Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which the sources of transmission may be challenging to elucidate. We describe the use of whole-genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-β-lactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read sequencing (Illumina) and long-read sequencing (MinION; Oxford Nanopore Technologies) were used to confirm species taxonomy, to identify antimicrobial resistance genes, and to determine phylogenetic relationships using single-nucleotide polymorphism profiling. A total of 21 organisms (10 patient-derived isolates and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from multiple detergent-dispensing bottles were either identical or closely related by single-nucleotide polymorphism comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk expression equipment. No new cases were identified once the detergent bottles were removed. Environmental reservoirs may be an important source in outbreaks of multidrug-resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses.
In 1896, a serendipitous laboratory accident led to the understanding that hookworms propagate infection by penetrating skin, a theory that was then confirmed with the first experimental human infection, reported in 1901. Experimental human infections undertaken in the 20th century enabled understanding of the natural history of infection and the immune response. More recently, experimental hookworm infection has been performed to investigate the immunomodulatory potential of hookworm infection and for the evaluation of hookworm vaccines and chemotherapeutic interventions. Experimental human hookworm infection has been proven to be safe, with no deaths observed in over 500 participants (although early reports predate systematic adverse event reporting) and no serious adverse events described in over 200 participants enrolled in contemporary clinical trials. While experimental human hookworm infection holds significant promise, as both a challenge model for testing anti-hookworm therapies and for treating various diseases of modernity, there are many challenges that present. These challenges include preparation and storage of larvae, which has not significantly changed since Harada and Mori first described their coproculture method in 1955. In vitro methods of hookworm larval culture, storage, and the development of meaningful potency or release assays are required. Surrogate markers of intestinal infection intensity are required because faecal egg counts or hookworm faecal DNA intensity lack the fidelity required for exploration of hookworm infection as a vaccine/drug testing platform or as a regulated therapy.
Prosthetic Joint Infection (PJI) causes significant morbidity and mortality for patients globally. Delivery of antibiotics to the site of infection has potential to improve the treatment outcomes and enhance biofilm eradication. These antibiotics can be delivered using an intra-articular catheter or combined with a carrier substance to enhance pharmacokinetic properties. Carrier options include non-resorbable polymethylmethacrylate (PMMA) bone cement and resorbable calcium sulphate, hydroxyapatite, bioactive glass, and hydrogels. PMMA allows for creation of structural spacers used in multi-stage revision procedures, however it requires subsequent removal and antibiotic compatibility and the levels delivered are variable. Calcium sulphate is the most researched resorbable carrier in PJI, but is associated with wound leakage and hypercalcaemia, and clinical evidence for its effectiveness remains at the early stage. Hydrogels provide a versatile combability with antibiotics and adjustable elution profiles, but clinical usage is currently limited. Novel anti-biofilm therapies include bacteriophages which have been used successfully in small case series.
We report 3 cases of koala bite wound infection with Lonepinella koalarum–like bacteria requiring antimicrobial and surgical management. The pathogens could not be identified by standard tests. Phylogenetic analysis of 16S rRNA and housekeeping genes identified the genus. Clinicians should isolate bacteria and determine antimicrobial susceptibilities when managing these infections.
Key points:• K. michiganensis can be misidentified as K. oxytoca and is probably underrecognized as a nosocomial pathogen• Whole genome sequencing of neonatal and environmental isolates during an outbreak of ESBL-producing K. michiganensis confirmed contaminated detergent and sinks to be the source Abstract Background: Klebsiella species are problematic pathogens in neonatal units and may cause outbreaks, for which sources of transmission can be challenging to elucidate. We describe the use of whole genome sequencing (WGS) to investigate environmental sources of transmission during an outbreak of extended-spectrum-βlactamase (ESBL)-producing Klebsiella michiganensis colonizing neonates. Methods: Ceftriaxone-resistant Klebsiella spp. isolated from neonates (or their mothers) and the hospital environment were included. Short-read (Illumina) and longread (MinION, Oxford Nanopore Technologies) sequencing was used to confirm species taxonomy, define antimicrobial resistance genes and determine phylogenetic relationships using single nucleotide polymorphism (SNP) profiling. Results: A total of 21 organisms (10 patient-derived and 11 environmental isolates) were sequenced. Standard laboratory methods identified the outbreak strain as an ESBL-producing Klebsiella oxytoca, but taxonomic assignment from WGS data suggested closer identity to Klebsiella michiganensis. Strains isolated from baby bath drains and multiple detergent dispensing bottles were either identical or closely related by SNP comparison. Detergent bottles contaminated by K. michiganensis had been used for washing milk-expressing equipment. No new cases were identified once the detergent bottles were removed and the baby baths decommissioned. Conclusions: Environmental reservoirs may be an important source in outbreaks of multi-drug resistant organisms. WGS, in conjunction with traditional epidemiological investigation, can be instrumental in revealing routes of transmission and guiding infection control responses. (214 words)
Paramecium bursaria Chlorella virus-1 is an icosahedrally shaped, 1,900-Å-diameter virus that infects unicellular eukaryotic green algae. A 5-fold symmetric, 3D reconstruction using cryoelectron microscopy images has now shown that the quasiicosahedral virus has a unique vertex, with a pocket on the inside and a spike structure on the outside of the capsid. The pocket might contain enzymes for use in the initial stages of infection. The unique vertex consists of virally coded proteins, some of which have been identified. Comparison of shape, size, and location of the spike with similar features in bacteriophages T4 and P22 suggests that the spike might be a cell-puncturing device. Similar asymmetric features may have been missed in previous analyses of many other viruses that had been assumed to be perfectly icosahedral.5-fold averaging ͉ cryoelectron microscopy reconstruction ͉ Paramecium bursaria Chlorella virus-1 ͉ cell entry ͉ specialized vertex protein
Background Although there is unprecedented interest in experimental human hookworm infection, details of hookworm manufacture and characterisation have been sparsely reported. In this report, we detail the production and characterisation of Necator americanus larvae for use in a recently published clinical trial. Methods Faeces was obtained from an experimentally infected donor. Faecal hookworm DNA was determined by quantitative PCR. Paired samples were incubated in either sterile water or sterile water mixed with antimicrobials (amphotericin and gentamicin). Coproculture was performed by modified Harada-Mori method. The harvested larvae were then processed in either sterile water or antiseptic solution. Larval yield was then calculated (larvae per gram), larval viability was determined by thermally induced motility assay and microbial burden was determined at the day of harvest, at 48 h and at 7 days. Results Twenty-eight faecal cultures were performed over 16 months. The faecal hookworm DNA content was variable over this time. There was no association of larval yield with faecal hookworm DNA content. Pre-treatment of faeces with antimicrobials did not influence larval yield. Larval motility was 85.3% (95% CI 79.3–91.3%). Incubation of larvae in antiseptics did not reduce viability at 14 days with a marginal mean of 68.6% (95% CI 59.1–78.1%) washed in water vs. 63.3% (95% CI 53.8 – 72.9%) when incubated in betadine (p = 0.38). Larvae washed in sterile water did not meet microbial bioburden criteria. Incubation in antiseptic resulted in acceptable microbial bioburden at 48 h but not at 7 days. Although the addition of gentamicin did reduce the microbial bio-burden acceptable levels, it was found to significantly lower larval motility at 7 days compared to incubation in sterile water and motility at 7 days 37.8% (95% CI 4.7–70.9%) vs. 67.3% (95% CI 35.2–99.3%, p < 0.001), respectively. Conclusions Despite standardised culture methodologies and the use of a single donor, larval yield varied considerably between batches and had no association with faecal hookworm DNA. Larval viability decreases over time and the age of larvae at time of use are likely to be important. Microbial bioburden maybe temporarily reduced by incubation in antiseptics and has little effect on viability. Incubation of larvae in gentamicin is effective at reducing microbial bioburden but is deleterious to larval viability. Graphical Abstract
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