Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence among emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. By creating and analyzing isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates than the historic strains. Via the creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found a high ratio of mucosal (i.e., pharyngeal) relative to invasive infections among emm4 GAS. Since ever-increasing virulence is unlikely to be evolutionarily advantageous for a microbial pathogen, our data further understanding of the well-described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

Hepatitis A occurrence and outbreaks in Europe over the past two decades: A systematic review.

Hepatitis A (HA) is a vaccine-preventable liver disease with >170 million new cases occurring yearly. In recent outbreaks in the USA, hospitalization and case-fatality ratios were >60% and ~1%, respectively. In Europe, endemicity persists and outbreaks continue to occur. We performed a systematic literature review to understand the changes in HA occurrence in Europe over the past two decades. PubMed and Embase were systematically searched for peer-reviewed articles published between 1 January 2001 and 14 April 2021 using terms covering HA, 11 selected European countries, outbreaks, outcomes and HA virus circulation. Here, we focus on HA occurrence and outbreaks in the five countries with the largest population and the most comprehensive vaccination recommendations: France, Germany, Italy, Spain and the UK; 118 reports included data for these five European countries. Notification rates (≤9.7/100,000 population) and percentages of men among cases (≤83.0%) peaked in 2017. The number of person-to-person-transmitted cases and outbreaks decreased in children but increased in other risk groups, such as men who have sex with men (MSM). Sexually transmitted outbreaks in MSM clustered around 2017. Travel-related outbreaks were few; the proportion of travel-related cases decreased during the past two decades, while the number of domestic cases increased. Despite the existing risk-based vaccination recommendations, HA transmission shifted in proportions from travelers and children to other risk groups, such as MSM and older age groups. Because a substantial proportion of the European population is susceptible to HA, adherence to existing recommendations should be monitored more closely, and enhanced vaccination strategies should be considered.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Activation of the innate signaling molecule MAVS by bunyavirus infection upregulates the adaptor protein SARM1, leading to neuronal death.

La Crosse virus (LACV), a zoonotic Bunyavirus, is a major cause of pediatric viral encephalitis in the United States. A hallmark of neurological diseases caused by LACV and other encephalitic viruses is the induction of neuronal cell death. Innate immune responses have been implicated in neuronal damage, but no mechanism has been elucidated. By using in vitro studies in primary neurons and in vivo studies in mice, we have shown that LACV infection induced the RNA helicase, RIG-I, and mitochondrial antiviral signaling protein (MAVS) signaling pathway, resulting in upregulation of the sterile alpha and TIR-containing motif 1 (SARM1), an adaptor molecule that we found to be directly involved in neuronal damage. SARM1-mediated cell death was associated with induced oxidative stress response and mitochondrial damage. These studies provide an innate-immune signaling mechanism for virus-induced neuronal death and reveal potential targets for development of therapeutics to treat encephalitic viral infections.

Improving vaccination rates in older adults and at-risk groups: focus on pertussis.

Despite the implementation of effective paediatric vaccination programmes, pertussis remains a global health problem. Disease epidemiology has changed over time, shifting towards the adolescent and adult populations. In adults, the true burden of pertussis is greatly underestimated and pertussis vaccine coverage rates are suboptimal, including individuals with chronic conditions. Here, we report the outcomes of a virtual international scientific workshop to assess the evidence on the burden of pertussis in older adults and identify potential solutions to improve uptake of pertussis vaccines. In adults, pertussis is underdiagnosed in part due to atypical or milder clinical presentation and the lack of testing and case confirmation. However, contemporary epidemiological data denoted an increase in the burden of pertussis among adolescents and adults. This might be related to a variety of reasons including the waning of immunity over time, the lack of booster vaccination, and the improved diagnostic methods that led to increased recognition of the disease in adults. Pertussis sequelae can be severe in older adults, particularly those with existing chronic medical conditions, and the vulnerability of these groups is further enhanced by low pertussis vaccine coverage. Possible measures to increase vaccine uptake include strengthening and harmonisation of immunisation guidelines, healthcare professionals taking a more active role in recommending pertussis vaccination, involvement of vaccination centres and pharmacies in the vaccination process, and improving knowledge of pertussis burden and vaccine efficacy among the general population.

Burden of Pertussis in COPD: A Retrospective Database Study in England.

Chronic obstructive pulmonary disease (COPD) may increase the risk and severity of pertussis infection. Health care resource utilization (HCRU) and direct medical costs (DMC) of treating pertussis among patients with COPD are unknown. Reported incidence of pertussis among individuals aged ≥ 50 years with COPD was assessed in Clinical Practice Research Datalink and Hospital Episode Statistics databases during 2009-2018 using a retrospective cohort design. HCRU and DMC from the National Health Service perspective were compared between patients with COPD and pertussis and propensity score-matched patients with COPD without pertussis. Seventy-eight new pertussis events were identified among 387 086 patients with COPD aged ≥ 50 years (incidence rate: 4.73; 95% confidence interval 3.74-5.91 per 100 000 person-years). HCRU and DMC were assessed among 67 patients with COPD and pertussis and 267 matched controls. During the month before the pertussis diagnosis, the rates of general practitioner (GP)/nurse visits (4289 vs. 1774 per 100 patient-years) and accident and emergency visits (182 vs. 18 per 100 patient-years) were higher in the pertussis cohort; GP/nurse visits (2935 vs. 1705 per 100 patient-years) were also higher during the following 2 months (all p < 0.001). During the month before the pertussis diagnosis, annualized per-patient total DMC were £2012 higher in the pertussis cohort (£3729 vs. £1717; p < 0.001); during the following 2 months, they were £2407 higher (£5498 vs. £3091; p < 0.001). In conclusion, a pertussis episode among individuals with COPD resulted in significant increases in HCRU and DMC around the pertussis event.

Seroprevalence of Bordetella pertussis Infection in Patients With Chronic Obstructive Pulmonary Disease in England: Analysis of the AERIS Cohort.

Pertussis is underdiagnosed and underreported in adults and patients with underlying conditions. Patients with chronic obstructive pulmonary disease (COPD) may be at increased risk of severe pertussis. Understanding the true prevalence of pertussis infections in such patients is important. We therefore evaluated the seroprevalence of anti-pertussis toxin (PT) antibodies in a cohort of 40-85-year-old patients diagnosed with moderate, severe or very severe COPD enrolled (between June 2011 and June 2012) in the prospective, observational "Acute Exacerbation and Respiratory InfectionS in COPD" (AERIS; NCT01360398) study, conducted in England. Serum anti-PT antibodies were measured in 104 patients using an enzyme-linked immunosorbent assay on samples collected 12 months (M12) and 24 months (M24) after enrollment. Overall, 14/104 (13.5%) patients had anti-PT concentrations ≥50 IU/mL at M12 or M24, indicative of exposure to Bordetella pertussis during the preceding 2-3 years. Of these, 6/104 (5.8%) had anti-PT ≥70 IU/mL, of whom 3/104 (2.9%) had anti-PT ≥120 IU/mL, indicative of exposure within 12 and 6 months, respectively. These results show a high circulation of B. pertussis in 40-85-year-old patients with moderate, severe or very severe COPD in England between 2012 and 2014, and call for enhanced immunization to prevent pertussis infections in such patients.

Antibacterial substances produced by pathogen inhibitory gut bacteria in Labeo rohita: Physico-chemical characterization, purification and identification through MALDI-TOF mass spectrometry.

Application of antibiotics to combat bacterial diseases in fish has been criticized due to likely emergence of drug resistance. Therefore, investigation of new bioactive compounds from natural sources has been taken into account. This study was designed to purify and characterize the bioactive compound in the cell free supernatant (CFSs) of autochthonous gut bacteria (Bacillus methylotrophicus KU556164, B. amyloliquefaciens KU556165, Pseudomonas fluorescens KU556166 and B. licheniformis KU556167) isolated from rohu, Labeo rohita. CFSs were antagonistic to fish pathogenic Aeromonas spp., moderately thermo-tolerant and active in wide range of pH (5-11). Antibacterial activity of the CFSs was reduced by the action of proteases (e.g., Proteinase K and Trypsin), indicating proteinaceous nature of the bioactive compound like the bacteriocins. Three-step purification procedure resulted in recovery of 16.97%, 18.04%, 33.33% and 6.38% activity of the antimicrobial protein produced by B. methylotrophicus, B. amyloliquefaciens, P. fluorescens and B. licheniformis, respectively. Purification at each step revealed decrease in protein content with gradual increase in the specific activity of the antimicrobial protein. The purified antibacterial compound ranged between 18.2 and 25.6 kDa. Identification through MALDI-TOF MS/MS and database search through Mascot search engine predicted that the bactericidal compound belonged to either alkaline proteases, or, transcriptional regulator and some hypothetical proteins. Apart from potential technological application of the antibacterial compound, the present study might show promise for application of gut-associated bacteriocinogenic bacteria to control diseases in fish caused by pathogenic bacteria.

Campylobacter jejuni Colonization in the Crow Gut Involves Many Deletions within the Cytolethal Distending Toxin Gene Cluster.

Campylobacter spp. are major causes of gastroenteritis worldwide. The virulence potential of Campylobacter shed in crow feces obtained from a roost area in Bothell, Washington, was studied and compared with that from isolates from other parts of Washington and from a different crow species 7,000 miles away in Kolkata, India. Campylobacter organisms were isolated from 61% and 69% of the fecal samples obtained from Washington and Kolkata, respectively, and were confirmed to be C. jejuni The cytolethal distending toxin (CDT) gene cluster from these isolates revealed a truncated sequence of approximately 1,350 bp. Sequencing of the gene cluster revealed two types of mutations: a 668-bp deletion across cdtA and cdtB and a 51-bp deletion within cdtB Some strains had additional 20-bp deletions in cdtB In either case, a functional toxin is not expected; a functional toxin is produced by the expression of three tandem genes, cdtA, cdtB, and cdtC Reverse transcriptase PCR with total RNA extracted from the isolates showed no expression of cdtB A toxin assay performed with these isolates on HeLa cells failed to show cytotoxic effects on the cells. However, the isolates were able to colonize the chicken ceca for a period of at least 4 weeks, similar to that of a clinical isolate. Other virulence gene markers, flagellin A and CadF, were present in 100% of the isolates. Our study suggests that crows carry the bacterium C. jejuni but with a dysfunctional toxin protein that is expected to drastically reduce its potential to cause diarrhea.IMPORTANCE Campylobacters are a major cause of gastroenteritis in humans. Since outbreaks have most often been correlated with poultry or unpasteurized dairy products, contact with farm animals, or contaminated water, historically, the majority of the studies have been with campylobacter isolates from poultry, domestic animals, and human patients. However, the bacterium has a broad host range that includes birds. These reservoirs need to be investigated, because the identification of the source and a determination of the transmission routes for a pathogen are important for the development of evidence-based disease control programs. In this study, two species of the human-commensal crow, from two different geographical regions separated by 7,000 miles of land and water, have been examined for their ability to cause disease by shedding campylobacters. Our results show that the crow may not play a significant role in campylobacteriosis, because the campylobacter organisms they shed produce a nonfunctional toxin.

SARM1, Not MyD88, Mediates TLR7/TLR9-Induced Apoptosis in Neurons.

Neuronal apoptosis is a key aspect of many different neurologic diseases, but the mechanisms remain unresolved. Recent studies have suggested a mechanism of innate immune-induced neuronal apoptosis through the stimulation of endosomal TLRs in neurons. TLRs are stimulated both by pathogen-associated molecular patterns as well as by damage-associated molecular patterns, including microRNAs released by damaged neurons. In the present study, we identified the mechanism responsible for TLR7/TLR9-mediated neuronal apoptosis. TLR-induced apoptosis required endosomal localization of TLRs but was independent of MyD88 signaling. Instead, apoptosis required the TLR adaptor molecule SARM1, which localized to the mitochondria following TLR activation and was associated with mitochondrial accumulation in neurites. Deficiency in SARM1 inhibited both mitochondrial accumulation in neurites and TLR-induced apoptosis. These studies identify a non-MyD88 pathway of TLR7/ TLR9 signaling in neurons and provide a mechanism for how innate immune responses in the CNS directly induce neuronal damage.

Emergent emm4 group A Streptococcus evidences a survival strategy during interaction with immune effector cells.

The major gram-positive pathogen group A Streptococcus (GAS) is a model organism for studying microbial epidemics as it causes waves of infections. Since 1980, several GAS epidemics have been ascribed to the emergence of clones producing increased amounts of key virulence factors such as streptolysin O (SLO). Herein, we sought to identify mechanisms underlying our recently identified temporal clonal emergence amongst emm4 GAS, given that emergent strains did not produce augmented levels of virulence factors relative to historic isolates. Through the creation and analysis of isoallelic strains, we determined that a conserved mutation in a previously undescribed gene encoding a putative carbonic anhydrase was responsible for the defective in vitro growth observed in the emergent strains. We also identified that the emergent strains survived better inside macrophages and killed macrophages at lower rates relative to the historic strains. Via creation of isogenic mutant strains, we linked the emergent strain "survival" phenotype to the downregulation of the SLO encoding gene and upregulation of the msrAB operon which encodes proteins involved in defense against extracellular oxidative stress. Our findings are in accord with recent surveillance studies which found high ratio of mucosal (i.e., pharyngeal) relative to invasive infections amongst emm4 GAS. Inasmuch as ever-increasing virulence is unlikely to be evolutionary advantageous for a microbial pathogen, our data furthers understanding of the well described oscillating patterns of virulent GAS infections by demonstrating mechanisms by which emergent strains adapt a "survival" strategy to outcompete previously circulating isolates.

An Arm-to-Disarm Strategy to Overcome Phenotypic AMR in Mycobacterium tuberculosis.

Most front-line tuberculosis drugs are ineffective against hypoxic non-replicating drug-tolerant Mycobacterium tuberculosis (Mtb) contributing to phenotypic antimicrobial resistance (AMR). This is largely due to the poor permeability in the thick and waxy cell wall of persister cells, leading to diminished drug accumulation and reduced drug-target engagement. Here, using an "arm-to-disarm" prodrug approach, we demonstrate that non-replicating Mtb persisters can be sensitized to Moxifloxacin (MXF), a front-line TB drug. We design and develop a series of nitroheteroaryl MXF prodrugs that are substrates for bacterial nitroreductases (NTR), a class of enzymes that are over-expressed in hypoxic Mtb. Enzymatic activation involves electron-transfer to the nitroheteroaryl compound followed by protonation via water that contributes to the rapid cleavage rate of the protective group by NTR to produce the active drug. Phenotypic and genotypic data are fully consistent with MXF-driven lethality of the prodrug in Mtb with the protective group being a relatively innocuous bystander. The prodrug increased intracellular concentrations of MXF than MXF alone and is more lethal than MXF in non-replicating persisters. Hence, arming drugs to improve permeability, accumulation and drug-target engagement is a new therapeutic paradigm to disarm phenotypic AMR.

LiaR-dependent gene expression contributes to antimicrobial responses in group A Streptococcus.

The ability to sense and respond to host defenses is essential for pathogen survival. Some mechanisms involve two-component systems (TCS) that respond to host molecules, such as antimicrobial peptides (AMPs) and activate specific gene regulatory pathways to aid in survival. Alongside TCSs, bacteria coordinate cell division proteins, chaperones, cell wall sortases and secretory translocons at discrete locations within the cytoplasmic membrane, referred to as functional membrane microdomains (FMMs). In Group A Streptococcus (GAS), the FMM or "ExPortal" coordinates protein secretion, cell wall synthesis and sensing of AMP-mediated cell envelope stress via the LiaFSR three-component system. Previously we showed GAS exposure to a subset of AMPs (α-defensins) activates the LiaFSR system by disrupting LiaF and LiaS co-localization in the ExPortal, leading to increased LiaR phosphorylation, expression of the transcriptional regulator SpxA2, and altered GAS virulence gene expression. The mechanisms by which LiaFSR integrates cell envelope stress with responses to AMP activity and virulence are not fully elucidated. Here, we show the LiaFSR regulon is comprised of genes encoding SpxA2 and three membrane-associated proteins: a PspC domain-containing protein (PCP), the lipoteichoic acid-modifying protein LafB and the membrane protein insertase YidC2. Our data show phosphorylated LiaR induces transcription of these genes via a conserved operator, whose disruption attenuates GAS virulence and increases susceptibility to AMPs in a manner primarily dependent on differential expression of SpxA2. Our work expands understanding of the LiaFSR regulatory network in GAS and identifies targets for further investigation of mechanisms of cell envelope stress tolerance contributing to GAS pathogenesis.

Toll-like receptor 7 suppresses virus replication in neurons but does not affect viral pathogenesis in a mouse model of Langat virus infection.

Toll-like receptor 7 (TLR7) recognizes guanidine-rich viral ssRNA and is an important mediator of peripheral immune responses to several ssRNA viruses. However, the role that TLR7 plays in regulating the innate immune response to ssRNA virus infections in specific organs such as the central nervous system (CNS) is not as clear. This study examined the influence of TLR7 on the neurovirulence of Langat virus (LGTV), a ssRNA tick-borne flavivirus. TLR7 deficiency did not substantially alter the onset or incidence of LGTV-induced clinical disease; however, it did significantly affect virus levels in the CNS with a log(10) increase in virus titres in brain tissue from TLR7-deficient mice. This difference in virus load was also observed following intracranial inoculation, indicating a direct effect of TLR7 deficiency on regulating virus replication in the brain. LGTV-induced type I interferon responses in the CNS were not dependent on TLR7, being higher in TLR7-deficient mice compared with wild-type controls. In contrast, induction of pro-inflammatory cytokines including tumour necrosis factor, CCL3, CCL4 and CXCL13 were dependent on TLR7. Thus, although TLR7 is not essential in controlling LGTV pathogenesis, it is important in controlling virus infection in neurons in the CNS, possibly by regulating neuroinflammatory responses.

The curious case of SARM1: Dr. Jekyll and Mr. Hyde in cell death and immunity?

Sterile alpha and toll/interleukin-1 receptor motif-containing protein 1 (SARM1) was first identified as a novel ortholog of Drosophila protein CG7915 and was subsequently placed as the fifth member of the human TIR-containing adaptor protein. SARM1 holds a unique position in this family where, unlike other members, it downregulates NFκB activity in response to immunogenic stimulation, interacts with another member of the family, TRIF, to negatively regulate its function, and it also mediates cell death responses. Over the past decade, SARM1 has emerged as one of the primary mediators of programmed axonal degeneration and this robust regulation of axonal degeneration-especially in models of peripheral neuropathy and traumatic injury-makes it an attractive target for therapeutic intervention. The TIR domain of SARM1 possesses an intrinsic NADase activity resulting in cellular energy deficits within the axons, a striking deviation from its other family members of human TLR adaptors. Interestingly, the TIR NADase activity, as seen in SARM1, is also observed in several prokaryotic TIR-containing proteins where they are involved in immune evasion once within the host. Although the immune function of SARM1 is yet to be conclusively discerned, this closeness in function with the prokaryotic TIR-domain containing proteins, places it at an interesting juncture of evolution raising questions about its origin and function in cell death and immunity. In this review, we discuss how a conserved immune adaptor protein like SARM1 switches to a pro-neurodegenerative function and the evolutionarily significance of the process.

Early loss of endogenous NAD+ following rotenone treatment leads to mitochondrial dysfunction and Sarm1 induction that is ameliorated by PARP inhibition.

Sarm1 is an evolutionary conserved innate immune adaptor protein that has emerged as a primary regulator of programmed axonal degeneration over the past decade. In vitro structural insights have revealed that although Sarm1 induces energy depletion by breaking down nicotinamide adenine dinucleotide+ (NAD+ ), it is also allosterically inhibited by NAD+ . However, how NAD+ levels modulate the activation of intracellular Sarm1 has not been elucidated so far. This study focuses on understanding the events leading to Sarm1 activation in both neuronal and non-neuronal cells using the mitochondrial complex I inhibitor rotenone. Here, we report the regulation of rotenone-induced cell death by loss of NAD+ that may act as a 'biological trigger' of Sarm1 activation. Our study revealed that early loss of endogenous NAD+ levels arising due to PARP1 hyperactivation preceded Sarm1 induction following rotenone treatment. Interestingly, replenishing NAD+ levels by the PARP inhibitor, PJ34 restored mitochondrial complex I activity and also prevented subsequent Sarm1 activation in rotenone-treated cells. These cellular data were further validated in Drosophila melanogaster where a significant reduction in rotenone-mediated loss of locomotor abilities, and reduced dSarm expression was observed in the flies following PARP inhibition. Taken together, these observations not only uncover a novel regulation of Sarm1 induction by endogenous NAD+ levels but also point towards an important understanding on how PARP inhibitors could be repurposed in the treatment of mitochondrial complex I deficiency disorders.

Molecularly Imprinted Polymer Interface on Screen-Printed ZnO Nanorod Field Effect Transistors for Serotonin Detection in Clinical Samples.

Ultrasensitive detection of serotonin is crucial for the early diagnosis of several diseases like Parkinson's and Alzheimer's. Most of the existing detection strategies are still not suitable for sensitive point-of-care applications. This study presents direct molecular imprinting of serotonin on the surface of three-dimensional zinc oxide (ZnO) nanorod devices connected in a field effect transistor (FET) configuration to achieve ultrasensitive, real-time, and rapid detection with a convenient and affordable approach, which has significant potential for translation to clinical settings. This strategy has enabled pushing the detection limit to 0.1 fM in a physiological analyte in real time with screen-printed electrodes, thereby resulting in the convenient batch fabrication of sensors for clinical validation. The response of the sensor with the clinical sample has been correlated with that of the gold standard and has been observed to be statistically similar.

Development and evaluation of a simple PCR assay and nested PCR for rapid detection of clarithromycin-resistant Helicobacter pylori from culture and directly from the biopsy samples in India.

BACKGROUND: Eradication of Helicobacter pylori provides the most effective treatment for gastroduodenal diseases caused by H. pylori infection. Clarithromycin, a member of the macrolide family, still remains the most important antibiotic used in H. pylori eradication treatment. But the increasing prevalence of clarithromycin resistant H. pylori strains due to point mutations in the V region of the 23S rRNA, poses a great threat in treating the ailing patients. So, we aimed for PCR-mediated rapid detection of the point mutation at 2143 position of 23S rRNA gene in H. pylori that is relevant to clarithromycin resistance from culture and simultaneously from biopsy specimens to avoid the empirical treatment. RESULTS: Newly developed PCR assay using DNA of pure culture detected point mutation in 23S rRNA gene in 21 (8.04%) of 261 clinical strains tested. The agar dilution method showed that all these 21 strains were resistant to clarithromycin indicating the perfect match of the PCR based results. Additionally, the sequencing study also identified the A to G mutation at 2143 position in 23S rRNA gene of the resistant strains only. Consequently, the newly developed Nested-ASP-PCR dealing directly with 50 biopsy specimens demonstrated 100% sensitivity and specificity with the findings of agar dilution method taken as Gold standard. Bioinformatics based analysis such as accessibility analysis and dot plot clearly stated that the base pairing probability has increased due to mutation. Computational studies revealed that the point mutation confers more stability in secondary structure due to conversion of loop to stem. Furthermore, interaction studies showed binding affinity of the CLR to the mutant type is weaker than that to the wild type. CONCLUSION: This assay outlines a rapid, sensitive and simple approach to identify point mutation that confers clarithromycin resistance as well as clarithromycin sensitive strains, providing rapid initiation of effective antibiotic treatment. Additionally, it is simple to adopt for hospital based diagnostic laboratories to evaluate the degree of regional clarithromycin resistance from biopsy specimens itself. Furthermore, in silico studies provide evidence or a signal that the prevalence of clarithromycin resistance may rise in the near future as a result of this point mutation.

Burden of Pertussis in Adults Aged 50 Years and Older: A Retrospective Database Study in England.

INTRODUCTION: Pertussis, a highly infectious respiratory disease caused by Bordetella pertussis, affects people of all ages. Older adults are particularly susceptible to its severe outcomes and complications. METHODS: In this retrospective cohort study, the incidence rate of pertussis among individuals aged ≥ 50 years was assessed during 2009-2018 using Clinical Practice Research Datalink and Hospital Episode Statistics databases, United Kingdom. Health care resource utilisation (HCRU) and direct medical costs (DMCs) were compared between patients with a pertussis diagnosis and propensity score-matched controls (matched on demographic and clinical variables). RESULTS: Among 5,222,860 individuals, 1638 had a pertussis diagnosis (incidence rate: 5.8 per 100,000 person-years; 95% confidence interval 5.5-6.0). Baseline (- 18 to - 6 months) HCRU and DMC were similar among 1480 pertussis patients and 1480 matched controls. However, there were increases in HCRU in the pertussis vs. matched cohort around the pertussis diagnosis (from months - 6 to - 1 to 5-11). The most notable increases (pertussis vs. controls) were in the rates of general practitioner (GP)/nurse visits (4.7-fold), clinical assessments (4.1-fold), and accident and emergency visits (3.0-fold) during the month before diagnosis and GP/nurse visits during the 2 months after diagnosis (2.5-fold) (all p < 0.001). DMCs were significantly higher in the pertussis cohort (p < 0.001). Total excess DMC in the pertussis cohort during months - 1 to + 11 was £318 per patient. CONCLUSION: A pertussis diagnosis among adults aged ≥ 50 years resulted in significant increases in HCRU and DMC across several months around diagnosis. These results highlight the need for increased awareness of pertussis infection among adults aged ≥ 50 years and suggest that pertussis booster doses among this population should be considered.

Identification of distinct impacts of CovS inactivation on the transcriptome of acapsular group A streptococci.

Group A streptococcal (GAS) strains causing severe, invasive infections often have mutations in the control of virulence two-component regulatory system (CovRS) which represses capsule production, and high-level capsule production is considered critical to the GAS hypervirulent phenotype. Additionally, based on studies in emm1 GAS, hyperencapsulation is thought to limit transmission of CovRS-mutated strains by reducing GAS adherence to mucosal surfaces. It has recently been identified that about 30% of invasive GAS strains lacks capsule, but there are limited data regarding the impact of CovS inactivation in such acapsular strains. Using publicly available complete genomes (n = 2,455) of invasive GAS strains, we identified similar rates of CovRS inactivation and limited evidence for transmission of CovRS-mutated isolates for both encapsulated and acapsular emm types. Relative to encapsulated GAS, CovS transcriptomes of the prevalent acapsular emm types emm28, emm87, and emm89 revealed unique impacts such as increased transcript levels of genes in the emm/mga region along with decreased transcript levels of pilus operon-encoding genes and the streptokinase-encoding gene ska. CovS inactivation in emm87 and emm89 strains, but not emm28, increased GAS survival in human blood. Moreover, CovS inactivation in acapsular GAS reduced adherence to host epithelial cells. These data suggest that the hypervirulence induced by CovS inactivation in acapsular GAS follows distinct pathways from the better studied encapsulated strains and that factors other than hyperencapsulation may account for the lack of transmission of CovRS-mutated strains. IMPORTANCE Devastating infections due to group A streptococci (GAS) tend to occur sporadically and are often caused by strains that contain mutations in the control of virulence regulatory system (CovRS). In well-studied emm1 GAS, the increased production of capsule induced by CovRS mutation is considered key to both hypervirulence and limited transmissibility by interfering with proteins that mediate attachment to eukaryotic cells. Herein, we show that the rates of covRS mutations and genetic clustering of CovRS-mutated isolates are independent of capsule status. Moreover, we found that CovS inactivation in multiple acapsular GAS emm types results in dramatically altered transcript levels of a diverse array of cell-surface protein-encoding genes and a unique transcriptome relative to encapsulated GAS. These data provide new insights into how a major human pathogen achieves hypervirulence and indicate that factors other than hyperencapsulation likely account for the sporadic nature of the severe GAS disease.