Keeping up with the pathogens: improved antimicrobial resistance detection and prediction from Pseudomonas aeruginosa genomes.

BACKGROUND: Antimicrobial resistance (AMR) is an intensifying threat that requires urgent mitigation to avoid a post-antibiotic era. Pseudomonas aeruginosa represents one of the greatest AMR concerns due to increasing multi- and pan-drug resistance rates. Shotgun sequencing is gaining traction for in silico AMR profiling due to its unambiguity and transferability; however, accurate and comprehensive AMR prediction from P. aeruginosa genomes remains an unsolved problem. METHODS: We first curated the most comprehensive database yet of known P. aeruginosa AMR variants. Next, we performed comparative genomics and microbial genome-wide association study analysis across a Global isolate Dataset (n = 1877) with paired antimicrobial phenotype and genomic data to identify novel AMR variants. Finally, the performance of our P. aeruginosa AMR database, implemented in our AMR detection and prediction tool, ARDaP, was compared with three previously published in silico AMR gene detection or phenotype prediction tools-abritAMR, AMRFinderPlus, ResFinder-across both the Global Dataset and an analysis-naïve Validation Dataset (n = 102). RESULTS: Our AMR database comprises 3639 mobile AMR genes and 728 chromosomal variants, including 75 previously unreported chromosomal AMR variants, 10 variants associated with unusual antimicrobial susceptibility, and 281 chromosomal variants that we show are unlikely to confer AMR. Our pipeline achieved a genotype-phenotype balanced accuracy (bACC) of 85% and 81% across 10 clinically relevant antibiotics when tested against the Global and Validation Datasets, respectively, vs. just 56% and 54% with abritAMR, 58% and 54% with AMRFinderPlus, and 60% and 53% with ResFinder. ARDaP's superior performance was predominantly due to the inclusion of chromosomal AMR variants, which are generally not identified with most AMR identification tools. CONCLUSIONS: Our ARDaP software and associated AMR variant database provides an accurate tool for predicting AMR phenotypes in P. aeruginosa, far surpassing the performance of current tools. Implementation of ARDaP for routine AMR prediction from P. aeruginosa genomes and metagenomes will improve AMR identification, addressing a critical facet in combatting this treatment-refractory pathogen. However, knowledge gaps remain in our understanding of the P. aeruginosa resistome, particularly the basis of colistin AMR.

Antibody-Mediated Serum Resistance Protects Pseudomonas aeruginosa During Bloodstream Infections.

BACKGROUND: Pseudomonas aeruginosa is a frequent pathogen isolated from bacterial bloodstream infection (BSI) and is associated with high mortality. To survive in the blood, P aeruginosa must resist the bactericidal action of complement (ie, serum killing). Antibodies usually promote serum killing through the classical complement pathway; however, "cloaking antibodies" (cAbs) have been described, which paradoxically protect bacteria from serum killing. The relevance of cAbs in P aeruginosa BSI is unknown. METHODS: Serum and P aeruginosa were collected from a cohort of 100 patients with BSI. Isolates were tested for sensitivity to healthy control serum (HCS). cAb prevalence was determined in sera. Patient sera were mixed with HCS to determine if killing of the matched isolate was inhibited. RESULTS: Overall, 36 patients had elevated titers of cAbs, and 34 isolates were sensitive to HCS killing. Fifteen patients had cAbs and HCS-sensitive isolates; of these patients, 14 had serum that protected their matched bacteria from HCS killing. Patients with cAbs were less likely to be neutropenic or have comorbidities. CONCLUSIONS: cAbs are prevalent in patients with P aeruginosa BSI and allow survival of otherwise serum-sensitive bacteria in the bloodstream. Generation of cAbs may be a risk factor for the development of BSI.

Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR.

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa.Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I (gyrA248) and GyrA D87N, D87Y and D87H (gyrA259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa.Methods. Following assay validation, the gyrA248 and gyrA259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing.Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes.Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa. An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Infection control behaviours, intra-household transmission and quarantine duration: a retrospective cohort analysis of COVID-19 cases.

OBJECTIVES: To study COVID-19 (Delta Variant) cases and close contacts co-located within households. Focusing on epidemiology of transmission of COVID-19, quarantine duration and utilisation of infection control behaviours under a telehealth model of care in an elimination setting. METHODS: A retrospective cohort analysis examined household spread of infection, duration of quarantine and change in PCR CT value during illness. A survey explored infection control behaviours used by household members during isolation and quarantine. RESULTS: The cohort was 141 individuals in 35 households. Thirty-seven were index cases, and 48 became positive during quarantine, most within 10 days. Whole-household infection occurred in 12 households with multiple members. Behaviours focused on fomite transmission reduction rather than preventing aerosol transmission. The median duration of close contact household quarantine was 25 days. The majority of COVID-19 cases were de-isolated after 14 days with no evidence of further community transmission. CONCLUSION: Intrahousehold transmission was not universal and, if it occurred, usually occurred quickly. Behaviours utilised focused on fomites, suggesting a need for improved education regarding the potential utilisation of strategies to prevention the transmission of aerosols. Households experienced long durations of home-based quarantine. IMPLICATIONS FOR PUBLIC HEALTH: The impact of long quarantine durations must be considered, particularly where most community benefit from quarantine is achieved within 10 days from exposure in the setting of the Delta Variant. Education of households regarding aerosol risk reduction is a potential strategy in the household setting of individuals at risk of disease progression.

Evaluating Mono- and Combination Therapy of Meropenem and Amikacin against Pseudomonas aeruginosa Bacteremia in the Hollow-Fiber Infection Model.

Debate continues as to the role of combination antibiotic therapy for the management of Pseudomonas aeruginosa infections. We studied the extent of bacterial killing by and the emergence of resistance to meropenem and amikacin as monotherapies and as a combination therapy against susceptible and resistant P. aeruginosa isolates from bacteremic patients using the dynamic in vitro hollow-fiber infection model. Three P. aeruginosa isolates (meropenem MICs of 0.125, 0.25, and 64 mg/L) were used, simulating bacteremia with an initial inoculum of ~1 × 105 CFU/mL and the expected pharmacokinetics of meropenem and amikacin in critically ill patients. For isolates susceptible to amikacin and meropenem (isolates 1 and 2), the extent of bacterial killing was increased with the combination regimen compared with the killing by monotherapy of either antibiotic. Both the combination and meropenem monotherapy were able to sustain bacterial killing throughout the 7-day treatment course, whereas regrowth of bacteria occurred with amikacin monotherapy after 12 h. For the meropenem-resistant P. aeruginosa isolate (isolate 3), only the combination regimen demonstrated bacterial killing. Given that tailored antibiotic regimens can maximize potential synergy against some isolates, future studies should explore the benefit of combination therapy against resistant P. aeruginosa. IMPORTANCE Current guidelines recommend that aminoglycosides should be used in combination with ß-lactam antibiotics as initial empirical therapy for serious infections, and otherwise, patients should receive ß-lactam antibiotic monotherapy. Given the challenges associated with studying the clinical effect of different antibiotic strategies on patient outcomes, useful data for subsequent informed clinical testing can be obtained from in vitro models like the hollow-fiber infection model (HFIM). Based on the findings of our HFIM, we propose that the initial use of combination therapy with meropenem and amikacin provides some bacterial killing against carbapenem-resistant P. aeruginosa isolates. For susceptible isolates, combination therapy may only be of benefit in specific patient populations, such as critically ill or immunocompromised patients. Therefore, clinicians may want to consider using the combination therapy for the initial management and ceasing the aminoglycosides once antibiotic susceptibility results have been obtained.

A contemporary 16-year review of Coxiella burnetii infective endocarditis in a tertiary cardiac center in Queensland, Australia.

BACKGROUND: Coxiella burnetii endocarditis can be difficult to diagnose leading to delays in treatment. This retrospective case series study was undertaken to understand the epidemiologic trends and clinical features of Q fever endocarditis in Southeast Queensland, Australia. METHODS: Clinical records of patients from a single center with coding diagnosis of C. burnetii, or serology consistent with chronic Q fever, were reviewed from 1999 to 2015. Data from patients with probable or confirmed Q fever endocarditis was abstracted. RESULTS: Thirteen patients had confirmed and 5 had probable Q fever endocarditis. Median age at diagnosis in confirmed cases was 60 years. In confirmed cases, 92% (12/13) of patients had an underlying valvular defect. Two patients in the confirmed cases had serology not consistent with a diagnosis of Q fever endocarditis. Eight patient records noted retrospective diagnosis of Q fever endocarditis after surgery. CONCLUSIONS: As pre-existing valve pathology is a major risk for developing endocarditis, prophylactic strategies such as targeted echocardiography and Q fever vaccination could be considered to reduce the incidence of Q fever endocarditis.

Molecular epidemiology of Pseudomonas aeruginosa bloodstream infection isolates in a non-outbreak setting.

PURPOSE: The molecular epidemiology of Pseudomonas aeruginosa bloodstream infection (BSI) isolates has received limited attention. This study aims to characterize the molecular relationship of P. aeruginosa BSI isolates in the non-outbreak setting at a single tertiary healthcare facility. METHODOLOGY: P. aeruginosa BSI isolates from patients who were admitted to the Royal Brisbane and Women's Hospital over a 13 month period from November 2009 were identified retrospectively from the Pathology Queensland Clinical and Scientific Information System. The isolates were typed by the iPLEX MassARRAY matrix assisted lazer desorption/isonisation time of flight (MALDI-TOF) MS genotyping. The DiversiLab automated rapid strain typing platform (bioMérieux) was used to assess the genotypic relationships between study isolates that showed indistinguishable iPLEX20SNP profiles. Clinical data was also collected retrospectively from patient notes. RESULTS: Fifty-three P. aeruginosa BSI episodes were available for study. Thirty-five different clones or clonal complexes were identified by the iPLEX MassARRAY MALDI-TOF MS genotyping. Seventeen BSI isolates with indistinguishable iPLEX20SNP profiles underwent further DiversiLab genotyping and were found to belong to a further 13 different genotypes. There was no relationship between clonality and acquisition type, source of infection or length of stay in the setting of hospital-acquired infection. CONCLUSION: The non-clonal population structure suggests that there is ongoing environmental exposure of inpatients to P. aeruginosa. In clinical areas dealing with at-risk patients, routine attention to mechanism of environmental colonization is important and should be addressed even in the non-outbreak setting.

Detection of carbapenemase activity in Enterobacteriaceae using LC-MS/MS in comparison with the neo-rapid CARB kit using direct visual assessment and colorimetry.

It has been described that the sensitivity of the Carba NP test may be low in the case of OXA-48-like carbapenamases and mass spectrometry based methods as well as a colorimetry based method have been described as alternatives. We evaluated 84 Enterobacteriaceae isolates including 31 OXA-48-like producing isolates and 13 isolates that produced either an imipenemase (IMP; n=8), New Delhi metallo-ß-lactamase (NDM; n=3), or Klebsiella pneumoniae carbapenemase (KPC; n=2), as well as 40 carbapenemase negative Enterobacteriaceae isolates. We used the Neo-Rapid CARB kit, assessing the results with the unaided eye and compared it with a colorimetric approach. Furthermore, we incubated the isolates in growth media with meropenem and measured the remaining meropenem after one and 2h of incubation, respectively, using liquid chromatography tandem mass spectrometry (LC-MS/MS). Whilst all carbapenemase producing isolates with the exception of the OXA-244 producer tested positive for both the Neo-rapid CARB test using the unaided eye or colorimetry, and the 13 isolates producing either IMP, NDM or KPC hydrolysed the meropenem in the media almost completely after 2h of incubation, the 31 OXA-48-like producing isolates exhibited very variable hydrolytic activity when incubated in growth media with meropenem. In our study, the Neo-Rapid CARB test yielded a sensitivity of 98% for both the traditional and the colorimetric approach with a specificity of 95% and 100% respectively. Our results indicate that the Neo-Rapid CARB test may have use for the detection of OXA-48 type carbapenemases and that it may be particularly important to ensure bacterial lysis for the detection of these weaker hydrolysers.

Severe photosensitivity causing multifocal squamous cell carcinomas secondary to prolonged voriconazole therapy.

A 32-year-old woman was treated with long-term voriconazole therapy for recurrent aspergillosis associated with chronic granulomatous disease. A short time after commencement of voriconazole therapy, a severe photosensitivity reaction developed. Continued voriconazole exposure led to the development of multifocal facial squamous cell carcinomas. The photosensitivity reaction resolved after the patient changed therapy to posaconazole.