Phenotypic and genotypic analysis of antimicrobial resistance in Nocardia species.

BACKGROUND: Antimicrobial resistance is common in Nocardia species but data regarding the molecular mechanisms beyond their resistance traits are limited. Our study aimed to determine the species distribution, the antimicrobial susceptibility profiles, and investigate the associations between the resistance traits and their genotypic determinants. METHODS: The study included 138 clinical strains of Nocardia from nine Israeli microbiology laboratories. MIC values of 12 antimicrobial agents were determined using broth microdilution. WGS was performed on 129 isolates of the eight predominant species. Bioinformatic analysis included phylogeny and determination of antimicrobial resistance genes and mutations. RESULTS: Among the isolates, Nocardia cyriacigeorgica was the most common species (36%), followed by Nocardia farcinica (16%), Nocardia wallacei (13%), Nocardia abscessus (9%) and Nocardia brasiliensis (8%). Linezolid was active against all isolates, followed by trimethoprim/sulfamethoxazole (93%) and amikacin (91%). Resistance to other antibiotics was species-specific, often associated with the presence of resistance genes or mutations: (1) aph(2″) in N. farcinica and N. wallacei (resistance to tobramycin); (ii) blaAST-1 in N. cyriacigeorgica and Nocardia neocaledoniensis (resistance to amoxicillin/clavulanate); (iii) blaFAR-1 in N. farcinica (resistance to ceftriaxone); (iv) Ser83Ala substitution in the gyrA gene in four species (resistance to ciprofloxacin); and (v) the 16S rRNA m1A1408 methyltransferase in N. wallacei isolates (correlating with amikacin resistance). CONCLUSIONS: Our study provides a comprehensive understanding of Nocardia species diversity, antibiotic resistance patterns, and the molecular basis of antimicrobial resistance. Resistance appears to follow species-related patterns, suggesting a lesser role for de novo evolution or transmission of antimicrobial resistance.

Clinical impact of the accelerate PhenoTest® BC system on patients with gram-negative bacteremia and high risk of antimicrobial resistance: a prospective before-after implementation study.

BACKGROUND: The Accelerate PhenoTest® BC system (AXDX) is a novel assay for rapid bacterial identification and antimicrobial susceptibility (AST). We report an evaluation of its impact on treatment of patients with Gram-negative bacteremia (GNB) with a high risk of antimicrobial resistance (AMR). METHODS: A prospective single-center evaluation before and after implementation of AXDX in addition to standard-of-care (SOC) microbiology and antimicrobial stewardship program (ASP). Patients with GNB reported during laboratory working hours and prespecified risk factors for AMR were included. The primary outcome was an ASP-oriented beneficial antimicrobial change, defined as either an escalation of an inappropriate empiric treatment or de-escalation of a broad-spectrum treatment of a susceptible organism. Main secondary outcomes were time to an appropriate treatment, antimicrobial treatment duration, length of stay (LOS) and mortality. RESULTS: Included were 46 and 57 patients in the pre- and post-intervention periods, respectively. The median time to an AST-oriented beneficial change was 29.2 h vs. 49.6 h, respectively (p < 0.0001). There were no significant differences in the time to appropriate treatment, LOS or mortality. Antimicrobial treatment duration was longer during the intervention period (10 vs. 8 days, p = 0.007). AXDX failed to correctly identify pathogens in all 6 cases of polymicrobial bacteremia. In two cases patient care was potentially compromised due to inappropriate de-escalation. CONCLUSIONS: AXDX implementation resulted in a 20.4-hour shorter time to an ASP-oriented beneficial antimicrobial change. This should be weighed against the higher costs, the lack of other proven clinical benefits and the potential harm from mis-identification of polymicrobial bacteremias.

Delayed respiratory syncytial virus epidemic in children after relaxation of COVID-19 physical distancing measures, Ashdod, Israel, 2021.

Following low incidence of respiratory syncytial virus (RSV) infections in 2020 during the COVID-19 pandemic, we noted a resurgence in hospitalised children in spring/summer 2021 following relaxation of public health measures. We compared this outbreak to previous autumn/winter seasons. We found higher weekly case numbers and incidence rates, more cases from urban neighbourhoods with lower socioeconomic status, and similar clinical presentation and severity. Public health implications include the re-evaluation of palivizumab administration and the need for surge capacity planning.

Statins and outcomes of hospitalized patients with laboratory-confirmed 2017-2018 influenza.

No studies evaluating the association between statins and outcomes of patients with seasonal influenza have been performed since the 2007-2008 and the 2009 pandemic H1N1 influenza seasons. All consecutive hospitalized patients between October 2017 and April 2018, diagnosed with laboratory-confirmed influenza A and B virus, were included. Patients were divided into two groups: statin and non-statin users. Outcomes were 30- and 90-day mortality, complications (pneumonia, myocarditis, encephalitis, intensive care unit (ICU) transfer, mechanical ventilation, vasopressor support), length of hospital stay, and readmission rates. A multivariate analysis was performed to adjust for mortality risk factors. To compare the groups, we matched patients to the nearest neighbor propensity score. Of the 526 patients ill with influenza A (201/526) and B (325/526), 36% (188/526) were statin users; 64% (338/526) were not. Statin users were older (78 vs.70; p = < 0.05) and suffered from more comorbidities (Charlson comorbidity scores of 6 vs.4; p < 0.005). The 30-day mortality rate among statin vs. non-statin users was 6% vs. 8% (p = 0.3). On multivariate analysis, statin use was not associated with mortality benefit (OR = 0.67 (0.29-1.36)). After propensity score matching, the results were unchanged (OR = 0.71 (0.29-1.71)). Statin users were diagnosed with less complicated diseases as they were less likely to receive vasopressor support, mechanical ventilation, and/or transfer to the ICU. Although statin users were significantly older and exhibited more comorbidities, 30-day mortality rates did not differ between statin users and non-users, which may signify a protective role of statins on seasonal influenza patients. Further studies performed during different influenza seasons and different subtypes are essential.

Clinical benefits of FilmArray meningitis-encephalitis PCR assay in partially-treated bacterial meningitis in Israel.

BACKGROUND: Management of partially-treated, community-acquired bacterial meningitis (PCBM) is commonly compromised by lack of microbiological diagnosis. We aimed to analyze the impact of FilmArray Meningitis-Encephalitis (FA-ME) PCR on the management of PCBM. METHODS: Comparison of treatment variables of PCBM cases between two periods, before (6.5 years, control group) and after (2 years, study group) the application of FA-ME PCR assay. RESULTS: The total duration of antimicrobial treatment in the study group (n = 8) was significantly shorter than the control group (n = 23) (9.5 ± 3.7 days vs. 15.2 ± 5 days, p = 0.007). The percentage of narrow-spectrum regimens was significantly higher in the study group (78 ± 11% vs. 40 ± 9%, p = 0.03). There was a significant difference in implementation of antimicrobial chemoprophylaxis for close contacts (4/8 (50%) vs. 1/23 (4%), p = 0.01). CONCLUSIONS: The use of FA-ME PCR provides significant benefits in the management of PCBM by shortening duration of antibiotic treatment, increasing the use of narrow-spectrum regimens, and allowing proper administration of antimicrobial chemoprophylaxis. TRIAL REGISTRATION: The study was approved and retrospectively registered by the Tel-Aviv Sourasky Medical Center ( 0378-17-TLV , 10/17/2017) and Rabin Medical Center ( 0270-18-RMC , 11/11/2018) Ethics committees and conforms to recognized standards.

Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1.

We performed a screen for genetic suppressors of cobra, an Arabidopsis mutant with defects in cellulose formation and an increased ratio of unesterified/esterified pectin. We identified a suppressor named mongoose1 (mon1) that suppressed the growth defects of cobra, partially restored cellulose levels, and restored the esterification ratio of pectin to wild-type levels. mon1 was mapped to the MEDIATOR16 (MED16) locus, a tail mediator subunit, also known as SENSITIVE TO FREEZING6 (SFR6). When separated from the cobra mutation, mutations in MED16 caused resistance to cellulose biosynthesis inhibitors, consistent with their ability to suppress the cobra cellulose deficiency. Transcriptome analysis revealed that a number of cell wall genes are misregulated in med16 mutants. Two of these genes encode pectin methylesterase inhibitors, which, when ectopically expressed, partially suppressed the cobra phenotype. This suggests that cellulose biosynthesis can be affected by the esterification levels of pectin, possibly through modifying cell wall integrity or the interaction of pectin and cellulose.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual ß1-4-linked glucan chains with a KD of 3.2 µm. Competition assays suggests that COBRA binds individual ß1-4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging ß1-4-glucan chains by acting as a "polysaccharide chaperone."

A Multi-Laboratory Evaluation of Commercial Monkeypox Virus Molecular Tests.

In this report, we describe the first national scale multi-laboratory evaluation of monkeypox virus (MPXV) DNA commercial PCR kits. The objective of this study was to evaluate 2 kits by different diagnostic laboratories across Israel. Ten standardized samples were tested simultaneously using the Novaplex (15 laboratories) and Bio-Speedy (seven laboratories) kits. An in-house assay based on previously published reactions was used as reference. Comparison of the results showed high intra-assay agreement between laboratories, with small variations for most samples. The in-house assay had an analytical detection limit of less than 10 copies per reaction. While the 2 commercial kits were able to detect specimens with low viral loads similarly to the in-house assay, significant differences were observed, in the Cq values and relative fluorescence (RF), between the assays. The RF signal of the in-house and Bio-Speedy assays ranged between 5,000 and 10,000 RFU, while the signal in the Novaplex assay was less than 600 RFU. Due to the kit measurement protocol, the Cq values of the Bio-Speedy kit were 5 to 7.5 cycles lower than those of the in-house assay. On the contrary, the Cq values of the Novaplex kit were significantly higher than those of the in-house assay, with differences of 3 to 5 cycles per sample. Our results suggest that while all assays were similar in their overall sensitivity, direct comparison of Cq values between them may be misleading. To our knowledge, this is the first methodical evaluation of commercial MPX test kits. We therefore anticipate that this study would help diagnostic laboratories in choosing a specific MPX detection assay. IMPORTANCE To the best of our knowledge, this study is the first methodical evaluation of commercial kits designed for Monkeypox virus detection. This was done by performing the same tests using the same sample set in multiple laboratories, simultaneously, on a national scale. It therefore provides important and unique information on the performance of such kits and provides a guideline for choosing the assay of choice for monkeypox virus diagnosis in a standard diagnostic laboratory. It also demonstrates potential complications when trying to compare the results of different assays, even when testing exactly the same samples, under identical conditions.

Differential effects of prenylation and s-acylation on type I and II ROPS membrane interaction and function.

Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.

Increase in Haemophilus influenzae Detection in 13-Valent Pneumococcal Conjugate Vaccine Immunized Children With Acute Otitis Media.

NTHi was the predominant pathogen in ear cultures from severe acute otitis media (AOM) episodes in PCV-13 vaccinated children, more commonly in girls. NTHi-AOM episodes were associated with more myringotomies due to a higher treatment failure incidence. The low rate of ß-lactamase NTHi isolates in middle ear fluid cultures from PCV-13 vaccinated children presenting with AOM strengthens to still use amoxicillin as the first-line antibiotics.

Morbidity and mortality of respiratory syncytial virus infection in hospitalized adults: Comparison with seasonal influenza.

INTRODUCTION: Respiratory syncytial virus (RSV) is considered a major pathogen that causes acute influenza-like illness. The objective of this study was to compare the clinical outcomes of patients with laboratory-confirmed RSV and patients with influenza infection. METHODS: Adults hospitalized in Beilinson Hospital (October 2017-April 2018) with laboratory-confirmed RSV or influenza were included. The primary outcome was the composite of RSV/influenza complications: 30-day mortality, pneumonia, mechanical ventilation, vasopressor support, intensive care unit admission, and myocarditis/encephalitis. Secondary outcomes were individual components of the primary outcome, 90-day mortality, 90-day readmission, and length of hospital stay. RESULTS: A total of 639 patients with RSV (n = 113) and influenza (n = 526) were included. The composite primary outcome was 21.4% (136/633), and was higher in RSV patients (30% (34/113) vs 19% (102/526), p = 0.002). Pneumonia was more common in RSV patients (21.2% (24/113) vs 9.1% (48/526), p = 0.001). On multivariable analysis, hypoalbuminemia (odds ratio (OR) 3.3, 95% confidence interval (CI) 2.1-5.3, p < 0.001), reduced room-air saturation (OR 1.1, 95% CI 1.02-1.1, p = 0.001), and infection with RSV (OR 1.67, 95% CI 1.01-2.76, p = 0.046) were predictors of complications. CONCLUSIONS: RSV infection in hospitalized adults resulted in serious respiratory illness with complications that are comparable to those caused by influenza.

Oropharyngeal shedding of herpesviruses before and after BNT162b2 mRNA vaccination against COVID-19.

INTRODUCTION: Concerns were raised over an increase in Bell's palsy, herpes simplex and herpes zoster after BNT162b2 vaccination, all are manifestations of herpesviruses reactivation. As herpesviruses commonly reactivate in the oropharynx, we have hypothesized that oropharyngeal shedding of herpesviruses will increase after vaccination. METHODS: Immune-competent Adults, excluding those using topical steroids or manifesting symptomatic herpesvirus infection, were sampled before BNT162b2 vaccination and one week after. Herpesviruses 1-7 shedding was tested with a multiplexed PCR. RESULTS: In 103 paired samples the prevalence of herpesviruses was similar before and after vaccination: HSV1, 3.9% vs. 5.8% (p = 0.75); HSV2, 0% vs. 1% (p = not applicable, NA); VZV, 0% vs. 0% (p = NA); EBV, 14.6% vs. 17.5% (p = 0.63); CMV, 0% vs. 0% (p = NA); HHV6, 4.9% vs. 7.8% (p = 0.55); HHV7, 71.8% vs. 72.8% (p = 1); any herpesvirus, 73.8% vs. 74.8% (p = 1). DISCUSSION: We did not find evidence for increased oropharyngeal reactivation of herpesviruses one week after BNT162b2.

A Novel ROP/RAC effector links cell polarity, root-meristem maintenance, and vesicle trafficking.

ROP/RAC GTPases are master regulators of cell polarity in plants, implicated in the regulation of diverse signaling cascades including cytoskeleton organization, vesicle trafficking, and Ca(2+) gradients [1-8]. The involvement of ROPs in differentiation processes is yet unknown. Here we show the identification of a novel ROP/RAC effector, designated interactor of constitutive active ROPs 1 (ICR1), that interacts with GTP-bound ROPs. ICR1 knockdown or silencing leads to cell deformation and loss of root stem-cell population. Ectopic expression of ICR1 phenocopies activated ROPs, inducing cell deformation of leaf-epidermis-pavement and root-hair cells [3, 5, 6, 9]. ICR1 is comprised of coiled-coil domains and forms complexes with itself and the exocyst vesicle-tethering complex subunit SEC3 [10-13]. The ICR1-SEC3 complexes can interact with ROPs in vivo. Plants overexpressing a ROP- and SEC3-noninteracting ICR1 mutant have a wild-type phenotype. Taken together, our results show that ICR1 is a scaffold-mediating formation of protein complexes that are required for cell polarity, linking ROP/RAC GTPases with vesicle trafficking and differentiation.

Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase.

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His(6)-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His(6)-GFP-Atrop6(CA) mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPgammaS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His(6)-GFP-Atrop6(CA)mS(156) in which cysteine(156) was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.

Full genome viral sequences inform patterns of SARS-CoV-2 spread into and within Israel.

Full genome sequences are increasingly used to track the geographic spread and transmission dynamics of viral pathogens. Here, with a focus on Israel, we sequence 212 SARS-CoV-2 sequences and use them to perform a comprehensive analysis to trace the origins and spread of the virus. We find that travelers returning from the United States of America significantly contributed to viral spread in Israel, more than their proportion in incoming infected travelers. Using phylodynamic analysis, we estimate that the basic reproduction number of the virus was initially around 2.5, dropping by more than two-thirds following the implementation of social distancing measures. We further report high levels of transmission heterogeneity in SARS-CoV-2 spread, with between 2-10% of infected individuals resulting in 80% of secondary infections. Overall, our findings demonstrate the effectiveness of social distancing measures for reducing viral spread.

Neisseria meningitidis and cytomegalovirus simultaneous detection in the filmarray meningitis/encephalitis panel and its clinical relevance.

A BioFire FilmArray Meningitis/Encephalitis test was performed on a 7-month old child suspected for bacterial meningitis. Two pathogens were detected, Neisseria meningitidis and cytomegalovirus (CMV). We verified the filmarray meningitis/encephalitis test by pan-bacterial assay to test for Neisseria meningitidis and CMV viral load test for the CMV detection. Pan-bacterial confirmed presence of N. meningitidis, but CMV was not detected by the CMV viral load test. Together with the manifestations of high fever, vomiting, diffuse petechial rash, bulging fontanel, and leukocytosis, it is a clear case of meningococcal meningitis, while CMV detection had no clinical relevance.

Predicting candidemia in the internal medicine wards: a comparison with gram-negative bacteremia-a retrospectives study.

INTRODUCTION: Risk factors for candidemia in the internal medicine wards (IMW) are poorly characterized. Their elucidation might assist in early diagnosis and treatment. OBJECTIVES: We aimed to elucidate predictors of candidemia in the IMWs comparing them to patients with gram-negative bacteremia (GNB). METHODS: A retrospective study of consecutive patients with candidemia in IMWs in Beilinson hospital (2007-2016) was performed. Patient demographics, comorbidities, and clinical characteristics were documented. The comparator group was GNB patients. RESULTS: Sixty-two patients with candidemia were compared with 178 patients with GNB. Candidemic patients were younger and with less body mass index > 20 kg/m2 (73 ±â€¯15 vs. 78 ±â€¯10, P = 0.01; 44% vs. 60%, P = <0.0001,respectively). In multivariate model, underweight, prior cephalosporin use, and central venous catheters (CVCs) were significantly associated with candidemia [odds ratio (OR) = 0.2, 95% confidence interval (CI) 0.07-0.4; OR = 4, 95% CI 1.3-11; and OR = 4, 95% CI 1.5-12, respectively]. CONCLUSION: Underweight, recent cephalosporin exposure, and CVCs were statistically significant predictors of candidemia in the IMW. Using these predictors might aid in recognizing high-risk patients for candidemia in the IMWs, leading to earlier appropriate empirical treatment.

Corrected and Republished from: Activation Status-Coupled Transient S-Acylation Determines Membrane Partitioning of a Plant Rho-Related GTPase.

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane via posttranslational lipid modifications. The relationships between ROP activation status and membrane localization has not been established. Here, we show that endogenous ROPs, as well as a transgenic His6-green fluorescent protein (GFP)-Arabidopsis thaliana ROP6 (AtROP6) fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, the His6-GFP-Atrop6CA activated mutant accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 proteins were purified from Arabidopsis plants, and in turn, their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, the wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids, identified as palmitic and stearic acids. Consistently, activated His6-GFP-Atrop6CAmS156, in which C156 was mutated into serine, accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6, and possibly other ROPs, are transiently S-acylated, inducing their partitioning into detergent-resistant membranes.