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We characterized two bacteriophages, ΦFG02 and ΦCO01, against clinical isolates of Acinetobacter baumannii and established that the bacterial capsule is the receptor for these phages. Phage-resistant mutants harboured loss-of-function mutations in genes responsible for capsule biosynthesis, resulting in capsule loss and disruption of phage adsorption. The phage-resistant strains were resensitized to human complement, beta-lactam antibiotics and alternative phages and exhibited diminished fitness in vivo. Using a mouse model of A. baumannii infection, we showed that phage therapy was effective. The authors characterize two phages against Acinetobacter baumannii , both in vitro and in vivo. A. baumannii strains develop resistance against both phages, but are in turn resensitized to different antimicrobial compounds.

The large and complex genome of Pseudomonas aeruginosa , which consists of significant portions (up to 20%) of transferable genetic elements contributes to the rapid development of antibiotic resistance. The whole genome sequences of 22 strains isolated from eye and cystic fibrosis patients in Australia and India between 1992 and 2007 were used to compare genomic divergence and phylogenetic relationships as well as genes for antibiotic resistance and virulence factors. Analysis of the pangenome indicated a large variation in the size of accessory genome amongst 22 stains and the size of the accessory genome correlated with number of genomic islands, insertion sequences and prophages. The strains were diverse in terms of sequence type and dissimilar to that of global epidemic P . aeruginosa clones. Of the eye isolates, 62% clustered together within a single lineage. Indian eye isolates possessed genes associated with resistance to aminoglycoside, beta-lactams, sulphonamide, quaternary ammonium compounds, tetracycline, trimethoprims and chloramphenicols. These genes were, however, absent in Australian isolates regardless of source. Overall, our results provide valuable information for understanding the genomic diversity of P . aeruginosa isolated from two different infection types and countries.

To determine whether Acanthamoeba keratitis (AK) patients have higher rates of Acanthamoeba and free-living amoeba (FLA) colonising domestic sinks than control contact lens (CL) wearers, and whether these isolates are genetically similar to the corneal isolates from their CL associated AK. 129 AK patients from Moorefield Eye Hospital, London and 64 control CL wearers from the Institute of Optometry were included in this study. The participants self-collected home kitchen and bathroom samples from tap-spouts, overflows and drains using an instructional kit. The samples were cultured by inoculating onto a non-nutrient agar plate seeded with Escherichia coli, incubated at 32°C and examined for amoebae by microscopy for up to 2 weeks. Partial sequences of mitochondrial cytochrome oxidase genes (coxA) of Acanthamoeba isolates from four AK patients were compared to Acanthamoeba isolated from the patient's home. The association between sampling sites was analysed with the chi-square test. A total of 513 samples from AK patients and 189 from CL controls were collected. The yield of FLA was significantly greater in patients' bathrooms (72.1%) than CL controls' bathrooms (53.4%) (p<0.05). Spouts (kitchen 6.7%, bathroom 11%) had the lowest rate of Acanthamoeba isolation compared to drains (kitchen 18.2%, bathroom 27.9%) and overflow (kitchen 39.1%, bathroom 25.9%) either in kitchens or bathrooms (p<0.05). There was no statistically significant difference between the average prevalence of Acanthamoeba in all three sample sites in kitchens (16.9%) compared to all three sample sites in bathrooms (21.5%) and no association for Acanthamoeba prevalence between AK patients and CL controls. All four corneal isolates had the same coxA sequence as at least one domestic water isolate from the patients' sink of the kitchen and the bathroom. The prevalence of Acanthamoeba and FLA was high in UK homes. FLA colonisation was higher in AK patients compared to controls but the prevalence of Acanthamoeba between AK patients and CL controls domestic sinks was similar. This study confirms that domestic water isolates are probably the source of AK infection. Advice about avoiding water contact when using CL's should be mandatory.

Virulent strains of Pseudomonas aeruginosa are often associated with an acquired cytotoxic protein, exoenzyme U (ExoU) that rapidly destroys the cell membranes of host cells by its phospholipase activity. Strains possessing the exoU gene are predominant in eye infections and are more resistant to antibiotics. Thus, it is essential to understand treatment options for these strains. Here, we have investigated the resistance profiles and genes associated with resistance for fluoroquinolone and beta-lactams. A total of 22 strains of P. aeruginosa from anterior eye infections, microbial keratitis (MK), and the lungs of cystic fibrosis (CF) patients were used. Based on whole genome sequencing, the prevalence of the exoU gene was 61.5% in MK isolates whereas none of the CF isolates possessed this gene. Overall, higher antibiotic resistance was observed in the isolates possessing exoU. Of the exoU strains, all except one were resistant to fluoroquinolones, 100% were resistant to beta-lactams. 75% had mutations in quinolone resistance determining regions (T81I gyrA and/or S87L parC) which correlated with fluoroquinolone resistance. In addition, exoU strains had mutations at K76Q, A110T, and V126E in ampC, Q155I and V356I in ampR and E114A, G283E, and M288R in mexR genes that are associated with higher beta-lactamase and efflux pump activities. In contrast, such mutations were not observed in the strains lacking exoU. The expression of the ampC gene increased by up to nine-fold in all eight exoU strains and the ampR was upregulated in seven exoU strains compared to PAO1. The expression of mexR gene was 1.4 to 3.6 fold lower in 75% of exoU strains. This study highlights the association between virulence traits and antibiotic resistance in pathogenic P. aeruginosa.

Dengue is a mosquito-borne viral infection. Since the first reported incidence in 2004, several sporadic outbreaks of dengue have been recorded from both tropical and subtropical regions of Nepal, including the capital city Kathmandu. However, in the last 5 years, the incidence of dengue cases has risen alarmingly. The largest-ever outbreak was reported in 2019, which killed six people. The global warming, unplanned urbanization, increased transportation, and lack of efficient mosquito control are presumably associated with the spread of dengue and its vector to the plane and hilly regions of this country. With the ongoing Nepalese government campaign “Visit Nepal Year 2020” to attract two million tourists in mind, effective dengue control measures must be implemented to control potential future outbreaks.

Pseudomonas aeruginosa (P. aeruginosa) is a common antibiotic-resistant pathogen, posing significant public health threats worldwide. It is a major cause of ocular infections, mostly linked to contact lens wear. P. aeruginosa often produces biofilm during infections, and these are also associated with antibiotic resistance. Bacteriophage (phage) therapy is emerging as a promising approach for treating multidrug-resistant P. aeruginosa. Objective: This study aimed to assess the antibiofilm effects of six phages against P. aeruginosa biofilms isolated from patients with corneal infections. Method: This study examined P. aeruginosa strains for their ability to form biofilms using crystal violet assay. Six P. aeruginosa bacteriophages (DiSu1 to DiSu6) were used, which were isolated from sewage water in Melbourne, Australia. Spot tests were used to assess phage sensitivity. The effect of phages against P. aeruginosa strains was determined using time–kill assay and efficiency of plating. The ability of phage to inhibit biofilm formation over 24 h or reduce preformed biofilms was also studied and confirmed using confocal laser scanning microscopy with Live/Dead staining. Result: After 24 h of incubation, all tested P. aeruginosa strains formed moderate to strong biofilms. All P. aeruginosa strains were sensitive to at least four of the six phages. The highest level of bacterial growth inhibition in the liquid infection model was observed when phages were applied at a multiplicity of infection (MOI) of 100. Certain bacteria/phage combinations were able to inhibit biofilm formation over 24 h, with the combination of strain PA235 and phage DiSu3 producing the highest inhibition (83%) at a MOI of 100. This was followed by the combinations of PA223/DiSu3 (56%), and PA225/DiSu5 (52%). For the reduction in preformed biofilms, the best combinations were PA235 (90%), PA221 (61%), and PA213 and PA225 (57% each), all with DiSu3 after 3 h. However, exposing the biofilm with phages for over 24 h appeared to promote phage resistance as there was evidence of biofilm growth, with the only combination still showing a significant reduction being PA221/DiSu3 (58%) at MOI of 100. Conclusions: This study showed that the effect of phages against P. aeruginosa is concentration (MOI) dependent. Phages at higher MOI have the ability to disrupt, inhibit, and reduce P. aeruginosa biofilms. However, prolonged exposure of the biofilm with phages appeared to promote phage resistance. To enhance phage efficacy and address this form of resistance, further studies utilizing phage cocktails or a combination of phages and antibiotics is warranted.

Introduction Acanthamoeba is an emerging pathogen, infamous for its resilience against antiprotozoal compounds, disinfectants and harsh environments. It is known to cause keratitis, a sight-threatening, painful and difficult to treat corneal infection which is often reported among contact lens wearers and patients with ocular trauma. Acanthamoeba comprises over 24 species and currently 23 genotypes (T1-T23) have been identified. Aims This retrospective study was designed to examine the Acanthamoeba species and genotypes recovered from patients with Acanthamoeba keratitis (AK), determine the presence of endosymbionts in ocular isolates of Acanthamoeba and review the clinical presentations. Methodology Thirteen culture-confirmed AK patients treated in a tertiary eye care facility in Hyderabad, India from February to October 2020 were included in this study. The clinical manifestations, medications and visual outcomes of all patients were obtained from medical records. The Acanthamoeba isolates were identified by sequencing the ribosomal nuclear subunit ( rns ) gene. Acanthamoeba isolates were assessed for the presence of bacterial or fungal endosymbionts using molecular assays, PCR and fluorescence in situ hybridization (FISH). Results The mean age of the patients was 33 years (SD ± 17.4; 95% CI 22.5 to 43.5 years). Six (46.2%) cases had AK associated risk factors; four patients had ocular trauma and two were contact lens wearers. A. culbertsoni (6/13, 46.2%) was the most common species, followed by A. polyphaga and A. triangularis . Most of the isolates (12/13) belonged to genotype T4 and one was a T12; three sub-clusters T4A, T4B, and T4F were identified within the T4 genotype. There was no significant association between Acanthamoeba types and clinical outcomes. Eight (61.5%) isolates harboured intracellular bacteria and one contained Malassezia restricta . The presence of intracellular microbes was associated with a higher proportion of stromal infiltrates (88.9%, 8/9), epithelial defect (55.6%, 5/9) and hypopyon (55.6%, 5/9) compared to 50% (2/4), 25% (1/4) and 25% (1/4) AK cases without intracellular microbes, respectively. Conclusions Genotype T4 was the predominant isolate in southern India. This is the second report of T12 genotype identified from AK patient in India, which is rarely reported worldwide. The majority of the Acanthamoeba clinical isolates in this study harboured intracellular microbes, which may impact clinical characteristics of AK.

T-series bacteriophages have been used throughout the world for various molecular biology researches, which were critical for establishing the fundamentals of molecular biology, from the structure of DNA to advanced gene-editing tools. These model bacteriophages help keep research data consistent and comparable between laboratories. T-series phages have been model organisms for molecular biology since the 1940s. Given that these phages have been stocked, distributed, and propagated for decades across the globe, there exists the potential for genetic drift to accumulate between stocks over time. Here, we compared the temporal stability and genetic relatedness of laboratory-maintained phage stocks with a T-series collection from 1972. Only the T-even phages produced viable virions. We obtained complete genomes of these T-even phages, along with two contemporary T4 stocks. Performing comparative genomics, we found 12 and 16 nucleotide variations, respectively, in the genomes of T2 and T6, whereas there were ∼172 nucleotide variations between T4 sublines compared with the NCBI RefSeq genome. To account for the possibility of artifacts in NCBI RefSeq, we used the 1972 T4 stock as a reference and compared genetic and phenotypic variations between T4 sublines. Genomic analysis predicted nucleotide variations in genes associated with DNA metabolism and structural proteins. We did not, however, observe any differences in growth characteristics or host range between the T4 sublines. Our study highlights the potential for genetic drift between individually maintained T-series phage stocks, yet after 48 years, this has not resulted in phenotypic alterations in these important model organisms. IMPORTANCE T-series bacteriophages have been used throughout the world for various molecular biology researches, which were critical for establishing the fundamentals of molecular biology, from the structure of DNA to advanced gene-editing tools. These model bacteriophages help keep research data consistent and comparable between laboratories. However, we observed genetic variability when we compared contemporary sublines of T4 phages to a 48-year-old stock of T4. This may have effects on the comparability of results obtained using T4 phage. Here, we highlight the genomic differences between T4 sublines and examined phenotypic differences in phage replication parameters. We observed limited genomic changes but no phenotypic variations between T4 sublines. Our research highlights the possibility of genetic drift in model bacteriophages.

Acanthamoeba, an opportunistic pathogen is known to cause an infection of the cornea, central nervous system, and skin. Acanthamoeba feeds different microorganisms, including potentially pathogenic prokaryotes; some of microbes have developed ways of surviving intracellularly and this may mean that Acanthamoeba acts as incubator of important pathogens. A systematic review of the literature was performed in order to capture a comprehensive picture of the variety of microbial species identified within Acanthamoeba following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. Forty-three studies met the inclusion criteria, 26 studies (60.5%) examined environmental samples, eight (18.6%) studies examined clinical specimens, and another nine (20.9%) studies analysed both types of samples. Polymerase chain reaction (PCR) followed by gene sequencing was the most common technique used to identify the intracellular microorganisms. Important pathogenic bacteria, such as E. coli, Mycobacterium spp. and P. aeruginosa, were observed in clinical isolates of Acanthamoeba, whereas Legionella, adenovirus, mimivirus, and unidentified bacteria (Candidatus) were often identified in environmental Acanthamoeba. Increasing resistance of Acanthamoeba associated intracellular pathogens to antimicrobials is an increased risk to public health. Molecular-based future studies are needed in order to assess the microbiome residing in Acanthamoeba, as a research on the hypotheses that intracellular microbes can affect the pathogenicity of Acanthamoeba infections.

Bacteria can acquire an accessory genome through the horizontal transfer of genetic elements from non-parental lineages. This leads to rapid genetic evolution allowing traits such as antibiotic resistance and virulence to spread through bacterial communities. The study of complete genomes of bacterial strains helps to understand the genomic traits associated with virulence and antibiotic resistance. We aimed to investigate the complete accessory genome of an ocular isolate of Pseudomonas aeruginosa strain PA34. We obtained the complete genome of PA34 utilising genome sequence reads from Illumina and Oxford Nanopore Technology followed by PCR to close any identified gaps. In-depth genomic analysis was performed using various bioinformatics tools. The susceptibility to heavy metals and cytotoxicity was determined to confirm expression of certain traits. The complete genome of PA34 includes a chromosome of 6.8 Mbp and two plasmids of 95.4 Kbp (pMKPA34-1) and 26.8 Kbp (pMKPA34-2). PA34 had a large accessory genome of 1,213 genes and had 543 unique genes not present in other strains. These exclusive genes encoded features related to metal and antibiotic resistance, phage integrase and transposons. At least 24 genomic islands (GIs) were predicated in the complete chromosome, of which two were integrated into novel sites. Eleven GIs carried virulence factors or replaced pathogenic genes. A bacteriophage carried the aminoglycoside resistance gene (AAC(3)-IId). The two plasmids carried other six antibiotic resistance genes. The large accessory genome of this ocular isolate plays a large role in shaping its virulence and antibiotic resistance.