Cysteine and resistance to oxidative stress: implications for virulence and antibiotic resistance.

Reactive oxygen species (ROS), including the superoxide radical anion (O2•-), hydrogen peroxide (H2O2), and the hydroxyl radical (•HO), are inherent components of bacterial metabolism in an aerobic environment. Bacteria also encounter exogenous ROS, such as those produced by the host cells during the respiratory burst. As ROS have the capacity to damage bacterial DNA, proteins, and lipids, detoxification of ROS is critical for bacterial survival. It has been recently recognised that low-molecular-weight (LMW) thiols play a central role in this process. Here, we review the emerging role of cysteine in bacterial resistance to ROS with a link to broader elements of bacterial lifestyle closely associated with cysteine-mediated oxidative stress response, including virulence and antibiotic resistance.

Staphylococcus aureus surface attachment selectively influences tolerance against charged antibiotics.

The threat of infection during implant placement surgery remains a considerable burden for millions of patients worldwide. To combat this threat, clinicians employ a range of anti-infective strategies and practices. One of the most common interventions is the use of prophylactic antibiotic treatment during implant placement surgery. However, these practices can be detrimental by promoting the resilience of biofilm-forming bacteria and enabling them to persist throughout treatment and re-emerge later, causing a life-threatening infection. Thus, it is of the utmost importance to elucidate the events occurring during the initial stages of bacterial surface attachment and determine whether any biological processes may be targeted to improve surgical outcomes. Using gene expression analysis, we identified a cellular mechanism of S. aureus which modifies its cell surface charge following attachment to a medical grade titanium surface. We determined the upregulation of two systems involved in the d-alanylation of teichoic acids and the lysylation of phosphatidylglycerol. We supported these molecular findings by utilizing synchrotron-sourced attenuated total reflection Fourier-transform infrared microspectroscopy to analyze the biomolecular properties of the S. aureus cell surface following attachment. As a direct consequence, S. aureus quickly becomes substantially more tolerant to the positively charged vancomycin, but not the negatively charged cefazolin. The present study can assist clinicians in rationally selecting the most potent antibiotic in prophylaxis treatments. Furthermore, it highlights a cellular process that could potentially be targeted by novel technologies and strategies to improve the outcome of antibiotic prophylaxis during implant placement surgery. STATEMENT OF SIGNIFICANCE: The antibiotic tolerance of bacteria in biofilm is a well-established phenomenon. However, the physiological adaptations employed by Staphylococcus aureus to increase its antibiotic tolerance during the early stages of surface attachment are poorly understood. Using multiple techniques, including gene expression analysis and synchrotron-sourced Fourier-transform infrared microspectroscopy, we generated insights into the physiological response of S. aureus following attachment to a medical grade titanium surface. We showed that this phenotypic transition enables S. aureus to better tolerate the positively charged vancomycin, but not the negatively charged cefazolin. These findings shed light on the antibiotic tolerance mechanisms employed by S. aureus to survive prophylactically administered antibiotics and can help clinicians to protect patients from infections.

Correction: Messele et al. Longitudinal Analysis of Antimicrobial Resistance among Enterococcus Species Isolated from Australian Beef Cattle Faeces at Feedlot Entry and Exit. Animals 2022, 12, 2690.

There was an error in the original publication [...].

Phylogeny, Virulence, and Antimicrobial Resistance Gene Profiles of Enterococcus faecium Isolated from Australian Feedlot Cattle and Their Significance to Public and Environmental Health.

The extent of similarity between E. faecium strains found in healthy feedlot beef cattle and those causing extraintestinal infections in humans is not yet fully understood. This study used whole-genome sequencing to analyse the antimicrobial resistance profile of E. faecium isolated from beef cattle (n = 59) at a single feedlot and compared them to previously reported Australian isolates obtained from pig (n = 60) and meat chicken caecal samples (n = 8), as well as human sepsis cases (n = 302). The E. faecium isolated from beef cattle and other food animal sources neither carried vanA/vanB responsible for vancomycin nor possessed gyrA/parC and liaR/liaS gene mutations associated with high-level fluoroquinolone and daptomycin resistance, respectively. A small proportion (7.6%) of human isolates clustered with beef cattle and pig isolates, including a few isolates belonging to the same sequence types ST22 (one beef cattle, one pig, and two human isolates), ST32 (eight beef cattle and one human isolate), and ST327 (two beef cattle and one human isolate), suggesting common origins. This provides further evidence that these clonal lineages may have broader host range but are unrelated to the typical hospital-adapted human strains belonging to clonal complex 17, significant proportions of which contain vanA/vanB and liaR/liaS. Additionally, none of the human isolates belonging to these STs contained resistance genes to WHO critically important antimicrobials. The results confirm that most E. faecium isolated from beef cattle in this study do not pose a significant risk for resistance to critically important antimicrobials and are not associated with current human septic infections.

The bacteriology of diabetic foot ulcers and infections and incidence of Staphylococcus aureus Small Colony Variants.

Introduction. Uninfected diabetes-related foot ulcer (DFU) progression to diabetes-related foot infection (DFI) is a prevalent complication for patients with diabetes. DFI often progresses to osteomyelitis (DFI-OM). Active (growing) Staphylococcus aureus is the most common pathogen in these infections. There is relapse in 40-60 % of cases even when the initial treatment at the DFI stage apparently clears infection.Hypothesis. S. aureus adopts the quasi-dormant Small Colony Variant (SCV) state during DFU and consequently infection, and when present in DFI cases also permits survival in non-diseased tissues as a reservoir to cause relapse.Aim. The aim of this study was to investigate the bacterial factors that facilitate persistent infections.Methodology. People with diabetes were recruited from two tertiary hospitals. Clinical and bacterial data was taken from 153 patients with diabetes (51 from a control group with no ulcer or infection) and samples taken from 102 patients with foot complications to identify bacterial species and their variant colony types, and then compare the bacterial composition in those with uninfected DFU, DFI and those with DFI-OM, of whom samples were taken both from wounds (DFI-OM/W) and bone (DFI-OM/B). Intracellular, extracellular and proximal 'healthy' bone were examined.Results. S. aureus was identified as the most prevalent pathogen in diabetes-related foot pathologies (25 % of all samples). For patients where disease progressed from DFU to DFI-OM, S. aureus was isolated as a diversity of colony types, with increasing numbers of SCVs present. Intracellular (bone) SCVs were found, and even within uninfected bone SCVs were present. Wounds of 24 % of patients with uninfected DFU contained active S. aureus. All patients with a DFI with a wound but not bone infection had previously had S. aureus isolated from an infection (including amputation), representing a relapse.Conclusion. The presence of S. aureus SCVs in recalcitrant pathologies highlights their importance in persistent infections through the colonization of reservoirs, such as bone. The survival of these cells in intracellular bone is an important clinical finding supporting in vitro data. Also, there seems to be a link between the genetics of S. aureus found in deeper infections compared to those only found in DFU.

The influence of biochar position in a leach bed system anaerobically digesting chicken litter.

As a consequence of the rapidly growing poultry industry, chicken litter is becoming an abundant and problematic waste. Anaerobic digestion of chicken litter can mitigate environmental issues while producing valuable by-products. Recent studies have shown that leach bed reactor (LBR) systems are suitable for processing chicken litter and that anaerobic digestion can be enhanced using biochar. This study investigates the influence of biochar position within an LBR system on anaerobic digestion of chicken litter. Compared to a system without biochar, application of biochar in both the LBR (mixed in with the feedstock or as a layer below the feedstock) and coupled leachate tank (LT) increased methane yield by 6 to 8% at 51 days and accelerated VFA degradation and methane production. More significant differences in methane yield were observed at shorter solid retention times. Biochar mixed in feedstock in addition to a filter in the LT performed best in terms of both methane and hydrogen sulfide production, with a 77% reduction in hydrogen sulfide yield and hydrogen sulfide contents maintained below 500 ppm. The enhanced rates of VFA degradation and methane production when applying biochar in both reactors corresponds with observed differences in the methanogen population. Biochar application in both reactors increased the abundance of Methanobacteriales in digestate and Methanosarcinaceae in leachate compared to the control. Microbial attachment and activity on biochar also increased when mixed in feedstock. Increased diversity of the methanogen population throughout the system, as well as increased activity on biochar, may have facilitated the syntrophic relationship between acetogenic bacteria and methanogens, thus accelerating VFA degradation and methane production. These results suggest mixing biochar in feedstock, in addition to a biochar filter in the LT, to enhance anaerobic digestion of chicken litter in this system.

Moving microcapillary antibiotic susceptibility testing (mcAST) towards the clinic: unravelling kinetics of detection of uropathogenic E. coli, mass-manufacturing and usability for detection of urinary tract infections in human urine.

Innovation in infection based point-of-care (PoC) diagnostics is vital to avoid unnecessary use of antibiotics and the development of antimicrobial resistance. Several groups including our research team have in recent years successfully miniaturised phenotypic antibiotic susceptibility tests (AST) of isolated bacterial strains, providing validation that miniaturised AST can match conventional microbiological methods. Some studies have also shown the feasibility of direct testing (without isolation or purification), specifically for urinary tract infections, paving the way for direct microfluidic AST systems at PoC. As rate of bacteria growth is intrinsically linked to the temperature of incubation, transferring miniaturised AST nearer the patient requires building new capabilities in terms of temperature control at PoC, furthermore widespread clinical use will require mass-manufacturing of microfluidic test strips and direct testing of urine samples. This study shows for the first-time application of microcapillary antibiotic susceptibility testing (mcAST) directly from clinical samples, using minimal equipment and simple liquid handling, and with kinetics of growth recorded using a smartphone camera. A complete PoC-mcAST system was presented and tested using 12 clinical samples sent to a clinical laboratory for microbiological analysis. The test showed 100% accuracy for determining bacteria in urine above the clinical threshold (5 out of 12 positive) and achieved 95% categorical agreement for 5 positive urines tested with 4 antibiotics (nitrofurantoin, ciprofloxacin, trimethoprim and cephalexin) within 6 h compared to the reference standard overnight AST method. A kinetic model is presented for metabolization of resazurin, demonstrating kinetics of degradation of resazurin in microcapillaries follow those observed for a microtiter plate, with time for AST dependent on the initial CFU ml-1 of uropathogenic bacteria in the urine sample. In addition, we show for the first time that use of air-drying for mass-manufacturing and deposition of AST reagents within the inner surface of mcAST strips matches results obtained with standard AST methods. These results take mcAST a step closer to clinical application, for example as PoC support for antibiotic prescription decisions within a day.

Phylogenetic Analysis of Escherichia coli Isolated from Australian Feedlot Cattle in Comparison to Pig Faecal and Poultry/Human Extraintestinal Isolates.

The similarity of commensal Escherichia coli isolated from healthy cattle to antimicrobial-resistant bacteria causing extraintestinal infections in humans is not fully understood. In this study, we used a bioinformatics approach based on whole genome sequencing data to determine the genetic characteristics and phylogenetic relationships among faecal Escherichia coli isolates from beef cattle (n = 37) from a single feedlot in comparison to previously analysed pig faecal (n = 45), poultry extraintestinal (n = 19), and human extraintestinal E. coli isolates (n = 40) from three previous Australian studies. Most beef cattle and pig isolates belonged to E. coli phylogroups A and B1, whereas most avian and human isolates belonged to B2 and D, although a single human extraintestinal isolate belonged to phylogenetic group A and sequence type (ST) 10. The most common E. coli sequence types (STs) included ST10 for beef cattle, ST361 for pig, ST117 for poultry, and ST73 for human isolates. Extended-spectrum and AmpC ß-lactamase genes were identified in seven out of thirty-seven (18.9%) beef cattle isolates. The most common plasmid replicons identified were IncFIB (AP001918), followed by IncFII, Col156, and IncX1. The results confirm that feedlot cattle isolates examined in this study represent a reduced risk to human and environmental health with regard to being a source of antimicrobial-resistant E. coli of clinical importance.

Subpopulations in Strains of Staphylococcus aureus Provide Antibiotic Tolerance.

The ability of Staphylococcus aureus to colonise different niches across the human body is linked to an adaptable metabolic capability, as well as its ability to persist within specific tissues despite adverse conditions. In many cases, as S. aureus proliferates within an anatomical niche, there is an associated pathology. The immune response, together with medical interventions such as antibiotics, often removes the S. aureus cells that are causing this disease. However, a common issue in S. aureus infections is a relapse of disease. Within infected tissue, S. aureus exists as a population of cells, and it adopts a diversity of cell types. In evolutionary biology, the concept of "bet-hedging" has established that even in positive conditions, there are members that arise within a population that would be present as non-beneficial, but if those conditions change, these traits could allow survival. For S. aureus, some of these cells within an infection have a reduced fitness, are not rapidly proliferating or are the cause of an active host response and disease, but these do remain even after the disease seems to have been cleared. This is true for persistence against immune responses but also as a continual presence in spite of antibiotic treatment. We propose that the constant arousal of suboptimal populations at any timepoint is a key strategy for S. aureus long-term infection and survival. Thus, understanding the molecular basis for this feature could be instrumental to combat persistent infections.

Mid-regional proadrenomedullin (MR-proADM), C-reactive protein (CRP) and other biomarkers in the early identification of disease progression in patients with COVID-19 in the acute NHS setting.

AIMS: There is a lack of biomarkers validated for assessing clinical deterioration in patients with COVID-19 on presentation to secondary or tertiary care. This evaluation looked at the potential clinical application of C reactive protein (CRP), procalcitonin, mid-regional proadrenomedullin (MR-proADM) and white cell count to support prediction of clinical outcomes. METHODS: 135 patients presenting to Hampshire Hospitals NHS Foundation Trust between April and June 2020 confirmed to have COVID-19 via reverse-transcription-qPCR were included. Biomarkers from within 24 hours of presentation were used to predict disease progression by Cox regression and area under the receiver operating characteristic curves. The endpoints assessed were 30-day all-cause mortality, intubation and ventilation, critical care admission and non-invasive ventilation (NIV) use. RESULTS: Elevated MR-proADM was shown to have the greatest ability to predict 30-day mortality adjusting for age, cardiovascular disease, renal disease and neurological disease. A significant association was also noted between raised MR-proADM and CRP concentrations and the requirement for critical care admission and NIV. CONCLUSIONS: The measurement of MR-proADM and CRP in patients with confirmed COVID-19 infection on admission shows significant potential to support clinicians in identifying those at increased risk of disease progression and need for higher level care, subsequently enabling prompt escalation in clinical interventions.

Longitudinal Analysis of Antimicrobial Resistance among Enterococcus Species Isolated from Australian Beef Cattle Faeces at Feedlot Entry and Exit.

Enterococcus faecium are commensal bacteria inhabiting the gastrointestinal tract of animals and humans and an important cause of drug-resistant nosocomial infections. This longitudinal study aimed to determine whether changes in the antimicrobial resistance (AMR) phenotype and genotype occurred among Enterococcus spp. isolated from cattle rectal samples obtained at the entry to and exit from an Australian feedlot. The samples obtained at the feedlot induction yielded enterococci (104/150; 69.3%), speciated as E. hirae (90/104; 86.5%), E. faecium (9/104; 8.7%), E. mundtii (3/104; 2.9%), E. durans, and E. casseliflavus (1/104; 1.0% each). AMR was observed to lincomycin (63/104; 60.6%), daptomycin (26/104; 25.0%), nitrofurantoin (9/104; 8.7%), ciprofloxacin (7/104; 6.7%), tetracycline (5/104; 4.8%), tigecycline (4/104; 3.9%), and quinupristin/dalfopristin (3/104; 2.9%). From the rectal swab samples collected at the abattoir from the same animals (i.e., the feedlot exit), the enterococci recovery was significantly higher (144/150; 96.0%), with a marked shift in species distribution dominated by E. faecium (117/144; 81.3%). However, the prevalence of AMR to individual antimicrobials remained largely static between the entry and exit except for the increased resistance to nitrofurantoin (77/144; 53.5%) and quinupristin/dalfopristin (26/144; 18.1%). Overall, 13 AMR genes were observed among the 62 E. faecium isolates. These included aac(6')Ii, aac(6')-Iid, and ant(6)-Ia (aminoglycosides); eatAv, lnu(G), vat(E), msr(C), and erm(B) (macrolides, lincosamides, and streptogramins); efmA (fluoroquinolones); and tet(45), tet(L), tet(M), and tet(S) (tetracyclines). The results confirm the presence of fluoroquinolone- and streptogramin-resistant enterococci in cattle faeces at the feedlot entry in the absence of antimicrobial selection pressure. E. faecium, exhibiting increased nitrofurantoin resistance, became the dominant Enterococcus spp. during the feeding period.

Phenotypic and Genotypic Analysis of Antimicrobial Resistance in Escherichia coli Recovered from Feedlot Beef Cattle in Australia.

This study investigated the antimicrobial resistance (AMR) profile of fecal Escherichia coli isolates from beef cattle (n = 150) at entry and exit from an Australian feedlot. Sample plating on MacConkey agar and Brilliance ESBL agar differentiated generic from extended-spectrum ß-lactamase (ESBL)-producing E. coli, respectively. Resistance profiles were determined by minimum inhibitory concentration (MIC) testing and further analyzed by whole-genome sequencing (WGS). At entry, the prevalence of antimicrobial resistance to amoxicillin/clavulanic acid, ampicillin, streptomycin, and trimethoprim/sulfamethoxazole was very low (0.7%, each). At the exit, the resistance prevalence was moderate to tetracycline (17.8%) and low to ampicillin (5.4%), streptomycin (4.7%), and sulfisoxazole (3.9%). The most common AMR genes observed in phenotypically resistant isolates were tet(B) (43.2%), aph(3″)-Ib and aph(6)-Id (32.4%), blaTEM-1B, and sul2 (24.3%, each), which are responsible for resistance to tetracyclines, aminoglycosides, ß-lactams, and sulfonamides, respectively. The ESBL-producing E. coli were recovered from one sample (0.7%) obtained at entry and six samples (4.0%) at the exit. The ESBL-producing E. coli harbored blaTEM (29.7%), blaCTX m(13.5%), and blaCMY (5.4%). The resistance phenotypes were highly correlated with resistance genotypes (r ≥ 0.85: p < 0.05). This study demonstrated that E. coli isolated from feedlot beef cattle can harbour AMR genes, but the low incidence of medically important resistance reflected the prudent antimicrobial use in the Australian industry.

Functional mgrA Influences Genetic Changes within a Staphylococcus aureus Cell Population over Time.

Prolonged survival in the host-bacteria microenvironment drives the selection of alternative cell types in Staphylococcus aureus, permitting quasi-dormant sub-populations to develop. These facilitate antibiotic tolerance, long-term growth, and relapse of infection. Small Colony Variants (SCV) are an important cell type associated with persistent infection but are difficult to study in vitro due to the instability of the phenotype and reversion to the normal cell type. We have previously reported that under conditions of growth in continuous culture over a prolonged culture time, SCVs dominated a heterogenous population of cell types and these SCVs harbored a mutation in the DNA binding domain of the gene for the transcription factor, mgrA. To investigate this specific cell type further, S. aureus WCH-SK2-ΔmgrA itself was assessed with continuous culture. Compared to the wild type, the mgrA mutant strain required fewer generations to select for SCVs. There was an increased rate of mutagenesis within the ΔmgrA strain compared to the wild type, which we postulate is the mechanism explaining the increased emergence of SCV selection. The mgrA derived SCVs had impeded metabolism, altered MIC to specific antibiotics and an increased biofilm formation compared to non-SCV strain. Whole genomic sequencing detected single nucleotide polymorphisms (SNP) in phosphoglucosamine mutase glmM and tyrosine recombinase xerC. In addition, several genomic rearrangements were detected which affected genes involved in important functions such as antibiotic and toxic metal resistance and pathogenicity. Thus, we propose a direct link between mgrA and the SCV phenotype. IMPORTANCE Within a bacterial population, a stochastically generated heterogeneity of phenotypes allows continual survival against current and future stressors. The generation of a sub-population of quasi-dormant Small Colony Variants (SCV) in Staphylococcus aureus is such a mechanism, allowing for persistent or relapse of infection despite initial intervention seemingly clearing the infection. The use of continuous culture under clinically relevant conditions has allowed us to introduce time to the growth system and selects SCV within the population. This study provides valuable insights into the generation of SCV which are not addressed in standard laboratory generated models and reveals new pathways for understanding persistent S. aureus infection which can potentially be targeted in future treatments of persistent S. aureus infection.

Identification of COVID-19 patients at risk of hospital admission and mortality: a European multicentre retrospective analysis of mid-regional pro-adrenomedullin.

BACKGROUND: Mid-Regional pro-Adrenomedullin (MR-proADM) is an inflammatory biomarker that improves the prognostic assessment of patients with sepsis, septic shock and organ failure. Previous studies of MR-proADM have primarily focussed on bacterial infections. A limited number of small and monocentric studies have examined MR-proADM as a prognostic factor in patients infected with SARS-CoV-2, however there is need for multicenter validation. An evaluation of its utility in predicting need for hospitalisation in viral infections was also performed. METHODS: An observational retrospective analysis of 1861 patients, with SARS-CoV-2 confirmed by RT-qPCR, from 10 hospitals across Europe was performed. Biomarkers, taken upon presentation to Emergency Departments (ED), clinical scores, patient demographics and outcomes were collected. Multiclass random forest classifier models were generated as well as calculation of area under the curve analysis. The primary endpoint was hospital admission with and without death. RESULTS: Patients suitable for safe discharge from Emergency Departments could be identified through an MR-proADM value of ≤ 1.02 nmol/L in combination with a CRP (C-Reactive Protein) of ≤ 20.2 mg/L and age ≤ 64, or in combination with a SOFA (Sequential Organ Failure Assessment) score < 2 if MR-proADM was ≤ 0.83 nmol/L regardless of age. Those at an increased risk of mortality could be identified upon presentation to secondary care with an MR-proADM value of > 0.85 nmol/L, in combination with a SOFA score ≥ 2 and LDH > 720 U/L, or in combination with a CRP > 29.26 mg/L and age ≤ 64, when MR-proADM was > 1.02 nmol/L. CONCLUSIONS: This international study suggests that for patients presenting to the ED with confirmed SARS-CoV-2 infection, MR-proADM in combination with age and CRP or with the patient's SOFA score could identify patients at low risk where outpatient treatment may be safe.

The central role of arginine in Haemophilus influenzae survival in a polymicrobial environment with Streptococcus pneumoniae and Moraxella catarrhalis.

Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis are bacterial species which frequently co-colonise the nasopharynx, but can also transit to the middle ear to cause otitis media. Chronic otitis media is often associated with a polymicrobial infection by these bacteria. However, despite being present in polymicrobial infections, the molecular interactions between these bacterial species remain poorly understood. We have previously reported competitive interactions driven by pH and growth phase between H. influenzae and S. pneumoniae. In this study, we have revealed competitive interactions between the three otopathogens, which resulted in reduction of H. influenzae viability in co-culture with S. pneumoniae and in triple-species culture. Transcriptomic analysis by mRNA sequencing identified a central role of arginine in mediating these interactions. Arginine supplementation was able to increase H. influenzae survival in a dual-species environment with S. pneumoniae, and in a triple-species environment. Arginine was used by H. influenzae for ATP production, and levels of ATP generated in dual- and triple-species co-culture at early stages of growth were significantly higher than the combined ATP levels of single-species cultures. These results indicate a central role for arginine-mediated ATP production by H. influenzae in the polymicrobial community.

Reverse-Transcription Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals.

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab CT values of ≤25 and ≤33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone.

A Human Osteocyte Cell Line Model for Studying Staphylococcus aureus Persistence in Osteomyelitis.

Infectious osteomyelitis associated with periprosthetic joint infections is often recalcitrant to treatment and has a high rate of recurrence. In the case of Staphylococcus aureus, the most common pathogen in all forms of osteomyelitis, this may be attributed in part to residual intracellular infection of host cells, yet this is not generally considered in the treatment strategy. Osteocytes represent a unique cell type in this context due to their abundance, their formation of a syncytium throughout the bone that could facilitate bacterial spread and their relative inaccessibility to professional immune cells. As such, there is potential value in studying the host-pathogen interactions in the context of this cell type in a replicable and scalable in vitro model. Here, we examined the utility of the human osteosarcoma cell line SaOS2 differentiated to an osteocyte-like stage (SaOS2-OY) as an intracellular infection model for S. aureus. We demonstrate that S. aureus is capable of generating stable intracellular infections in SaOS2-OY cells but not in undifferentiated, osteoblast-like SaOS2 cells (SaOS2-OB). In SaOS2-OY cells, S. aureus transitioned towards a quasi-dormant small colony variant (SCV) growth phenotype over a 15-day post-infection period. The infected cells exhibited changes in the expression of key immunomodulatory mediators that are consistent with the infection response of primary osteocytes. Thus, SaOS2-OY is an appropriate cell line model that may be predictive of the interactions between S. aureus and human osteocytes, and this will be useful for studying mechanisms of persistence and for testing the efficacy of potential antimicrobial strategies.

Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations.

OBJECTIVES: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 pandemic. We investigated the use of LamPORE, where loop-mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. METHODS: In an asymptomatic prospective cohort, for 3 weeks in September 2020, health-care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza-like illness from March 2020 to June 2020 were similarly tested from nasopharyngeal swabs. RESULTS: In the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. CONCLUSIONS: LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

How are rapid diagnostic tests for infectious diseases used in clinical practice: a global survey by the International Society of Antimicrobial Chemotherapy (ISAC).

Novel rapid diagnostic tests (RDTs) offer huge potential to optimise clinical care and improve patient outcomes. In this study, we aim to assess the current patterns of use around the world, identify issues for successful implementation and suggest best practice advice on how to introduce new tests. An electronic survey was devised by the International Society of Antimicrobial Chemotherapy (ISAC) Rapid Diagnostics and Biomarkers working group focussing on the availability, structure and impact of RDTs around the world. It was circulated to ISAC members in December 2019. Results were collated according to the UN human development index (HDI). 81 responses were gathered from 31 different countries. 84% of institutions reported the availability of any test 24/7. In more developed countries, this was more for respiratory viruses, whereas in high and medium/low developed countries, it was for HIV and viral hepatitis. Only 37% of those carrying out rapid tests measured the impact. There is no 'one-size fits all' solution to RDTs: the requirements must be tailored to the healthcare setting in which they are deployed and there are many factors that should be considered prior to this.

Preliminary optimisation of a simplified sample preparation method to permit direct detection of SARS-CoV-2 within saliva samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

We describe the optimisation of a simplified sample preparation method which permits rapid and direct detection of SARS-CoV-2 RNA within saliva, using reverse-transcription loop-mediated isothermal amplification (RT-LAMP). Treatment of saliva samples prior to RT-LAMP by dilution 1:1 in Mucolyse™, followed by dilution in 10 % (w/v) Chelex© 100 Resin and a 98 °C heat step for 2 min enabled detection of SARS-CoV-2 RNA in positive saliva samples. Using RT-LAMP, SARS-CoV-2 RNA was detected in as little as 05:43 min, with no amplification detected in 3097 real-time reverse transcription PCR (rRT-PCR) negative saliva samples from staff tested within a service evaluation study, or for other respiratory pathogens tested (n = 22). Saliva samples can be collected non-invasively, without the need for skilled staff and can be obtained from both healthcare and home settings. Critically, this approach overcomes the requirement for, and validation of, different swabs and the global bottleneck in obtaining access to extraction robots and reagents to enable molecular testing by rRT-PCR. Such testing opens the possibility of public health approaches for effective intervention during the COVID-19 pandemic through regular SARS-CoV-2 testing at a population scale, combined with isolation and contact tracing.