamination of Cronobacter spp. The presence of diverse genotypes of Cronobacter spp. in dairy farms in Jiangsu and Shandong provinces indicates that surveillance of Cronobacter spp. on dairy farms should be strengthened, to prevent and control transmission and ensure the quality and safety of raw dairy products.

Metagenomics revealed a correlation of gut phageome with autism spectrum disorder.

The human gut bacteriome is believed to have pivotal influences on human health and disease while the particular roles associated with the gut phageome have not been fully characterized yet with few exceptions. It is argued that gut microbiota can have a potential role in autism spectrum disorders (ASD). The public microbiota database of ASD and typically developing (TD) Chinese individuals were analyzed for phage protein-coding units (pPCU) to find any link between the phageome and ASD. The gut phageome of ASD individuals showed a wider diversity and higher abundance compared to TD individuals. The ASD phageome was associated with a significant expansion of Caudoviricetes bacteriophages. Phages infecting Bacteroidaceae and prophages encoded within Faecalibacterium were more frequent in ASD than in TD individuals. The expansion and diversification of ASD phageome can influence the bacterial homeostasis by imposing pressure on the bacterial communities. In conclusion, the differences of phages community in in ASD and TD can be used as potential diagnosis biomarkers of ASD. Further investigations are needed to verify the role of gut phage communities in the pathogenesis of ASD.

Genome Sequence of Salmonella enterica Serovar Typhimurium Phage SAP12.

Here, we report the genome of phage SAP012, which was isolated against Salmonella enterica serovar Typhimurium. The SAP012 genome is 59,618 bp, with a G+C content of 56.2% and with no antibiotic resistance or virulence genes, and is quite similar at the nucleotide level to a number of previously sequenced Salmonella phage genomes, e.g., GenBank accession numbers KM366098.1 and KC139515.1.

Clinical and experimental bacteriophage studies: Recommendations for possible approaches for standing against SARS-CoV-2.

In 2019, the world faced a serious health challenge, the rapid spreading of a life-threatening viral pneumonia, coronavirus disease 2019 (COVID-19) caused by a betacoronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of January 2022 WHO statistics shows more than 5.6 million death and about 350 million infection by SARS-CoV-2. One of the life threatening aspects of COVID-19 is secondary infections and reduced efficacy of antibiotics against them. Since the beginning of COVID-19 many researches have been done on identification, treatment, and vaccine development. Bacterial viruses (bacteriophages) could offer novel approaches to detect, treat and control COVID-19. Phage therapy and in particular using phage cocktails can be used to control or eliminate the bacterial pathogen as an alternative or complementary therapeutic agent. At the same time, phage interaction with the host immune system can regulate the inflammatory response. In addition, phage display and engineered synthetic phages can be utilized to develop new vaccines and antibodies, stimulate the immune system, and elicit a rapid and well-appropriate defense response. The emergence of SARS-CoV-2 new variants like delta and omicron has proved the urgent need for precise, efficient and novel approaches for vaccine development and virus detection techniques in which bacteriophages may be one of the plausible solutions. Therefore, phages with similar morphology and/or genetic content to that of coronaviruses can be used for ecological and epidemiological modeling of SARS-CoV-2 behavior and future generations of coronavirus, and in general new viral pathogens. This article is a comprehensive review/perspective of potential applications of bacteriophages in the fight against the present pandemic and the post-COVID era.

A rapid competitive method for bacteriophage genomic DNA extraction.

The bacteriophage (phage) DNA extraction methods for genomics analysis is a critical and time-consuming process. Hence, a rapid and cost-effective method for DNA extraction of phages is favorable for phage biologists. In the present study, a cost-effective, simple and rapid procedure for phage genome extraction in less than 10 min is introduced. Highly concentrated phage lysates were prepared using acetone precipitation followed by extraction using various methods such as commercial kits, TES lysis buffer, potassium iodide, and sodium iodide. The quality of the extracted DNA was analyzed by agarose gel electrophoresis and UV absorbance of DNA at 260 and 280 nm. Finally, the extracted DNA was subjected to restriction digestion and next-generation sequencing to approve the efficiency of the method. Based on the time, cost, and quality of obtained DNA, the acetone precipitation of phages and extraction by potassium iodide or sodium iodide method was determined to be the best method for phage DNA extraction tested in this study. Moreover, the extracted genomic DNA using this method is suitable for phage genomic analysis such as restriction enzyme studies, preparation of DNA library, and also next-generation sequencing.

vB_EfaS-DELF1, a novel Siphoviridae bacteriophage with highly effective lytic activity against vancomycin-resistant Enterococcus faecalis.

Enterococcus faecalis is an environmental agent of bovine mastitis in cows and has many cytopathic effects on the urinary tract in both humans and animals. In this study, a novel lytic bacteriophage, vB_EfaS-DELF1, was isolated against 21 E. faecalis isolated from bovine mastitis, including vancomycin-resistant E. faecalis (VRE). vB_EfaS-DELF1 bacteriophage was specific for E. faecalis and showed no lytic effects against other tested Enterococcus spp., Gram-negative, or Gram-positive bacteria. Moreover, no activity was observed against yogurt starters. The phage suspension was stable in a wide range of pH, salinity, and temperature. It retained its activity in 3.5 % fat milk. vB_EfaS-DELF1 has the common phenotypic features of Siphoviridae with a double-strand DNA of 40,248 bp in length and a G + C content of 34.9 %. The genome encodes 62 putative ORFs and no tRNA. No undesirable genes such as lysogenic mediators, antibiotic resistance, or virulence factor genes were detected in the genome. The comparative genomic analysis demonstrated similarity to the other available phage genomes. The highest similarity was observed with two other phages (50 % coverage and 82.38 % identities with IME-EFm1; 35 % coverage and 86.22 % identities with IME-EFm5) that were placed in the same clade. The differences with the other aligned phages were high and were placed in distant clusters. Regarding the specificity of this new bacteriophage against all of the tested E. faecalis isolates and, in particular, against the vancomycin-resistant ones, and also the absence of antibiotic resistance or virulence genes in its genome, vB_EfaS-DELF1 is suggested as a potential candidate for biocontrol of E. faecalis infections.

Resistotyping, phenotyping and genotyping of New Delhi metallo-ß-lactamase (NDM) among Gram-negative bacilli from Iranian patients.

PURPOSE: The purpose of this study was to investigate New Delhi metallo-ß-lactamase (NDM) production among Gram-negative bacilli. METHODOLOGY: Antibiogram-resistotyping and detection of New Delhi metallo-ß-lactamase (NDM) in clinical isolates of Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and comparative evaluation of the diagnostic performance of three phenotypic methods for NDM detection, with PCR considered as the gold standard, were performed. Minimum inhibitory concentration (MIC) of antibiotics against NDM-positive strains using E-tests and clonal relationship analysis using enterobacterial repetitive intergenic consensus (ERIC)-PCR in these strains were determined. RESULTS: The most effective antibiotics against strains of the species K. pneumoniae were Colistin, Chloramphenicol and Tigecycline; against P. aeruginosa were Fosfomycin and Polymyxins, and against A. baumannii were Polymyxins, Ampicillin/Sulbactam and Minocycline. Overall, 66, 31 and 40 different resistotypes were observed among K. pneumoniae, A. baumannii and P. aeruginosa strains, respectively. The blaNDM-1 gene was detected in 28 (8.5 %) strains of the bacteria investigated. The sensitivities and specificities of the Meropenem-EDTA combined disk test, the meropenem-dipicolinic acid combined disk test and the modified Hodge test methods for NDM detection were 96.43, 55.15; 96.43, 54.85; and 89.29, 35.15, respectively. Additionally, in spite of the low positive predictive values of these tests, their negative predictive values were high. ERIC-PCR results revealed two main clusters in NDM-positive strains of each of the species P. aeruginosa and A. baumannii, and ten main clusters in K. pneumoniae. In all the NDM-positive strains maximum MIC rates (>256) were observed for all beta-lactam antibiotics. CONCLUSION: There were high levels of antibiotic resistance and a high frequency of multi-drug resistance and extensive-drug resistance profiles, as well as highly prevalent blaNDM-1 genes in the bacteria investigated.

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages.

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Isolation of Dickeya dadantii strains from potato disease and biocontrol by their bacteriophages

One of the most economically important bacterial pathogens of plants and plant products is Dickeya dadantii. This bacterium causes soft rot disease in tubers and other parts of the potato and other plants of the Solanaceae family. The application of restricted host range bacteriophages as biocontrol agents has recently gained widespread interest. This study purposed to isolate the infectious agent of the potato and evaluate its biocontrol by bacteriophages. Two phytopathogenic strains were isolated from infected potatoes, identified based on biochemical and 16S rRNA gene sequencing, and submitted to GenBank as D. dadantii strain pis3 (accession no. HQ423668) and D. dadantii strain sip4 (accession no. HQ423669). Their bacteriophages were isolated from Caspian Sea water by enriching the water filtrate with D. dadantii strains as hosts using spot or overlay methods. On the basis of morphotypes, the isolated bacteriophages were identified as members of the Myoviridae and Siphoviridae families and could inhibit the growth of antibiotic resistant D. dadantii strains in culture medium. Moreover, in Dickeya infected plants treated with bacteriophage, no disease progression was detected. No significant difference was seen between phage-treated and control plants. Thus, isolated bacteriophages can be suggested for the biocontrol of plant disease caused by Dickeya strains.

Isolation and Identification of Two Novel Escherichia coli Bacteriophages and Their Application in Wastewater Treatment and Coliform's Phage Therapy.

BACKGROUND: Phage therapy or use of lytic bacteriophages for eliminating bacterial populations has been developed for several aspects of human affairs such as medicine, agriculture and food industries. OBJECTIVES: The high load of coliforms of treated wastewater effluents that are discharged into the rivers or agricultural lands is a serious concern of the Iran Department of Environment and the reduction of coliforms using phages to overcome this problem is an asset. This research aimed to isolate and identify specific lytic coliphages and investigate their effects on native and standard Escherichia coli strains as well as coliform populations in municipal wastewater. MATERIALS AND METHODS: The wastewater sample was cultured on selective culture media to isolate a native coliform strain and characterized using molecular methods. River water was centrifuged and passed through a 0.45 µm filter and its lytic coliphages were enriched and purified against a native E. coli as well as a standard E. coli strain. Municipal wastewater was treated with isolated lytic coliphages and most probable number (MPN) reduction was examined. RESULTS: E. coli SBSWF27, which is a native strain of E. coli from Isfahan municipal wastewater treatment plant, was isolated and characterized. Also two novel bacteriophages related to Myoviridae and Podoviridae families of bacteriophages from Zayandehrood River (Isfahan, Iran) were isolated. These coliphages had lytic effects on E. coli PTCC1399 and E. coli SBSWF27 as coliform's index. The myovirus had a hexagonal head measuring 27.28 nm and a noncontractile tail measuring 204.5 × 13.63 nm. The podovirus had an oval head measuring 98 × 35 nm and a tail, 14 nm in diameter. The treatment of municipal sewage with the coliphage mixture resulted in a 22-fold decrease of the coliform's MPN from 2400 to 110 after two hours of incubation. CONCLUSIONS: This is the first report on isolation and identification of two novel lytic myovirus and podovirus from Zayandehrood River in Isfahan that had lytic effects on E. coli PTCC1399 and E. coli SBSWF27 strains as well as coliform's population of Isfahan municipal wastewater. We suggest that the use of these lytic coliphages for reduction of coliform's population in sewage could be considered as an effective and simple alternative for costly replacement of instruments and establishments of the old wastewater treatment plants.