Studies on the epidemiology of dry surface biofilms within healthcare has shown an almost universal distribution across frequently touched items.  Despite a growing body of evidence for dry surface biofilms in hospitals little attention has been paid to the recovery capacity of techniques used to detect these microbial communities. Biofilms are inherently difficult to remove from surfaces due to adhesive substances within their matrix and may act as sources of infection but to what extent is largely unknown. In this study we evaluate the recovery efficiencies of commonly used environmental swabs against dry surface biofilms containing 7.24-Log₁₀ Acinetobacter baumannii/cm2 using a drip flow reactor and desiccation cycle. Biofilm presence was visually confirmed using episcopic differential interference contrast microscopy combined with epifluorescence and quantified using sonicated viable plate counts. The swab materials used comprised of foam viscose and cotton all of which were pre-moistened using a buffer solution. The surfaces were vigorously swabbed by each material type and the resultant microbe populations for both swabs and remaining dry surface biofilms were quantified. Our results found foam tipped swabs to be superior detecting on average 30% of the original dry surface biofilm contamination; followed by viscose (6%) and cotton (3%). However no distinct difference was revealed in the concentration of microbes remaining on the surface after swabbing for each swab type suggesting there is variation in the capacity for each swab to release biofilm associated microorganisms. We conclude whilst environmental swabs do possess the ability to detect biofilms on dry surfaces; the reduced efficiencies are likely to cause an underestimation of the microbes present and should be considered during clinical application.

Removing the swab after collection can speed up diagnosis and improve the quality of laboratory procedures. This study investigates the impact of swab removal on SARS-CoV-2 detection in clinical specimens with a focus on high Cycle Threshold (Ct) samples (Cts ≥32). The method assessed pairs of SARS-CoV-2 samples mimicking combined throat and nose swabs and tested them on two real-time-PCR platforms; the Applied Biosystems 7500 and the Abbott Alinity. Swab removal did not significantly affect detection rates of SARS-CoV-2 samples with Ct values <32 regardless of the PCR platform. However reduced reproducibility was seen at the endpoint limit of detection of the platforms which meant that fewer samples with Ct values ≥32 were detected in the swab removal group. .

We investigated the resistance genes pilus islet biofilm formation ability and sequence types of multidrug-resistant Streptococcus pneumoniae (MDRSP) isolated from the healthy children below 5 years of age in Indonesia. In all 104 archived MDRSP isolates from previous carriage study in Indonesia in 2016-2019 were screened for the presence of antibiotic resistance genes and the rrgC (pilus islet 1) and pitB (pilus islet 2) genes. Multilocus sequence typing and biofilm formation was determined by PCR-Sequencing and the ability of cells to adhere to the walls respectively. Results have shown that the mefA ermB and tetM genes were found in 93% 52% and 100% of MDRSP isolates respectively. The insertion of arginine proline and Ile-100-Leu were the most common mutations in the folA and folP genes. Pilus islet 1 and type 2 were discovered in 93% and 82% of MDRSP isolates respectively. The MDRSP isolates showed no biofilm formation ability (64%) and five out of ten strains of MDRSP strains were ST1464. This finding can be used to provide further considerations in implementing and monitoring pneumococcal vaccination in Indonesia.

The Hollow-fibre Infection Model (HFIM) is a valuable in vitro platform for emulating antimicrobial drug (AMD) pharmacokinetic (PK) profiles. Despite its potential standardized protocols for HFIM operation especially concerning fastidious organisms are lacking. This study addresses this gap by examining challenges in culturing Pasteurella multocida and Actinobacillus pleuropneumoniae two fastidious organisms in the HFIM.

Our findings reveal effective strategies to prevent system clogging involving multiple freeze-thaw cycles of horse blood centrifugation and cell straining to enhance the clarity of the Mueller-Hinton fastidious (MH-F) medium defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI). Additionally we demonstrate that the provision of a CO2 atmosphere along with the utilization of gas-permeable tubing and gas vent filters significantly facilitates the growth of fastidious organisms. Remarkably both P. multocida and A. pleuropneumoniae were sustained for a period of up to 10 days under these optimized conditions.

This study provides crucial insights into the modifications necessary to successfully culture fastidious organisms in the HFIM paving the way for more accurate and representative in vitro models for antimicrobial drug testing. These advancements hold promise for advancing research in the field of antimicrobial pharmacokinetics and efficacy against challenging pathogens.

SStudies investigating environmental hotspots of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in Nigeria are limited. This study was designed to assess various environmental sources and commonly touched surfaces as potential carriers of ARB and ARGs with implications for public health. A total of 392 samples including sewage (36) sludge (36) diapers (20) plastics (20) water sachet polythene bags (20) food wastes (20) soil beneath dump sites (20) and frequently touched surfaces like restroom floors (80) corridors (24) door handles (56) and room floors and walls (60) were collected and screened for the presence of resistant bacteria carrying genes such as bla_KPC bla_NDM-1 bla_CMY-2 bla_IMP bla_OXA66 and MecA. Additionally we employed standard techniques to detect methicillin-resistant Staphylococcus aureus (MRSA) and extended-spectrum β-lactamase-producing Escherichia coli Klebsiella pneumoniae Pseudomonas aeruginosa and Acinetobacter baumannii. We also evaluated the effectiveness of routine disinfection procedures in eliminating ARB from restroom floors. Our findings revealed that sewage sludge diapers food wastes and restroom floors are frequently contaminated with highly and moderately resistant strains of E. coli K. pneumoniae P. aeruginosa and MRSA. Notably we identified two variants of the bla_OXA51-like gene (bla_OXA-66 & bla_OXA-180) in A. baumannii isolated from these environmental sources. Furthermore we detected seven ESBL-K. pneumoniae five ESBL-A. baumannii two ESBL-E. coli and one ESBL-P. aeruginosa all carrying one or more ARGs (bla_KPC bla_NDM-1 bla_CMY-2) in isolates recovered from sewage sludge restroom floors and plastics. It is worth noting that ARB persisted on restroom floors even after disinfection procedures. In conclusion this study highlights that environmental wastes indiscriminately discarded in residential areas and shared surfaces among individuals are heavily colonized by ARB carrying ARGs of significant public health importance.

Background Klebsiella pneumoniae is the second most frequently isolated bacterium in medical bacteriology laboratory after Escherichia coli. It can be responsible for many infections including urinary tract infections and pneumonia. The treatment of these infections is often prolonged because of the resistance of this bacterium to antibiotics. The production of aminoglycoside-modifying enzymes and the production of RNA methylases confer resistance to aminoglycosides. In Burkina Faso studies on bacterial resistance to aminoglycosides an antibiotic regularly prescribed and consumed by patients are limited hence the purpose of this study was to determine the prevalence of aminoglycoside resistance genes in K. pneumoniae isolated from urine and pus samples.

Methods and Findings A total of 150 Klebsiella strains from pus and urine cultures were collected from October 2018 to June 2019 in two hospitals in Ouagadougou and were included in this study. After plating on Muller Hinton (MH) medium to obtain pure colonies antibiotic susceptibility testing was performed with aminoglycosides β-lactams fluoroquinolones and sulfonamides. PCR testing for the phoE gene allowed genotypic identification of Klebsiella pneumoniae. PCRs were then performed on strains with at least one aminoglycoside resistance for identification of the aac(6')-Ib aac(3)-IIc and armA resistance genes.Antibiotic susceptibility testing showed that 38.67% (58/150) of the strains were resistant to Tobramycin 28.67% (43/150) to Gentamicin 10.00% (15/150) to Netilmicin 8.67% (13/150) to Kanamycin 4.67% (7/150) to Amikacin and 0.67% (1/150) to Neomycin. Of the 63 strains (42.66%) with at least one aminoglycoside resistance the resistance genes aac(3)-IIc and aac(6')-Ib were detected in 49 strains (77.77%) and 39 strains (61.90%) respectively; and 34 strains (53.97%) had both genes. Among the 16S rRNA genes the armA gene was present in 19.04% of the strains (12/63).

Conclusion Aminoglycoside resistance in K. pneumoniae strains in this study is primarily due to aac(3)-IIc and aac(6')-Ib acetyltransferases. Although armA RNA methylases genes are less frequent increased surveillance is necessary due to the high level of aminoglycoside resistance conferred by this gene.

To streamline the analysis and visualization of bacterial growth and gene expression data obtained by microtiter plate readers we developed BactEXTRACT an intuitive easy-to-use R-Shiny application. BactEXTRACT simplifies the transition from raw optical density fluorescence and luminescence measurements to publication-ready plots. This package offers a user-friendly interface that reduces the complexity involved in growth curve and gene expression analysis and is generally applicable. BactEXTRACT is available at https://veeninglab.shinyapps.io/BactEXTRACT/.

Vancomycin-resistant Enterococcus (VRE) is an increasingly identified cause of human disease with most infections resulting from the vanA and vanB genotypes; less is known about other clinically relevant genotypes. Here we report a genomic exploration of a vanD VRE faecium (VREfm) which arose de novo during a single infectious episode. The genomes of the vancomycin-susceptible E. faecium (VSEfm) recipient and resulting VREfm were subjected to long-read sequencing and closed with whole genome alignments and cross-mapping used to identified genomic variation. Three key differences were identified. (i) The VREfm chromosome gained a 142.6kb integrative conjugative element (ICE) harbouring the vanD locus. (ii) The native ligase (ddl) was disrupted by an ISEfm1 insertion. (iii) A large 1.74Mb chromosomal inversion of unknown consequence occurred. Alignment and phylogenetic-based comparisons of the VREfm with a global collection of vanD-harbouring genomes identified strong similarities in the 120-160kb genomic region surrounding vanD suggestive of a common mobile element and integration site irrespective of the diverse taxonomic geographic and host origins of the isolates. This isolate diversity revealed that this putative ICE (and its source) is globally disseminated and is capable of being acquired by different genera. Although the incidence of vanD VREfm is low understanding its emergence and potential for spread is crucial for the ongoing efforts to reduce antimicrobial resistance.

Invasive fungal infections (IFI) such as mucormycosis are causing devastating morbidity and mortality in immunocompromised patients as anti-fungal agents do not work in the setting of suppressed immune system. COVID-19 pandemic has created a novel landscape for IFI in post-pandemic patients which results from severe immune suppression caused by COVID-19 infection comorbidities (diabetes obesity) and immunosuppressive treatments such as steroids. The antigen-antibody interaction has been employed in radioimmunotherapy (RIT) to deliver lethal doses of ionizing radiation emitted by radionuclides to targeted cells and has demonstrated efficacy in several cancers. One of the advantages of RIT is its independence of immune status of a host which is crucial for immunosuppressed post-COVID-19 patients. In present work we targeted with RIT fungal pan-antigens 13-beta-glucan and melanin pigment which are present in the majority of pathogenic fungi thus making such targeting pathogen-agnostic. We demonstrated in experimental murine mucormycosis in immunocompetent and immunocompromised mice that 177Lutetium-labeled antibodies to these two antigens effectively decreased the fungal burden in major organs including the brain. These results are encouraging because they show the effectiveness of pathogen-agnostic RIT in significantly decreasing fungal burden in vivo while they can be also potentially applied to treat the broad range of invasive fungal infections which express pan-antigens 13-beta-glucan or melanin.

Gymnopus fusipes is an understudied root rot pathogen associated with multiple tree species and is linked to episodes of oak decline across the UK and Europe. The reported distribution of G. fusipes potentially spans most of the northern hemisphere however many of these observations are based on visual identification of fruiting bodies only which might not always be correct and there is a lack of confirmatory molecular and/or isolation data to validate this ecological range. Given the paucity of information regarding the true ecological distribution of G. fusipes it is difficult to predict and model the potential distribution of the species under both current and future climate scenarios. In this study to determine the growth capabilities of G. fusipes across a range of ecologically relevant temperatures five geographically diverse isolates of G. fusipes were grown at five different temperatures (ranging from 4˚C to 37˚C) in order to determine the optimal incubation temperature for G. fusipes growth and to establish whether geographically diverse isolates showed any signs of local adaptation to temperature tolerance by having different growth rates at different temperatures. G. fusipes isolates grew with varying success at 4˚C 10˚C 20˚C and 25˚C with minimal growth observed with incubation at 37˚C. Statistical analysis and a generalised linear mixed-effects model fitted to the growth data illustrated that incubation temperature had a significant effect on G. fusipes growth rate with 25˚C found to be the optimum (P < 0.001). Isolates were found to have different growth rates at each of the temperatures with a UK isolate (originating from the south of England) having the highest overall growth rate across all five temperatures tested (P < 0.001) increasing by a mean value of over 4915 mm2 over the 28-day experimental period (at the optimal growth temperature of 25˚C). Local adaptation to temperature was not found in the isolates tested as there was no significant interaction between temperature and isolate. These data demonstrate the optimal incubation temperature for future laboratory studies on G. fusipes and provide the first data on the growth rate of this pathogen across ecologically relevant climate ranges that may inform land managers modellers and policy makers in predicting the current and potentially future geographical limits of this widespread root rot pathogen.