Preclinical characterization and in silico safety assessment of three virulent bacteriophages targeting carbapenem-resistant uropathogenic Escherichia coli.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20 min, the outbreak period ~ 50 min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37 °C for up to 180 min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163 kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951 bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.

Genomic characterization of three bacteriophages targeting multidrug resistant clinical isolates of Escherichia, Klebsiella and Salmonella.

Application of bacteriophages (phages) to treat complex multidrug-resistant bacterial infection is gaining traction because of its efficacy and universal availability. However, as phages are specific to their host, a diverse collection of locally isolated phage from various geographical locations is required to formulate a wide host range phage cocktail. Here, we report morphological and genomic features of three newly isolated phages from river water of the urban region in Kathmandu, Nepal, targeting three different bacteria (Escherichia coli, Klebsiella pneumoniae and Salmonella enterica.) from the Enterobacteriaceae family. Morphological identification and genome analysis indicated that two phages (Escherichia phage vB_EcoM_TU01 and Klebsiella phage vB_KpnP_TU02) were strictly lytic and free from integrases, virulence factors, toxins and known antimicrobial resistance genes, whereas Salmonella phage vB_SalS_TU03 was possibly a temperate phage. The genomic features of these phages indicate that natural phages are capable of lysing pathogenic bacteria and may have potential in bacterial biocontrol.

Isolation and Characterization of Lytic Bacteriophage Against Multi-drug Resistant Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections. METHODS: In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling. RESULTS: Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other. CONCLUSIONS: As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.

Pharmacokinetics and Pharmacodynamics of a Novel Virulent Klebsiella Phage Kp_Pokalde_002 in a Mouse Model.

Phage therapy is one of the most promising alternatives to antibiotics as we face global antibiotic resistance crisis. However, the pharmacokinetics (PK) and pharmacodynamics (PD) of phage therapy are largely unknown. In the present study, we aimed to evaluate the PK/PD of a locally isolated virulent novel øKp_Pokalde_002 (Podoviridae, C1 morphotype) that infects carbapenem-resistant Klebsiella pneumoniae (Kp56) using oral and intraperitoneal (IP) route in a mouse model. The result showed that the øKp_Pokalde_002 rapidly distributed into the systemic circulation within an hour via both oral and IP routes. A higher concentration of phage in plasma was found after 4 h (2.3 x 105 PFU/ml) and 8 h (7.3 x 104 PFU/ml) of administration through IP and oral route, respectively. The phage titer significantly decreased in the blood and other tissues, liver, kidneys, and spleen after 24 h and completely cleared after 72 h of administration. In the Kp56 infection model, the bacterial count significantly decreased in the blood and other organs by 4-7 log10 CFU/ml after 24 h of øKp_Pokalde_002 administration. Elimination half-life of øKp_Pokalde_002 was relatively shorter in the presence of host-bacteria Kp56 compared to phage only, suggesting rapid clearance of phage in the presence of susceptible host. Further, administration of the øKp_Pokalde_002 alone in healthy mice (via IP or oral) did not stimulate pro-inflammatory cytokines (TNF-α and IL-6). Also, treatment with øKp_Pokalde_002 resulted in a significant reduction of pro-inflammatory cytokines (TNF-α and IL-6) caused by bacterial infection, thereby reducing the tissue inflammation. In conclusion, the øKp_Pokalde_002 possess good PK/PD properties and can be considered as a potent therapeutic candidate for future phage therapy in carbapenem-resistant K. pneumoniae infections.

Therapeutic Efficacy of Bacteriophage Therapy to Treat Carbapenem Resistant Klebsiella Pneumoniae in Mouse Model.

BACKGROUND: Global emergence of carbapenem-resistant Klebsiella pneumoniae is a major public health concern. Phage therapy - application of lytic phage to kill pathogenic bacteria - is considered as one of the promising alternatives to tackle this antibiotic crisis in recent days. This study aimed to isolate, characterize and evaluate therapeutic efficacy of a novel K. pneumoniae phage in mouse model. METHODS: A novel lytic bacteriophage (phage) Kp_Pokalde_002 was isolated against carbapenem-resistant K. pneumoniae (Kp56) and characterized. Safety parameters of the phage were evaluated by bioinformatic analysis of its genome. A lethal dose (~1×107 CFU/mouse) of Kp56 was determined and administrated in the mice. The infected mice were treated with phage Kp_Pokalde_002 at a multiplicity of infection (MOI) 1.0 (~1×107 PFU/mouse) via both oral and intraperitoneal (IP) routes. RESULTS: Isolated phage comprised an icosahedral capsid with a short tail. Based on genome analysis, the phage was strictly lytic belonging the Podoviridae family (T7-like viruses) and free from any virulent and antibiotic-resistant genes. The phage was stable up to 60 °C for 30 minutes and effective between pH 4 to 11 (optimum pH 9). The phage exhibited a short latent period (20 minutes) with burst size of 121 phage particles per infected cell. The infected mice were rescued with the phage therapy via both oral and IP route. Significant reduction of bacterial load (3-7 log10 CFU/ml) in the blood and lung was observed in the treatment group. CONCLUSIONS: We provide an evidence of successful phage therapy against carbapenem-resistant K. pneumoniae infected mouse model using locally isolated lytic phage.

Intestinal Carriage of Extended-Spectrum ß-Lactamase- (ESBL-) Possessing Escherichia coli and Klebsiella Species among Nepalese Health Science and Non-Health Science Students.

Infections due to extended-spectrum ß-lactamase- (ESBL-) producing Gram-negative bacteria have led to increased mortality, morbidity, and economic burden worldwide. These bacteria can colonize the healthy intestine of human beings and can disseminate in communities and hospital. This study aimed to investigate the prevalence of fecal carriage of ESBL-producing Escherichia coli and Klebsiella species among health science (HS) and non-health science (NHS) students. This descriptive cross-sectional study was conducted on 104 HS and 104 NHS students in which one stool sample from each student was collected and processed for bacterial culture and sensitivity testing according to standard bacteriological procedures. Each morphotype was identified and characterized phenotypically. The antimicrobial sensitivity profile of bacterial isolates was determined by the Kirby-Bauer disk diffusion technique. ESBL production was tested by combination disk method as recommended by the Clinical and Laboratory Standards Institute. Out of 208 stool samples, E. coli and Klebsiella spp. were recovered from 203 (86.8%) and 31 (13.2%) stool samples, respectively. Among those 234 isolates, 69 were positive for ESBL which included E. coli (n = 66, 95.7%) and Klebsiella spp. (n = 3, 4.3%). Fifty (42.4%) out of 118 isolates from HS students and 19 (16.4%) out of 116 from NHS students were colonized by ESBL-producers. Compared to non-ESBL producers, a higher number of ESBL-producing isolates were resistant to ciprofloxacin (14.5% vs. 1.8%, p < 0.001), cotrimoxazole (59.4% vs. 16.4%, p < 0.001), and amikacin (10.1% vs 4.2%, p < 0.001). All E. coli and Klebsiella species isolates were susceptible to meropenem. The prevalence of fecal carriage of ESBL-producing bacteria was higher in HS students; however, there was a considerable number of these strains colonizing NHS students as well. This "iceberg phenomenon" of asymptomatic carriage of ESBL-producing pathogens might act as a source of infection in both the community and hospitals. Therefore, surveillance of carriage of drug-resistant bacteria should be performed regularly.

Fungal Infection among Diabetic and Nondiabetic Individuals in Nepal.

BACKGROUND: Low immunity, comorbid clinical conditions, and metabolic disorders may be the underlying factors that determine the severity of infection. Diabetes increases the risk of infection and multiple organ damage. In Nepal, the actual burden of fungal infections has not been estimated or is in a limited progress. This study aimed to investigate the status of fungal infection in diabetic and nondiabetic individuals in Bhaktapur, Nepal. MATERIALS AND METHODS: A total of 670 samples were collected from 134 participants. From each participant, five samples were collected from different sites like an oral wash, toe swab, midstream urine, hair shaft, and nail scrapings. All samples were cultured on Sabouraud dextrose agar. Gram stain was used to observe yeast cells and lactophenol cotton blue stain was used for hyphae. Chlamydospore production by Candida species was observed in cornmeal agar medium by Dalmau Plate method. Candida species isolated were characterized by germ-tube test and differentiated using CHROM agar Candida medium. Candida species isolates were tested for antibiotic susceptibility. RESULTS: Overall, 19.4% of the samples showed fungal growth. The prevalence of fungal infection was higher in diabetic (34.0%) than nondiabetic individuals (4.7%). Fungal growth was found to be higher in oral wash followed by toe, urine, hair, and nail samples. Predominant fungi were Candida species (57.5%), Aspergillus species (28.4%), and Trichophyton species (10.7%). Oral wash, toe, and urine samples in diabetics had a significantly higher fungal prevalence when compared between both groups, p value < 0.05. In Candida isolates, higher resistance was seen against fluconazole 36.8% and ketoconazole 28.9%, whereas other drugs showed low resistance. CONCLUSION: Diabetic participants are more susceptible to fungal infection than the nondiabetics. Overall, Candida species and Aspergillus species are highly predominant fungi. Candida species are highly resistant to fluconazole and ketoconazole.

Complete Genome Sequence of Myophage Ec_Makalu_002, Which Infects Uropathogenic Escherichia coli.

We isolated phage Ec_Makalu_002, which infects uropathogenic strains of Escherichia coli Here, we report its complete genome sequence, annotated features, and relatedness to other phages.

Multidrug-Resistant Bacteria from Raw Meat of Buffalo and Chicken, Nepal.

Antimicrobial resistance is a major global issue for human and animals. Increased use of antimicrobials in livestock and poultry has become one of the causes of antimicrobial resistance development in microorganisms. The aim of the study was to characterize antimicrobial resistant bacteria from raw buffalo and chicken meat in standard in vitro condition. A total of 140 raw meat samples were collected from different retail shops of Bhaktapur Metropolitan City, Nepal. Among them, 70 were raw buffalo meat and 70 were raw chicken meat samples. Bacterial growth, identification, and antimicrobial susceptibility test were performed according to Clinical & Laboratory Standards Institute (CLSI) guidelines. Out of 140 samples, bacterial growth was seen in 67 raw buffalo meat and 59 raw chicken meat samples, i.e., bacterial growth was observed in 90.0% of the samples. A total of 161 bacterial isolates were detected. Escherichia coli (35.4%) and Klebsiella spp. (30.4%) were found to be the most prevalent bacteria followed by Citrobacter spp. (11.8%), Staphylococcus aureus (9.3%), Salmonella spp. (7.4%), and Proteus spp. (5.5%). Chicken meat isolates showed higher antimicrobial resistance rates in comparison to buffalo meat isolates, particularly against antimicrobials like Amoxicillin, Tetracycline, Cotrimoxazole and Nalidixic acid, p value<0.05 when compared between buffalo and chicken meat. Overall, 32.7% Multidrug-Resistant (MDR) isolates were found, in which 50.0% MDR isolates were found from chicken raw meat and 21.9% were found from buffalo raw meat. MDR isolates of Escherichia coli, Proteus spp. and Staphylococcus aureus constituted 52.5%, 77.7% and 40.0%, respectively, of both buffalo and chicken raw meat. This study indicates antimicrobials resistant bacteria existing at an alarming rate, higher in chicken meat than in buffalo meat.