Abstract
Background
Enteric fever is common in southeast Asia. However, there is little information on the circulating Salmonella enterica strains causing enteric fever in Myanmar.
Methods
We ...performed antimicrobial susceptibility testing and whole genome sequencing on S. enterica bloodstream isolates from febrile patients aged ≥12 y attending two hospitals in Yangon, Myanmar, from 5 October 2015 through 4 October 2016. We identified the serovar of S. enterica, determined antimicrobial susceptibility and the molecular mechanisms of resistance. We analysed phylogenetic relationships among Myanmar S. enterica isolates and those with isolates from neighbouring countries.
Results
Of 73 S. enterica isolated, 39 (53%) were serovar Typhi and 34 (47%) were Paratyphi A. All isolates were susceptible to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole but resistant to ciprofloxacin. We identified mutations in chromosomal genes gyrA, gyrB and parC as responsible for fluoroquinolone resistance. All S. enterica Typhi isolates were of 4.3.1 subclade (formerly known as H58) and formed two closely related genotypic clusters; both clusters were most closely related to isolates from India from 2012. All S. enterica Paratyphi A were lineage C, clade C4 and were closely related.
Conclusion
Our study describes currently circulating S. enterica serovars in Myanmar, the genetic basis of their antimicrobial resistance and provides a genotypic framework for epidemiologic study.
Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in ...response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.
Immunopathology contributes to high mortality in tuberculous meningitis (TBM) but little is known about the blood and cerebrospinal fluid (CSF) immune response. We prospectively characterised the ...immune response of 160 TBM suspects in an Indonesian cohort, including 67 HIV-negative probable or definite TBM cases. TBM patients presented with severe disease and 38% died in 6 months. Blood from TBM patients analysed by flow cytometry showed lower αβT and γδT cells, NK cells and MAIT cells compared to 26 pulmonary tuberculosis patients (2.4-4-fold, all p < 0.05) and 27 healthy controls (2.7-7.6-fold, p < 0.001), but higher neutrophils and classical monocytes (2.3-3.0-fold, p < 0.001). CSF leukocyte activation was higher than in blood (1.8-9-fold). CSF of TBM patients showed a predominance of αβT and NK cells, associated with better survival. Cytokine production after ex-vivo stimulation of whole blood showed a much broader range in TBM compared to both control groups (p < 0.001). Among TBM patients, high ex-vivo production of TNF-α, IL-6 and IL-10 correlated with fever, lymphocyte count and monocyte HLA-DR expression (all p < 0.05). TBM patients show a strong myeloid blood response, with a broad variation in immune function. This may influence the response to adjuvant treatment and should be considered in future trials of host-directed therapy.
COVID-19 will be with us through the remainder of 2020 and almost certainly beyond. New Zealand needs a viable strategy to protect its populace until a vaccine is developed and in wide use. Until ...that time, it makes sense to protect the population by putting in place treatments that will be safe and effective, such as the use of convalescent sera and the use of direct-acting anti-virals. These treatments should be sourced externally or made locally, but steps in this direction must now begin as the lockdown ends. New Zealand has the scientists, the facilities and the will to make this happen, but the support of the government and the population will be needed if this plan is to succeed.
Carbapenem resistant organisms (CROs) such as Acinetobacter baumannii (CRAb), Pseudomonas aeruginosa (CRPa), Escherichia coli (CREc), and Klebsiella pneumoniae (CRKp) have been identified by the ...World Health Organization (WHO) as global priority pathogens. The dissemination of these pathogens and clonal outbreaks within healthcare facilities are of serious concern, particularly in regions with limited resources. In Fiji, where healthcare services are primarily provided by public hospitals, understanding the extent and nature of this problem is essential for the development of effective patient management, prevention interventions and control strategies.
CROs isolated from 211 (77.3%) non-sterile (urinary catheters, urine, sputum, wound swab, and endotracheal tube) and 62 (22.7%) normally sterile (blood, cerebrospinal fluid, intravascular catheter, and aspirates) body sites of 272 patients treated at the three major hospitals in Fiji, the Colonial War Memorial Hospital (CWMH), Lautoka Hospital (LTKH), and Labasa Hospital (LBSH), and outer peripheral health centres around Fiji, were analysed. Clinical and demographic patient data such as age, sex, admission diagnosis, admission and discharge dates, patient outcomes, date of death, start and end date of meropenem and colistin treatment were reviewed. These CRO isolates comprised A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae, that were prospectively collected at the microbiology laboratory of CWMH and LBSH from January 2020 through August 2021 and at the LTKH from January 2020 to December 2021. In addition, 10 retrospectively stored CRPa isolates collected from patients at the CWMH from January through December 2019, were also included in the study. All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole genome sequencing. Phylogenetic relationships among the CROs were assessed through core genome single nucleotide polymorphism (SNP) analysis. The CRAb isolates were also compared to the CRAb isolates from CWMH isolated in 2016/2017 and 2019, along with CRAb isolates obtained from Fijian patients admitted to New Zealand hospitals in 2020 and 2021 from our retrospective study.
Of 272 patients, 140 (51.5%) were male, the median (range) age of patients was 45 (<1–89) years, 161 (59.2%) were I-Taukei, 104 (38.2%) Fijians of Indian descent, and 7 (2.6%) were from other ethnic backgrounds. 234 (86.0%) of these 272 patients, had their first positive CRO sample collected ≥72 h following admission and the remaining 38 (14.0%) were isolated within 72 h following admission. Of the 273 CROs, 146 (53.5%) were collected at the CWMH, 66 (24.2%) LTKH, and 61 (22.3%) LBSH, while 62 (22.7%) were isolated from normally sterile sites and 211 (77.3%) from sites that are not sterile. Of 273 isolates, 131 (48.0%) were CRAb, 90 (33.0%) CRPa, 46 (16.8%) CREc, and 6 (2.2%) CRKp. Of 131 CRAb, 108 (82.4%) were ST2, with three distinct clones, all encoding blaOXA-23 and blaOXA-66, while clone 3 also encoded blaNDM-1; blaOXA-23 was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006. The first two CRAb ST2 clones were genetically linked to those isolated at CMWH 2016 through 2019, while the third was genetically linked to isolates from Fijian patients admitted to New Zealand hospitals in 2020 and 2021. Of CRPa, 65 (72.2%) were ST773 and carried β-lactamase genes blaNDM-1, blaOXA-50, and blaOXA-395. Of 10 retrospective CRPa isolates, all belonged to CRPa ST773 and carried blaNDM-1, blaOXA-50, and blaOXA-395. Of 46 CREc, 44 (95.7%) were ST410 and encoded blaNDM-7 on an IncX3 plasmid. Of 6 CRKp, 4 (66.7%) were ST16 and carried blaNDM-5 on an IncX3 plasmid. Other sequence types of CRPa (ST9, ST357, ST654, ST664), CRAb (ST25, ST374, ST499), CREc (ST167), and CRKp (ST45, ST336) were also detected. Of those receiving meropenem treatment in the prospective study, 30 (57.7%) received it inappropriately. Of 272 patients, 65 (23.9%) died within the 30 days after first positive CRO isolation.
We identified nosocomial transmission of distinct clones of CRAb ST2, CRPa ST773, CREc ST410, and CRKp ST16 within and between the three major hospitals in Fiji. Moreover, community onset infections associated with CRPa, CREc, and CRAb were also detected. Of note, cross-border transmission of CRAb ST2 clone 3 strain between Fiji and New Zealand was also detected. These clones encoded an array of carbapenem resistance genes associated with mobile genetic elements, including plasmids, transposons, and integrative and conjugative elements, signifying their potential for increased mobility, further acquisition of resistance genes, and spread. Inappropriate use of meropenem was common. Of note, the majority of patients who died had acquired CRO during their hospital stay. These findings highlight the need for stringent IPC strategies focusing on catheter and ventilator management, meticulous wound care, rigorous sepsis control, consistent hand hygiene, effective use of disinfectants, and thorough sanitisation of both hospital environments and medical equipment in the three major hospitals in Fiji. Additionally, diligent surveillance of AMR and robust antimicrobial stewardship are crucial for effectively managing nosocomial infections.
This project was funded by the Otago Medical School Foundations Trust (Dean's Bequest Fund) and a Fiji National University seed grant. The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.
Abstract
Objectives
The incidence of infections with ESBL-producing Escherichia coli (ESBL-Ec) in New Zealand is increasing. ESBL-Ec most commonly cause urinary tract infections and are seen in both ...community and hospitalized patients. The reason for the increasing incidence of ESBL-Ec infections is unknown.
Methods
In this study, 65 urinary ESBL-Ec isolates from the Otago region in 2015 were fully genetically characterized to understand the mechanisms of transmission. The ESBL gene, E. coli STs, plasmid types and genetic context (e.g. insertion sequences) of ESBL genes were determined by a combination of whole genome and plasmid sequencing. The phylogenetic relationships of the isolates were compared with ESBL-Ec isolates sequenced as part of the 2016 nationwide survey.
Results
Significant diversity of E. coli strains, plasmids, and the genetic context of ESBL genes was seen. However, there was evidence of common mobile genetic elements in unrelated ESBL-Ec.
Conclusions
Multiple introductions of ESBL resistance genes or resistant bacterial strains with limited horizontal transmission of mobile genetic elements accounts for the increased incidence of ESBL-Ec in this low prevalence area. Future studies should investigate modes of transmission of ESBL-Ec in the Otago region.
Carbapenem resistant
(CR
) is categorised by the World Health Organization (WHO) as a pathogen of critical concern. However, little is known about CR
transmission within the Oceania region. This ...study addresses this knowledge gap by using molecular epidemiology to characterise the phylogenetic relationships of CR
isolated in hospitals in Fiji, Samoa, and other countries within the Oceania region including Australia and New Zealand, and India from South Asia.
In this multicountry cohort study, we analysed clinical isolates of CR
collected from the Colonial War Memorial Hospital (CWMH) in Fiji from January through December 2019 (n = 64) and Tupua Tamasese Mea'ole Hospital (TTMH) in Samoa from November 2017 through June 2021 (n = 32). All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole-genome sequencing. For CWMH, data were collected on clinical and demographic characteristics of patients with CR
, duration of hospital stay, mortality and assessing the appropriateness of meropenem use from the treated patients who had CR
infections. To provide a broader geographical context, CR
strains from Fiji and Samoa were compared with CR
sequences from Australia collected in 2016-2018 (n = 22), New Zealand in 2018-2021 (n = 13), and India in 2019 (n = 58), a country which has close medical links with Fiji. Phylogenetic relationships of all these CR
isolates were determined using differences in core genome SNPs.
Of CR
isolates, 49 (77%) of 64 from Fiji and all 32 (100%) from Samoa belonged to CR
sequence type 2 (ST2). All ST2 isolates from both countries harboured
,
and
genes, mediating resistance to β-lactam antimicrobials, including cephalosporins and carbapenems. The
gene was associated with two copies of IS
insertion element, forming the composite transposon Tn
on the chromosome. Two distinct clusters (group 1 and group 2) of CR
ST2 were detected in Fiji. The first group shared common ancestral linkage to all CR
ST2 collected from Fiji's historic outbreak in 2016/2017, Samoa, Australia and 54% of total New Zealand isolates; they formed a single cluster with a median (range) SNP difference of 13 (0-102). The second group shared common ancestral linkage to 3% of the total CR
ST2 isolated from India. Fifty eight of the 64 patients with CR
infections at the CWMH had their first positive CR
sample collected 72 h or more following admission. Meropenem use was deemed inappropriate in 15 (48%) of the 31 patients that received treatment with meropenem in Fiji. Other strains of CR
ST1, ST25, ST107, and ST1112 were also detected in Fiji.
We identified unrecognised outbreaks of CR
ST2 in Fiji and Samoa that linked to strains in other parts of Oceania and South Asia. The existence of Tn
, containing the
and IS
insertion element, within CR
ST2 from Fiji and Samoa indicates the potential for high mobility and dissemination. This raises concerns about unmitigated prolonged outbreaks of CR
ST2 in the two major hospitals in Fiji and Samoa. Given the magnitude of this problem, there is a need to re-evaluate the current strategies used for infection prevention and control, antimicrobial stewardship, and public health measures locally and internationally. Moreover, a collaborative approach to AMR surveillance within the Oceania region with technical, management and budgetary support systems is required to prevent introduction and control transmission of these highly problematic strains within the island nation health systems.
This project was funded by an Otago Global Health Institute seed grant and Maurice Wilkins Centre of Research Excellence (CoREs) grant (SC0000169653, RO0000002300).
Dendritic cells are the key antigen-presenting cells involved in the initiation of the adaptive immune response. Recombinant adeno-associated viruses (rAAVs) can transduce dendritic cells and have ...gained attention as potential vaccines capable of stimulating T cell immunity. Here we show that rAAV2 pseudotyped with type 6 capsid (rAAV2/6) exhibits significantly higher tropism for human monocyte-derived dendritic cells (MoDCs) than other serotypes and variants. Transduction was abolished by a single lysine-to-alanine mutation within the AAV6 capsid previously shown to inhibit binding to heparin. However, unlike rAAV2, soluble heparin did not inhibit rAAV2/6 transduction of MoDCs. Further enhancement of MoDC transduction was observed after mutation of Tyr-731 in the capsid of AAV6 consistent with a report that tyrosine residues are phosphorylated, leading to ubiquitination of capsids during uptake. Pseudotyped rAAV2/6 vectors containing a Y731F mutation minimally altered the immunophenotype of MoDCs, which retained their immunostimulatory ability and were able to stimulate an antigen-specific CD8(+) T cell clone. These findings should assist in the development of rAAV2/6 as a vaccine vector.
Mucosal-associated invariant T (MAIT) cells are an abundant antibacterial innate-like lymphocyte population. There are conflicting reports as to their fate in HIV infection. The objective of this ...study was to determine whether MAIT cells are truly depleted in HIV infection. In this case-control study of HIV-positive patients and healthy controls, quantitative real-time polymerase chain reaction was used to assess the abundance of messenger RNA (mRNA) and genomic DNA (gDNA) encoding the canonical MAIT cell T cell receptor (Vα7.2-Jα33). Comparison was made with flow cytometry. Significant depletion of both Vα7.2-Jα33 mRNA and gDNA was seen in HIV infection. Depletion of Vα7.2+CD161++ T cells was confirmed by flow cytometry. In HIV infection, the abundance of Vα7.2-Jα33 mRNA correlated most strongly with the frequency of Vα7.2+CD161++ cells. No increase was observed in the frequency of Vα7.2+CD161- cells among CD3+CD4- lymphocytes. MAIT cells are depleted from blood in HIV infection as confirmed by independent assays. Significant accumulation of a CD161- MAIT cell population is unlikely. Molecular approaches represent a suitable alternative to flow cytometry-based assays for tracking of MAIT cells in HIV and other settings.
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