Respiratory carriage of hypervirulent Klebsiella pneumoniae by indigenous populations of Malaysia.

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low.

Multicentre case-control study of pneumococcal infections among children with pneumonia in Peninsular Malaysia (MY-Pneumo): a study protocol.

BACKGROUND: S. pneumoniae (SPN) is the most common cause of pneumonia. The disease can be effectively prevented through immunisation. Since December 2020, the Malaysian Government has included the 10-valent pneumococcal conjugate vaccine (PCV10) for all infants born on or after 1 January 2020 as part of the National Immunisation Programme (NIP). However, the epidemiology of pneumonia remains poorly understood. To fill the knowledge gap, we established a multicentre surveillance study to understand the burden of pneumococcal pneumonia among young children in Peninsular Malaysia. METHODS: MY-Pneumo is a multicentre prospective case-control study conducted in three sentinel sites located in three different states of Peninsular Malaysia - Kuala Lumpur, Pahang, and Kelantan. A cohort of at least 500 incident cases and 500 controls is enrolled beginning in October 2021 and matched for age. Cases are hospitalised children < 5 years with radiologically confirmed pneumonia, and the controls are children without any features suggestive of pneumonia. Clinical samples, including nasopharyngeal swabs (NPS) and urine, are collected according to the study protocol. Biological fluids such as blood, cerebrospinal fluid (CSF) and pleural fluid are obtained from invasive pneumonia disease (IPD) patients, if available. All children are tested for SPN using polymerase chain reaction (PCR) and pneumococcal urine antigen test (PUAT) using BinaxNow. DISCUSSION: Surveillance data, including carriage rate, serotype variations and the phylogeny data structure of SPN among young children in Malaysia during PCV implementation, will be generated from this study. Trends and patterns of pneumococcal serotypes by different regions are important for targeted public health strategies. Our data will provide baseline information for estimating the impact of PCV10 implementation and will influence policymakers' decisions regarding the upgrade from PCV10 to a higher-valency conjugate vaccine in Malaysia. TRIAL REGISTRATION: This project was registered at ClinicalTrials.gov (NCT04923035) on 2021, June 11. The study protocol was approved by the International Medical University Joint-Committee on Research & Ethics (4.15/JCM-216/2021) and the Institutional Review Board at sentinel sites (USM/JEPeM/21020190, IREC 2021-114, MREC ID No: 2021128-9769) and University of Southampton's Ethics and Research Governance (ERGo II 64844).

Nontypeable Haemophilus influenzae infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) is a respiratory tract pathobiont that chronically colonizes the airways of asthma patients and is associated with severe, neutrophilic disease phenotypes. The mechanism of NTHi airway persistence is not well understood, but accumulating evidence suggests NTHi can persist within host airway immune cells such as macrophages. We hypothesized that NTHi infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma. METHODS: Bronchoalveolar lavage (BAL) samples from 25 severe asthma patients were assessed by fluorescence in situ hybridisation to quantify NTHi presence. Weighted gene correlation network analysis (WGCNA) was performed on RNASeq data from NTHi-infected monocyte-derived macrophages to identify transcriptomic networks associated with NTHi infection. RESULTS: NTHi was detected in 56% of BAL samples (NTHi+) and was associated with longer asthma duration (34 vs 22.5 years, p = .0436) and higher sputum neutrophil proportion (67% vs 25%, p = .0462). WGCNA identified a transcriptomic network of immune-related macrophage genes significantly associated with NTHi infection, including upregulation of T17 inflammatory mediators and neutrophil chemoattractants IL1B, IL8, IL23 and CCL20 (all p < .05). Macrophage network genes SGPP2 (p = .0221), IL1B (p = .0014) and GBP1 (p = .0477) were more highly expressed in NTHi+ BAL and moderately correlated with asthma duration (IL1B; rho = 0.41, p = .041) and lower prebronchodilator FEV1/FVC% (GBP1; rho = -0.43, p = .046 and IL1B; rho = -0.42, p = .055). CONCLUSIONS: NTHi persistence with pulmonary macrophages may contribute to chronic airway inflammation and T17 responses in severe asthma, which can lead to decreased lung function and reduced steroid responsiveness. Identifying therapeutic strategies to reduce the burden of NTHi in asthma could improve patient outcomes.

Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons.

The development of sensors for the detection of pathogen-specific DNA, including relevant species/strain level discrimination, is critical in molecular diagnostics with major impacts in areas such as bioterrorism and food safety. Herein, we use electrochemically driven denaturation assays monitored by surface-enhanced Raman spectroscopy (SERS) to target single nucleotide polymorphisms (SNPs) that distinguish DNA amplicons generated from Yersinia pestis, the causative agent of plague, from the closely related species Y. pseudotuberculosis. Two assays targeting SNPs within the groEL and metH genes of these two species have been successfully designed. Polymerase chain reaction (PCR) was used to produce Texas Red labeled single-stranded DNA (ssDNA) amplicons of 262 and 251 bases for the groEL and metH targets, respectively. These amplicons were used in an unpurified form to hybridize to immobilized probes then subjected to electrochemically driven melting. In all cases electrochemically driven melting was able to discriminate between fully homologous DNA and that containing SNPs. The metH assay was particularly challenging due to the presence of only a single base mismatch in the middle of the 251 base long PCR amplicon. However, manipulation of assay conditions (conducting the electrochemical experiments at 10 °C) resulted in greater discrimination between the complementary and mismatched DNA. Replicate data were collected and analyzed for each duplex on different days, using different batches of PCR product and different sphere segment void (SSV) substrates. Despite the variability introduced by these differences, the assays are shown to be reliable and robust providing a new platform for strain discrimination using unpurified PCR samples.

Microbiome alterations are associated with apolipoprotein E mutation in Octodon degus and humans with Alzheimer's disease.

Gut microbiome dysbiosis is linked to many neurological disorders including Alzheimer's disease (AD). A major risk factor for AD is polymorphism in the apolipoprotein E (APOE) gene, which affects gut microbiome composition. To explore the gut-brain axis in AD, long-lived animal models of naturally developing AD-like pathologies are needed. Octodon degus (degu) exhibit spontaneous AD-like symptoms and ApoE mutations, making them suitable for studying the interplay between AD genetic determinants and gut microbiome. We analyzed the association between APOE genotype and gut microbiome in 50 humans and 32 degu using16S rRNA gene amplicon sequencing. Significant associations were found between the degu ApoE mutation and gut microbial changes in degu, notably a depletion of Ruminococcaceae and Akkermansiaceae and an enrichment of Prevotellaceae, mirroring patterns seen in people with AD. The altered taxa were previously suggested to be involved in AD, validating the degu as an unconventional model for studying the AD/microbiome crosstalk.

Non-pharmaceutical interventions for COVID-19 transiently reduced pneumococcal and Haemophilus influenzae carriage in a cross-sectional pediatric cohort in Southampton, UK.

The Southampton pneumococcal carriage study of children under 5 years old continued during the coronavirus disease 2019 (COVID-19) pandemic. Here, we present data from October 2018 to March 2023 describing prevalence of pneumococci and other pathobionts during the winter seasons before, during, and after the introduction of non-pharmaceutical interventions (NPIs) to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Nasopharyngeal swabs were collected from children attending outpatient clinics at a secondary care hospital and community healthcare sites. Pre-NPIs, in 2019/2020, the carriage prevalence of pneumococci at the hospital site was 32% (n = 161 positive/499 participants). During NPIs, this fell to 19% (n = 12/64), although based on fewer participants compared to previous years due to COVID-19 restrictions on health-care attendance. In 2021/2022, after NPIs had eased, prevalence rebounded to 33% (n = 15/46) [compared to NPIs period, χ2 (1, N = 110) =2.78, P = 0.09]. Carriage prevalence at community healthcare sites fell significantly from 27% (n = 127/470) in 2019/2020 to 19% during the NPI period (n = 44/228) in 2020/2021 [χ2 (1, N = 698) =4.95, P = 0.026]. No rebound was observed in 2021/2022 [19% (n = 56/288)]. However, in a multivariate logistic regression model, neither site had a significantly lower carriage prevalence during the NPI period compared to the post NPI period. A reduction in serotype diversity was observed in 2020/2021. Carriage of Haemophilus influenzae was particularly affected by NPIs with a significant reduction observed. In conclusion, among children under 5 years of age, transient, modest, and statistically non-significant alterations in carriage of both Streptococcus pneumoniae and H. influenzae were associated with SARS-CoV-2 NPIs.IMPORTANCEStreptococcus pneumoniae (the pneumococcus) continues to be a major contributor to global morbidity and mortality. Using our long-running pediatric study, we examined changes in pneumococcal carriage prevalence in nearly 3,000 children under the age of 5 years between the winters of 2018/2019 and 2022/2023. This period coincided with the severe acute respiratory syndrome coronavirus 2 pandemic and, in particular, the implementation of national strategies to limit disease transmission in the UK. We observed a transient reduction of both Streptococcus pneumoniae and Haemophilus influenzae in these populations during this period of non-pharmaceutical interventions. This aligned with the reduction in invasive pneumococcal disease seen in the UK and is therefore a likely contributor to this phenomenon.

A brief history of and future prospects for pneumococcal vaccination in Malaysia.

Pneumococcal pneumonia remains a significant global public health issue. Malaysia has recently added the 10 valent pneumococcal conjugate vaccine to its national immunisation programme. Data on pneumococcal serotype epidemiology is vital for informing national vaccination policy. However, there remains a lack of representative population-based pneumococcal surveillance in Malaysia to help both the assessment of vaccine effectiveness in the country and to shape future vaccine policy. This review explores the history of pneumococcal vaccination, the burden of pneumococcal disease in Malaysia, and offers an insight into the prospects for reducing pneumococcal disease in Malaysia.

Oral and gut microbial biomarkers of susceptibility to respiratory tract infection in adults: A feasibility study.

We conducted a feasibility cohort study which aimed to recruit and retain adults from the community to collect saliva (oral) and stool (gut) samples at three time points, at the start of the study (baseline), during a respiratory tract infection (RTI) and post-RTI. Community RTIs place a huge burden on health care services, and a non-invasive microbial diagnostic tool to predict the most vulnerable to respiratory infection would be ideal. To this aim, we analysed oral-gut baseline samples comparing those who reported RTI symptoms to those who remained healthy throughout the study for microbial biomarkers of respiratory susceptibility. Amplicon sequence variants (ASV) were identified by 16S sequence profiling to reveal oral-gut microbes. Reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to target common respiratory microbes. Two general practices were recruited, and the participant recruitment rate was 1.3%. A total of 40 adult participants were retained, of which 19 acquired an RTI whereas 21 remained healthy. In healthy baseline oral and gut samples, ASVs from participants with RTI symptoms compared to those who remained healthy were similar with a high relative abundance of Streptococcus sp., and Blautia sp., respectively. Linear discriminant analysis effect size (LEfSe) revealed baseline oral microbes differed, indicating participants who suffered RTI symptoms had enhanced Streptococcus sobrinus and Megamonas sp., and depletion of Lactobacillus salivarius, Synergistetes, Verrucomicrobia and Dethiosulfovibrio. Furthermore, a random forest model ranked Streptococcus (4.13) as the highest mean decrease in accuracy (MDA) and RT-PCR showed a higher level of carriage of coagulase-negative Staphylococcus. Baseline core gut microbes were similar in both participant groups whereas LEfSe analysis revealed enhanced Veillonella, Rikenellaceae, Enhydobacteria, Eggerthella and Xanthomonsdales and depleted Desulfobulbus and Coprobacillus. Sutterella (4.73) had a high MDA value. Overall, we demonstrated the feasibility of recruiting and retaining adult participants from the community to provide multiple biological samples for microbial profiling. Our analyses identified potential oral-gut microbial biomarkers of respiratory infection susceptibility in otherwise healthy participants.

Comparative genomic epidemiology of serotype 3 IPD and carriage isolates from Southampton, UK between 2005 and 2017.

Serotype 3 pneumococci remains a significant cause of disease despite its inclusion in PCV13. Whilst clonal complex 180 (CC180) represents the major clone, recent studies have refined the population structure into three clades: Iα, Iß and II, with the last being a recent divergent and more antibiotic-resistant. We present a genomic analysis of serotype 3 isolates from paediatric carriage and all-age invasive disease, collected between 2005 and 2017 in Southampton, UK. Forty-one isolates were available for analysis. Eighteen were isolated during the annual cross-sectional surveillance of paediatric pneumococcal carriage. The remaining 23 were isolated from blood/cerebrospinal fluid specimens at the University Hospital Southampton NHS Foundation Trust laboratory. All carriage isolates were CC180 GPSC12. Greater diversity was seen with invasive pneumococcal disease (IPD) with three GPSC83 (ST1377: n=2, ST260: n=1) and one GPSC3 (ST1716). For both carriage and IPD, Clade Iα was dominant (94.4 and 73.9 % respectively). Two isolates were Clade II with one from carriage (a 34-month-old, October 2017) and one invasive isolate (49-year-old, August 2015). Four IPD isolates were outside the CC180 clade. All isolates were genotypically susceptible to penicillin, erythromycin, tetracycline, co-trimoxazole and chloramphenicol. Two isolates (one each from carriage and IPD; both CC180 GPSC12) were phenotypically resistant to erythromycin and tetracycline; the IPD isolate was also resistant to oxacillin.In the Southampton area, carriage and invasive disease associated with serotype 3 is predominantly caused by Clade Iα CC180 GPSC12.

The characterization of Moraxella catarrhalis carried in the general population.

Moraxella catarrhalis is a common cause of respiratory tract infection, particularly otitis media in children, whilst it is also associated with the onset of exacerbation in chronic obstructive pulmonary disease in adults. Despite the need for an efficacious vaccine against M. catarrhalis, no candidates have progressed to clinical trial. This study, therefore, aimed to characterize the diversity of M. catarrhalis isolated from the upper respiratory tract of healthy children and adults, to gain a better understanding of the epidemiology of M. catarrhalis and the distribution of genes associated with virulence factors, to aid vaccine efforts. Isolates were sequenced and the presence of target genes reported. Contrary to prevailing data, this study found that lipooligosaccharide (LOS) B serotypes are not exclusively associated with 16S type 1. In addition, a particularly low prevalence of LOS B and high prevalence of LOS C serotypes was observed. M. catarrhalis isolates showed low prevalence of antimicrobial resistance and a high gene prevalence for a number of the target genes investigated: ompB2 (also known as copB), ompCD, ompE, ompG1a, ompG1b, mid (also known as hag), mcaP, m35, tbpA, lbpA, tbpB, lbpB, msp22, msp75 and msp78, afeA, pilA, pilQ, pilT, mod, oppA, sbp2, mcmA and mclS.

Controlled human infection with Neisseria lactamica in late pregnancy to measure horizontal transmission and microbiome changes in mother-neonate pairs: a single-arm interventional pilot study protocol.

INTRODUCTION: Infant upper respiratory microbiota are derived partly from the maternal respiratory tract, and certain microbiota are associated with altered risk of infections and respiratory disease. Neisseria lactamica is a common pharyngeal commensal in young children and is associated with reduced carriage and invasive disease by Neisseria meningitidis. Nasal inoculation with N. lactamica safely and reproducibly reduces N. meningitidis colonisation in healthy adults. We propose nasal inoculation of pregnant women with N. lactamica, to establish if neonatal pharyngeal colonisation occurs after birth, and to characterise microbiome evolution in mother-infant pairs over 1 month post partum. METHODS AND ANALYSIS: 20 healthy pregnant women will receive nasal inoculation with N. lactamica (wild type strain Y92-1009) at 36-38 weeks gestation. Upper respiratory samples, as well as optional breastmilk, umbilical cord blood and infant venous blood samples, will be collected from mother-infant pairs over 1 month post partum. We will assess safety, N. lactamica colonisation (by targeted PCR) and longitudinal microevolution (by whole genome sequencing), and microbiome evolution (by 16S rRNA gene sequencing). ETHICS AND DISSEMINATION: This study has been approved by the London Central Research Ethics Committee (21/PR/0373). Findings will be published in peer-reviewed open-access journals as soon as possible. TRIAL REGISTRATION NUMBER: NCT04784845.

Draft Genome Sequences of Two Acinetobacter soli Clinical Isolates from a Tertiary Hospital in Terengganu, Malaysia.

We report the draft genome sequences of Acinetobacter soli AC1511 and AC15148, which were isolated from a tertiary hospital in Terengganu, Malaysia, in 2015. AC1511 was assembled into 43 contigs with a total genome size of 3,320,693 bp, whereas AC15148 was 3,260,687 bp over 47 contigs.

Impact of bacterial strain acquisition in the lung of patients with COPD: the AERIS study.

BACKGROUND: Bacterial infections are associated with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but the mechanism is incompletely understood. METHOD: In a COPD observational study (NCT01360398), sputum samples were collected monthly at the stable state and exacerbation. Post-hoc analyses of 1307 non-typeable Haemophilus influenzae (NTHi) isolates from 20 patients and 756 Moraxella catarrhalis isolates from 38 patients in one year of follow-up were conducted by multilocus sequence typing (MLST). All isolates came from cultured sputum samples that were analyzed for bacterial species presence, apparition (infection not detected at the preceding visit), or acquisition (first-time infection), with the first study visit as a baseline. Strain apparition or new strain acquisition was analyzed by MLST. The odds ratio (OR) of experiencing an exacerbation vs. stable state was estimated by conditional logistic regression modelling, stratified by patient. RESULTS: The culture results confirmed a significant association with exacerbation only for NTHi species presence (OR 2.28; 95% confidence interval [CI]: 1.12-4.64) and strain apparition (OR 2.38; 95% CI: 1.08-5.27). For M. catarrhalis, although confidence intervals overlapped, the association with exacerbation for first-time species acquisition (OR 5.99; 2.75-13.02) appeared stronger than species presence (OR 3.67; 2.10-6.40), new strain acquisition (OR 2.94; 1.43-6.04), species apparition (OR 4.18; 2.29-7.63), and strain apparition (OR 2.78; 1.42-5.42). This may suggest that previous M. catarrhalis colonization may modify the risk of exacerbation associated with M. catarrhalis infection. CONCLUSIONS: The results confirm that NTHi and M. catarrhalis infections are associated with AECOPD but suggest different dynamic mechanisms in triggering exacerbations.

Comparative genomic analysis of clinical Acinetobacter nosocomialis isolates from Terengganu, Malaysia led to the discovery of a novel tetracycline-resistant plasmid.

OBJECTIVES: To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance. METHODS: Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform. RESULTS: The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes. CONCLUSIONS: Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

Changes in serotype prevalence of Streptococcus pneumoniae in Southampton, UK between 2006 and 2018.

Streptococcus pneumoniae continues to cause significant disease burden. Whilst pneumococcal conjugate vaccines (PCV) have substantially reduced this burden, serotype replacement partially negates this success due to increased disease associated with non-vaccine serotypes (NVTs). Continued surveillance is therefore essential to provide crucial epidemiological data. Annual cross-sectional surveillance of paediatric pneumococcal carriage was started in Southampton, UK following PCV7 roll-out in 2006. Nasopharyngeal swabs were collected from children < 5 years old each winter (October to March) from 2006/07 and for each consecutive year until 2017/18. Pneumococcal serotype was inferred from whole genome sequencing data. A total of 1429 (32.5%) pneumococci were isolated from 4093 children. Carriage ranged from 27.8% (95%CI 23.7-32.7) in 2008/09 to 37.9% (95%CI 32.8-43.2) in 2014/15. Analyses showed that carriage increased in children aged 24-35 months (p < 0.001) and 47-60 months (p < 0.05). Carriage of PCV serotypes decreased markedly following PCV7 and/or PCV13 introduction, apart from serotype 3 where the relative frequency was slightly lower post-PCV13 (pre-PCV13 n = 7, 1.67%; post-PCV13 n = 13, 1.27%). Prevalence of NVTs implicated in increased disease was low with 24F (n = 19, 1.4%) being the most common followed by 9N (n = 11, 0.8%), 8 (n = 7, 0.5%) and 12F (n = 3, 0.2%).

Preclinical evaluation method for total knees designed to restore normal knee mechanics.

The objective was to develop a simple, rapid, and low-cost method for evaluating proposed new total knee arthroplasty (TKA) models and then to evaluate 3 different TKA models with different kinematic characteristics. A "desktop" knee testing rig was used to apply forces and moments over a full flexion range, representing a spectrum of positions and activities; and the positions of the femur on the tibia were measured. The average neutral path of motion (for compressive force only) and the laxities about the neutral path (for superimposed shear and torque) were determined from 8 knee specimens to be used as a benchmark for the TKA evaluations. A typical posterior-stabilized TKA did not display the normal external femoral rotation with flexion and also showed abnormal anterior sliding on the medial side. A medial-pivot type of guided-motion design showed medial stability comparable to anatomical but still did not produce external femoral rotation and posterior lateral displacement with flexion. The addition of a central cam-post produced the rotation and displacement but only after 75° of flexion. It was concluded that the test method satisfied the objective and could be used as a design tool for evaluating new and existing designs, as well as for formulating a TKA with anatomical characteristics.

Manipulating the infant respiratory microbiomes to improve clinical outcomes: A review of the literature.

BACKGROUND: The association between infant respiratory microbiota and disease (including respiratory tract infections and asthma) is increasingly recognised, although the mechanism remains unclear. Respiratory infections and asthma account for a large proportion of infant morbidity and mortality, so the possibility of preventing disease or modifying clinical outcomes by manipulating microbiome development warrants investigation. OBJECTIVES AND METHODS: We identified studies that investigated the efficacy of live bacteria (probiotics or human challenge) or their substrates to modify respiratory colonisation or clinical outcomes in infants. ELIGIBILITY CRITERIA: Interventional studies involving infants under one year of age, administration of live bacteria or their substrates, and outcome measures including bacterial colonisation, microbiome profile, or respiratory disease phenotypes. RESULTS AND LIMITATIONS: Some bacterial interventions can reduce infant respiratory infections, although none have been shown to reduce asthma incidence. The literature is heterogeneous in design and quality, precluding meaningful meta-analysis. CONCLUSIONS: Upper respiratory tract infant microbiome manipulation may alter outcomes in respiratory tract infection, but further well-conducted research is needed to confirm this. Improved regulation of proprietary bacterial products is essential for further progress.

Serotype distribution of invasive, non-invasive and carried Streptococcus pneumoniae in Malaysia: a meta-analysis.

BACKGROUND: Pneumococcal pneumonia is the leading cause of under-five mortality globally. The surveillance of pneumococcal serotypes is therefore vital for informing pneumococcal vaccination policy and programmes. Pneumococcal conjugate vaccines (PCVs) have been available as an option in the private healthcare setting and beginning December 2020, PCV10 was incorporated as part of routine national immunisation programme (NIP) in Malaysia. We searched existing literature on pneumococcal serotype distribution across Malaysia to provide an overall view of this distribution before the implementation of PCV10. METHODS: Online databases (PubMed, Ovid MEDLINE and Scopus), reference lists of articles identified, and grey literature (Malaysian Ministry of Health website, WHO website) were systematically searched for relevant literature on pneumococcal serotype distribution across Malaysia up to 10th November 2020. No lower date limit was set to maximise the number of target reports returned. Results of serotypes were split by age categories, including ≤5 years, > 5 years and unreported for those that did not specify. RESULTS: The search returned 18 relevant results, with a total of 2040 isolates. The most common serotypes across all disease types were 19F (n = 313, 15.3% [95%CI: 13.8-17.0]), 23F (n = 166, 8.1% [95%CI: 7.0-9.4]), 14 (n = 166, 8.1% [95%CI: 7.0-9.4]), 6B (n = 163, 8.0% [95%CI: 6.9-9.2]) and 19A (n = 138, 6.8% [95%CI: 5.8-7.9]). CONCLUSION: Four of the most common serotypes across all isolate sources in Malaysia are covered by PCV10, while PCV13 provides greater serotype coverage in comparison to PCV10. There is still a need for surveillance studies, particularly those investigating serotypes in children under 5 years of age, to monitor vaccine effectiveness and pneumococcal population dynamic following implementation of PCV10 into routine immunisation.

Dual RNASeq Reveals NTHi-Macrophage Transcriptomic Changes During Intracellular Persistence.

Nontypeable Haemophilus influenzae (NTHi) is a pathobiont which chronically colonises the airway of individuals with chronic respiratory disease and is associated with poor clinical outcomes. It is unclear how NTHi persists in the airway, however accumulating evidence suggests that NTHi can invade and persist within macrophages. To better understand the mechanisms of NTHi persistence within macrophages, we developed an in vitro model of NTHi intracellular persistence using human monocyte-derived macrophages (MDM). Dual RNA Sequencing was used to assess MDM and NTHi transcriptomic regulation occurring simultaneously during NTHi persistence. Analysis of the macrophage response to NTHi identified temporally regulated transcriptomic profiles, with a specific 'core' profile displaying conserved expression of genes across time points. Gene list enrichment analysis identified enrichment of immune responses in the core gene set, with KEGG pathway analysis revealing specific enrichment of intracellular immune response pathways. NTHi persistence was facilitated by modulation of bacterial metabolic, stress response and ribosome pathways. Levels of NTHi genes bioC, mepM and dps were differentially expressed by intracellular NTHi compared to planktonic NTHi, indicating that the transcriptomic adaption was distinct between the two different NTHi lifestyles. Overall, this study provides crucial insights into the transcriptomic adaptations facilitating NTHi persistence within macrophages. Targeting these reported pathways with novel therapeutics to reduce NTHi burden in the airway could be an effective treatment strategy given the current antimicrobial resistance crisis and lack of NTHi vaccines.

Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases.

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-ß-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.