Highly accurate-single chromosomal complete genomes using IonTorrent and MinION sequencing of clinical pathogens.

Oxford Nanopore MinION sequencing technology has been gaining immense importance in identification of pathogen and antimicrobial resistance, though with 10-15% error rate. Short read technologies generates high accurate genome but with multiple fragments of genome. This study proposes a novel workflow to reduce the indels resulted from MinION long read sequencing by overlaying short read sequences from IonTorrent in the clinical isolates. Best of both techniques were employed which generated highly accurate-single chromosomal microbial genomes with increase in completeness of genomes from 44.5%, 30% and 43% to 98.6%, 98.6% and 96.6% for P. aeruginosa, A. veronii and B. pertussis respectively. To the best of our knowledge, this is the first study to generate a hybrid of IonTorrent and MinION reads to obtain single chromosomal genomes. This would enable to precisely infer both structural arrangement of genes and SNP based analysis for phylogenetic information.

Activity of cefepime/zidebactam against MDR Escherichia coli isolates harbouring a novel mechanism of resistance based on four-amino-acid inserts in PBP3.

BACKGROUND: Recent reports reveal the emergence of Escherichia coli isolates harbouring a novel resistance mechanism based on four-amino-acid inserts in PBP3. These organisms concomitantly expressed ESBLs or/and serine-/metallo-carbapenemases and were phenotypically detected by elevated aztreonam/avibactam MICs. OBJECTIVES: The in vitro activities of the investigational antibiotic cefepime/zidebactam and approved antibiotics (ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/relebactam and others) were determined against E. coli isolates harbouring four-amino-acid inserts in PBP3. METHODS: Whole-genome sequenced E. coli isolates (n = 89) collected from a large tertiary care hospital in Southern India (n = 64) and from 12 tertiary care hospitals located across India (n = 25) during 2016-18, showing aztreonam/avibactam MICs ≥1 mg/L (≥4 times the aztreonam epidemiological cut-off) were included in this study. The MICs of antibiotics were determined using the reference broth microdilution method. RESULTS: Four-amino-acid inserts [YRIK (n = 30) and YRIN (n = 53)] were found in 83/89 isolates. Among 83 isolates, 65 carried carbapenemase genes [blaNDM (n = 39), blaOXA-48-like (n = 11) and blaNDM + blaOXA-48-like (n = 15)] and 18 isolates produced ESBLs/class C ß-lactamases only. At least 16 unique STs were noted. Cefepime/zidebactam demonstrated potent activity, with all isolates inhibited at ≤1 mg/L. Comparator antibiotics including ceftazidime/avibactam and imipenem/relebactam showed limited activities. CONCLUSIONS: E. coli isolates concurrently harbouring four-amino-acid inserts in PBP3 and NDM are an emerging therapeutic challenge. Assisted by the PBP2-binding action of zidebactam, the cefepime/zidebactam combination overcomes both target modification (PBP3 insert)- and carbapenemase (NDM)-mediated resistance mechanisms in E. coli.

Identification of plasmids by PCR based replicon typing in bacteremic Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is a notorious pathogen with plasmid mediated resistance to all classes of antibiotics. It is important to determine the plasmid profile coding for resistance genes. Plasmid profile varies among geographical regions and tracking the types helps in determining the MDR and XDR K. pneumoniae spread especially in hospital setting. Aim of the present study was to determine the plasmid profile and types among bacteraemic K. pneumoniae. MATERIALS AND METHODS: Ninety consecutive K. pneumoniae collected over a period of three months from blood cultures were characterised by PCR for plasmid profile. Inc plasmid types were determined by PCR based replicon typing (PBRT) and carbapenemases were determined by multiplex PCR. For a subset of isolates hybrid assemblies were developed by sequencing with Ion Torrent and MinIon. RESULTS: Overall, PBRT showed 29% of isolates carried four plasmids including IncHI1B, IncFIA, IncFII(K) and IncR. The most common type of plasmid was IncHI1B (93%) followed by IncFIIK (89%) and IncR (82%). IncFIA was predominant among carbapenem resistant isolates. Almost all plasmids identified in K. pneumoniae were AMR plasmids, except two isolates which had virulence plasmids. IncX3 plasmid observed in this study was previously reported to be self-disseminating. Furthermore, the hybrid genome sequencing revealed complete structural arrangements of plasmids, which would be missed in short-read sequencing. NDM and OXA48-like were co-produced in 59% of the carbapenem resistant isolates. BlaOXA-232 was present on ColKP3; aac(6')-lb3 and rmtF on IncFIB. CONCLUSION: Diverse plasmid profile among the successive K. pneumoniae isolates indicates the transfer of resistance genes through different types of plasmids. IncHI1B, IncFIA, IncFIIK and IncR were the prevalent plasmid types. Hybrid assembly revealed blaOXA-232 was present on ColKP3 unlike global reports of IncL/M. Hybrid assemblies provide better plasmid structure that long and short read assemblies. There was no significant association of ß-lactamases with specific Inc groups in this study.

Accurate identification and epidemiological characterization of Burkholderia cepacia complex: an update.

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are among the most important pathogens isolated from cystic fibrosis (CF) patients and in hospital acquired infections (HAI). Accurate identification of Bcc is questionable by conventional biochemical methods. Clonal typing of Burkholderia is also limited due to the problem with identification. Phenotypic identification methods such as VITEK2, protein signature identification methods like VITEK MS, Bruker Biotyper, and molecular targets such as 16S rRNA, recA, hisA and rpsU were reported with varying level of discrimination to identify Bcc. rpsU and/or 16S rRNA sequencing, VITEK2, VITEK MS and Bruker Biotyper could discriminate between Burkholderia spp. and non-Burkholderia spp. Whereas, Bcc complex level identification can be given by VITEK MS, Bruker Biotyper, and 16S rRNA/rpsU/recA/hisA sequencing. For species level identification within Bcc hisA or recA sequencing are reliable. Identification of Bcc is indispensable in CF patients and HAI to ensure appropriate antimicrobial therapy.

Horizontal Transfer of Antimicrobial Resistance Determinants Among Enteric Pathogens Through Bacterial Conjugation.

Multi-drug resistance and transfer of mobile genetic elements among enteric pathogens is being reported to have increased rapidly. Commensal Escherichia coli was previously known to acquire mobile genetic elements from other genus/species. E. coli is also capable of disseminating these elements containing antimicrobial resistance determinants through horizontal transfer. Similarly, for Shigellae the antimicrobial resistance are on rise for fluoroquinolones and cephalosporins due to accumulation of mobile elements. Thus the study was hypothesized to investigate the role of transferable plasmids in commensal MDR E. coli vs Salmonella spp, and MDR Shigella flexneri vs Salmonella spp. pKP3-A plasmid containing qnrS1 was successfully transferred from E. coli to Salmonella spp. Similarly, a plasmid containing qnrS1 and blaCTX-M-15 was transferred from Shigella to Salmonella spp. However, blaCTX-M-15 was not transferred from E. coli as it was integrated into chromosome that was revealed by next-generation sequencing. This might be a reason that fluoroquinolone-resistant determinants are more frequently transferred than the cephalosporin resistant determinants. Findings from the study emphasize that mobile elements with AMR determinants are significant public health concern that has potential to rapidly disseminate.

Early treatment with fluvoxamine, bromhexine, cyproheptadine, and niclosamide to prevent clinical deterioration in patients with symptomatic COVID-19: a randomized clinical trial.

Background: Repurposed drugs with host-directed antiviral and immunomodulatory properties have shown promise in the treatment of COVID-19, but few trials have studied combinations of these agents. The aim of this trial was to assess the effectiveness of affordable, widely available, repurposed drugs used in combination for treatment of COVID-19, which may be particularly relevant to low-resource countries. Methods: We conducted an open-label, randomized, outpatient, controlled trial in Thailand from October 1, 2021, to June 21, 2022, to assess whether early treatment within 48-h of symptoms onset with combinations of fluvoxamine, bromhexine, cyproheptadine, and niclosamide, given to adults with confirmed mild SARS-CoV-2 infection, can prevent 28-day clinical deterioration compared to standard care. Participants were randomly assigned to receive treatment with fluvoxamine alone, fluvoxamine + bromhexine, fluvoxamine + cyproheptadine, niclosamide + bromhexine, or standard care. The primary outcome measured was clinical deterioration within 9, 14, or 28 days using a 6-point ordinal scale. This trial is registered with ClinicalTrials.gov (NCT05087381). Findings: Among 1900 recruited, a total of 995 participants completed the trial. No participants had clinical deterioration by day 9, 14, or 28 days among those treated with fluvoxamine plus bromhexine (0%), fluvoxamine plus cyproheptadine (0%), or niclosamide plus bromhexine (0%). Nine participants (5.6%) in the fluvoxamine arm had clinical deterioration by day 28, requiring low-flow oxygen. In contrast, most standard care arm participants had clinical deterioration by 9, 14, and 28 days. By day 9, 32.7% (110) of patients in the standard care arm had been hospitalized without requiring supplemental oxygen but needing ongoing medical care. By day 28, this percentage increased to 37.5% (21). Additionally, 20.8% (70) of patients in the standard care arm required low-flow oxygen by day 9, and 12.5% (16) needed non-invasive or mechanical ventilation by day 28. All treated groups significantly differed from the standard care group by days 9, 14, and 28 (p < 0.0001). Also, by day 28, the three 2-drug treatments were significantly better than the fluvoxamine arm (p < 0.0001). No deaths occurred in any study group. Compared to standard care, participants treated with the combination agents had significantly decreased viral loads as early as day 3 of treatment (p < 0.0001), decreased levels of serum cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1ß) as early as day 5 of treatment, and interleukin-8 (IL-8) by day 7 of treatment (p < 0.0001) and lower incidence of post-acute sequelae of COVID-19 (PASC) symptoms (p < 0.0001). 23 serious adverse events occurred in the standard care arm, while only 1 serious adverse event was reported in the fluvoxamine arm, and zero serious adverse events occurred in the other arms. Interpretation: Early treatment with these combinations among outpatients diagnosed with COVID-19 was associated with lower likelihood of clinical deterioration, and with significant and rapid reduction in the viral load and serum cytokines, and with lower burden of PASC symptoms. When started very soon after symptom onset, these repurposed drugs have high potential to prevent clinical deterioration and death in vaccinated and unvaccinated COVID-19 patients. Funding: Ped Thai Su Phai (Thai Ducks Fighting Danger) social giver group.

Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission.

This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

Ca-EDTA restores the activity of ceftazidime-avibactam or aztreonam against carbapenemase-producing Klebsiellapneumoniae infections.

Developing an effective therapy to overcome carbapenemase-positive Klebsiella pneumoniae (CPKp) is an important therapeutic challenge that must be addressed urgently. Here, we explored a Ca-EDTA combination with aztreonam or ceftazidime-avibactam in vitro and in vivo against diverse CPKp clinical isolates. The synergy testing of this study demonstrated that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combination was significantly effective in eliminating planktonic and mature biofilms in vitro, as well as eradicating CPKp infections in vivo. Both combinations revealed significant therapeutic efficacies in reducing bacterial load in internal organs and protecting treated mice from mortality. Conclusively, this is the first in vitro and in vivo study to demonstrate that novel aztreonam-Ca-EDTA or ceftazidime-avibactam-Ca-EDTA combinations provide favorable efficacy and safety for successful eradication of carbapenemase-producing Klebsiella pneumoniae planktonic and biofilm infections.

COVID-19 monitoring with sparse sampling of sewered and non-sewered wastewater in urban and rural communities.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Diagnostic methods and identification challenges experienced in a Burkholderia contaminans outbreak occurred in a tertiary care centre.

BACKGROUND: Recently, a novel species contaminans belonging to the family Burkholderia cepacia complex (Bcc) is rising as a hospital pathogen. Detection of Burkholderia contaminans, a member of Bcc can be done only by MALDI TOF and sequencing techniques. We report the diagnostic challenges faced in an outbreak of bacteremia due to B. contaminans grown in diltiazem vials. METHOD: The department of microbiology notified the infection control team about a cluster of eleven patients with B. contaminans isolated from blood culture. An outbreak investigation was initiated by performing environmental surveillance and sterility testing of solutions given for the patients. Routine phenotypical methods for identification of species followed by MALDI-TOF and sequencing was performed to identify the pathogen. RESULTS: All the patients detected with B. contaminans were having cardiac disease and received diltiazem. Sterility testing of diltiazem vials given for the patient and an unopened vial of same batch has grown B. contaminans. Clonal typing has confirmed the sequence similarities between patient and solution isolates. CONCLUSION: Due to diagnostic challenge in identifying the species of Bcc, MALDI TOF and clonal typing remains the key diagnostic tools available to detect Bcc species at an earliest especially in an outbreak.

Genetic Diversity of Clinical Bordetella Pertussis ST2 Strains in comparison with Vaccine Reference Strains of India.

Objectives: Pertussis is a highly contagious disease of the respiratory tract caused by Bordetella pertussis, a bacterium that lives in the mouth, nose, and throat. Current study reports the highly accurate complete genomes of two clinical B. pertussis strains from India for the first time. Methods: Complete genome sequencing was performed for two B. pertussis strains using Ion Torrent PGM and Oxford nanopore sequencing method. Data was assembled de novo and the sequence annotation was performed through PATRIC and NCBI server. Downstream analyses of the isolates were performed using CGE server databases for antimicrobial resistance genes, plasmids, and sequence types. The phylogenetic analysis was performed using Roary. Results: The analysis revealed insertional elements flanked by IS481, which has been previously regarded as the important component for bacterial evolution. The two B. pertussis clinical strains exhibited diversity through genome degradation when compared to whole-cell vaccine reference strains of India. These isolates harboured multiple genetic virulence traits and toxin subunits, which belonged to sequence type ST2. Conclusion: The genome information of Indian clinical B. pertussis strains will serve as a baseline data to decipher more information on the genome evolution, virulence factors and their role in pathogenesis for effective vaccine strategies.

Divergent evolution of Corynebacterium diphtheriae in India: An update from National Diphtheria Surveillance network.

Diphtheria is caused by a toxigenic bacterium Corynebacterium diphtheria which is being an emerging pathogen in India. Since diphtheria morbidity and mortality continues to be high in the country, the present study aimed to study the molecular epidemiology of C. diphtheriae strains from India. A total of 441 diphtheria suspected specimens collected as part of the surveillance programme between 2015 and 2020 were studied. All the isolates were confirmed as C. diphtheriae with standard biochemical tests, ELEK's test, and real-time PCR. Antimicrobial susceptibility testing for the subset of isolates showed intermediate susceptibility to penicillin and complete susceptible to erythromycin and cefotaxime. Isolates were characterized using multi locus sequence typing method. MLST analysis for the 216 C. diphtheriae isolates revealed major diversity among the sequence types. A total of 34 STs were assigned with majority of the isolates belonged to ST466 (30%). The second most common ST identified was ST405 that was present in 14% of the isolates. The international clone ST50 was also seen. The identified STs were grouped into 8 different clonal complexes (CC). The majority belongs to CC5 followed by CC466, CC574 and CC209, however a single non-toxigenic strain belongs to CC42. This epidemiological analysis revealed the emergence of novel STs and the clones with better dissemination properties. This study has also provided information on the circulating strains of C. diphtheriae among the different regions of India. The molecular data generated through surveillance system can be utilized for further actions in concern.

First Indian report on B4/H24RxC ST410 multidrug-resistant Escherichia coli from bloodstream infection harbouring blaOXA-181 and blaCTX-M-15.

OBJECTIVES: Escherichia coli is regarded as one of the most commonly isolated Gram-negative pathogens from bloodstream infections. Increasing antimicrobial resistance (AMR) among E. coli is a threat to disease management as well as further dissemination of AMR genes to other clinically important pathogens. Here we report the genome of a multidrug-resistant (MDR) E. coli (BA22372) from a bloodstream infection belonging to ST410 B4/H24RxC subtype from India. METHODS: Genomic DNA of E. coli BA22372 was sequenced using Ion Torrent™ PGM™ and MinION™ sequencing. Hybrid genome assembly was performed using short and long reads from both methods to achieve accurate and complete genome data. RESULTS: Here we report the genome of MDR E. coli BA22372 harbouring blaOXA-181 and blaCTX-M-15 in two individual plasmids, namely pOXA181_22372 (IncX3) and pCTX-M-15_22372 (IncF). The pCTX-M-15 plasmid is well known to co-harbour blaNDM-5, which was not seen in the studied isolate here. CONCLUSION: To the best of our knowledge, this is the first report of B4/H24RxC MDR E. coli from India co-harbouring blaCTX-M-15 and blaOXA-181 along with other AMR genes. Information from this genome data revealed the possession of AMR genes in two individual plasmids and their potential for rapid dissemination. This isolate is of high health concern as it harbours a plasmid with replicatory mechanisms capable of acquiring blaNDM-5, which is a great threat for rapid dissemination of AMR. This study enhances our understanding of the AMR mechanisms among different clones of E. coli.

Differential gene expression profile of Shigella dysenteriae causing bacteremia in an immunocompromised individual.

AIM: Shigella species has varying levels of virulence gene expression with respect to different sites of infection. In this study, the differential gene expression of S. dysenteriae in response to its site of infection was analyzed by transcriptomics. METHODS: This study includes four clinical Shigella isolates. Transcriptomics was done for the stool and blood samples of a single patient. Isolates were screened for the presence of antimicrobial resistance genes. RESULTS: The majority of genes involved in invasion were highly expressed in the strain isolated from the primary site of infection. Additionally, antimicrobial resistance (dhfr1A, sulII, bla OXA. bla CTX-M-1 and qnrS) genes were identified. CONCLUSION: This study provides a concise view of the transcriptional expression of clinical strains and provides a basis for future functional studies on Shigella spp.

The Influence of Biofilms on Carbapenem Susceptibility and Patient Outcome in Device Associated K. pneumoniae Infections: Insights Into Phenotype vs Genome-Wide Analysis and Correlation.

Klebsiella pneumoniae is one of the leading causes of nosocomial infections. Carbapenem-resistant K. pneumoniae are on the rise globally. The biofilm forming ability of K. pneumoniae further complicates patient management. There is still a knowledge gap on the association of biofilm formation with patient outcome and carbapenem susceptibility, which is investigated in present study. K. pneumoniae isolates from patients admitted in critical care units with catheters and ventilators were included. K. pneumoniae (n = 72) were subjected to 96-well plate biofilm formation assay followed by MBEC assay for subset of strong biofilm formers. Whole genome sequencing and a core genome phylogenetic analysis in comparison with global isolates were performed. Phenotypic analyses showed a positive correlation between biofilm formation and carbapenem resistance. Planktonic cells observed to be susceptible in vitro exhibited higher MICs in biofilm structure, hence MICs cannot be extrapolated for treatment. The biofilm forming ability had a significant association with morbidity/mortality. Infections by stronger biofilm forming pathogens significantly (p < 0.05) resulted in fewer "average days alive" for the patient (3.33 days) in comparison to those negative for biofilms (11.33 days). Phylogenetic analysis including global isolates revealed clear association of sequence types with genes for biofilm formation and carbapenem resistance. Known hypervirulent clone-ST23 with wcaG, magA, rmpA, rmpA2, and wzc with lack of mutation for hyper-capsulation might be poor biofilm formers. ST15, ST16, ST307, and ST258 (reported global high-risk clones) were wcaJ negative indicating the high potential of biofilm forming capacity. Genes wabG and treC for CPS, bcsA and pgaC for adhesins, luxS for quorum sensing were common in all clades in addition to genes for aerobactin (iutA), allantoin (allS), type I and III fimbriae (fimA, fimH, and mrkD) and pili (pilQ and ecpA). This study is the first of its kind to compare genetic features of antimicrobial resistance with a spectrum covering most of the genetic factors for K. pneumoniae biofilm. These results highlight the importance of biofilm screening to effectively manage nosocomial infections by K. pneumoniae. Further, data obtained on epidemiology and associations of biofilm and resistance genetic factors will serve to enhance our understanding on biofilm mechanisms in K. pneumoniae.

First Indian report on genome-wide comparison of multidrug-resistant Escherichia coli from blood stream infections.

BACKGROUND: Multidrug-resistant (MDR) E. coli with extended-spectrum ß-lactamases (ESBLs) is becoming endemic in health care settings around the world. Baseline data on virulence and antimicrobial resistance (AMR) of specific lineages of E. coli circulating in developing countries like India is currently lacking. METHODS: Whole-genome sequencing was performed for 60 MDR E. coli isolates. The analysis was performed at single nucleotide polymorphism (SNP) level resolution to identify the presence of their virulence and AMR genes. RESULTS: Genome comparison revealed the presence of ST-131 global MDR and ST410 as emerging-MDR clades of E. coli in India. AMR gene profile for cephalosporin and carbapenem resistance differed between the clades. Genotypes blaCTX-M-15 and blaNDM-5 were common among cephalosporinases and carbapenemases, respectively. For aminoglycoside resistance, rmtB was positive for 31.7% of the isolates, of which 95% were co-harboring carbapenemases. In addition, the FimH types and virulence gene profile positively correlated with the SNP based phylogeny, and also revealed the evolution of MDR clones among the study population with temporal accumulation of SNPs. The predominant clone was ST167 (blaNDM lineage) followed by ST405 (global clone ST131 equivalent) and ST410 (fast spreading high risk clone). CONCLUSIONS: This is the first report on the whole genome analysis of MDR E. coli lineages circulating in India. Data from this study will provide public health agencies with baseline information on AMR and virulent genes in pathogenic E. coli in the region.

Complete genome analysis of clinical Shigella strains reveals plasmid pSS1653 with resistance determinants: a triumph of hybrid approach.

Shigella is ranked as the second leading cause of diarrheal disease worldwide. Though infection occurs in people of all ages, most of the disease burden constitutes among the children less than 5 years in low and middle income countries. Recent increasing incidence of drug resistant strains make this as a priority pathogen under the antimicrobial resistance surveillance by WHO. Despite this, only limited genomic studies on drug resistant Shigella exists. Here we report the first complete genome of clinical S. flexneri serotype 2a and S. sonnei strains using a hybrid approach of both long-read MinION (Oxford Nanopore Technologies) and short-read Ion Torrent 400 bp sequencing platforms. The utilization of this novel approach in the present study helped to identify the complete plasmid sequence of pSS1653 with structural genetic information of AMR genes such as sulII, tetA, tetR, aph(6)-Id and aph(3'')-Ib. Identification of AMR genes in mobile elements in this human-restricted enteric pathogen is a potential threat for dissemination to other gut pathogens. The information on Shigella at genome level could help us to understand the genome dynamics of existing and emerging resistant clones.

IncFII plasmid carrying antimicrobial resistance genes in Shigella flexneri: Vehicle for dissemination.

OBJECTIVES: Plasmids harbouring antimicrobial resistance determinants in clinical strains are a significant public-health concern worldwide. The present study investigated such plasmids in clinical isolates of Shigella flexneri. METHODS: A total of 162 Shigella isolates were obtained from stool specimens in the year 2015. Among the 70 multidrug-resistant (MDR) Shigella spp., 27 S. flexneri isolates were randomly selected for further characterisation. Antimicrobial resistance genes (ARGs) and plasmid incompatibility (Inc) types were analysed. RESULTS: IncFII plasmids were found in 63% (17/27) of the studied S. flexneri isolates. ARGs such as dhfr1a (81%), sulII (74%), blaOXA (74%), blaTEM (33%), blaAmpC (30%), qnrS (15%) and qnrB (4%) were identified by PCR, whereas blaCTX-M was not detected. Next-generation sequencing of a representative S. flexneri IncFII-type plasmid (pSF470) revealed the presence of blaTEM1-B, blaDHA-1, qnrB10, mphA, sulI, sulII, strA, strB and tetR ARGs along with the intI1 integrase gene. In addition, pMLST analysis showed that the replicon belonged to F2:A-:B- type. CONCLUSIONS: This study helps to know the prevalent plasmid types in MDR Shigella isolates and will improve our understanding of resistance dissemination among enteric bacteria. ARGs in plasmids further highlight the importance of such studies in enteric bacteria.

Current strategy for local- to global-level molecular epidemiological characterisation of global antimicrobial resistance surveillance system pathogens.

The prime goal of molecular epidemiology is to identify the origin and evolution of pathogens, which can potentially influence the public health worldwide. Traditional methods provide limited information which is not sufficient for outbreak investigation and studying transmission dynamics. The recent advancement of next-generation sequencing had a major impact on molecular epidemiological studies. Currently, whole-genome sequencing (WGS) has become the gold standard typing method, especially for clinically significant pathogens. Here, we aimed to describe the application of appropriate molecular typing methods for global antimicrobial resistance surveillance system pathogens based on the level of discrimination and epidemiological settings. This shows that sequence-based methods such as multi-locus sequence typing (MLST) are widely used due to cost-effectiveness and database accessibility. However, WGS is the only method of choice for studying Escherichia coli and Shigella spp. WGS is shown to have higher discrimination than other methods in typing Klebsiella pneumoniae, Acinetobacter baumannii and Salmonella spp. due to its changing accessory genome content. For Gram positives such as Streptococcus pneumoniae, WGS would be preferable to understand the evolution of the strains. Similarly, for Staphylococcus aureus, combination of MLST, staphylococcal protein A or SCCmec typing along with WGS could be the choice for epidemiological typing of hospital- and community-acquired strains. This review highlights that combinations of different typing methods should be used to get complete information since no one standalone method is sufficient to study the varying genome diversity.

Ultrasound gel as a source of hospital outbreaks: Indian experience and literature review.

Purpose: Hospital outbreaks are observed increasingly worldwide with various organisms from different sources such as contaminated ultrasound gel, intravenous (IV) fluids and IV medications. Among these, ultrasound gel is one of the most commonly reported sources for Burkholderia cepacia complex (Bcc) outbreaks. In this study, we describe our experience on investigation and the management of Bcc bacteraemia outbreak due to contaminated ultrasound gel from a tertiary care centre, South India. Materials and Methods: Over a 10-day period in October 2016, seven children in our Paediatric intensive care unit (ICU) were found to have bacteraemia with Bcc isolated from their blood culture. Repeated isolation of the same organism with similar antimicrobial susceptibility pattern over a short incubation period from the same location, confirmed the outbreak. An active outbreak investigation, including environmental surveillance, was carried out to find the source and control the outbreak. Isolates were subjected to multi-locus sequence typing (MLST) and global eBURST (goeBURST) analysis. Results: Environmental surveillance revealed contaminated ultrasound gel as the source of infection. MLST and goeBURST analysis confirmed that the outbreak was caused by a novel sequence type 1362 with the same clonal complex CC517. The outbreak was controlled by stringent infection control measures, withdrawal of contaminated ultrasound gel from regular usage and implementing the practice of using ultrasonogram (USG) probe cover for USG screening and guided procedures. Conclusion: This report highlights the importance of early identification of an outbreak, prompt response of the ICU and infection control teams, sound environmental and epidemiological surveillance methods to identify the source and stringent infection control measures to control the outbreak. Contaminated ultrasound gel can be a potential source for healthcare-associated infection, which cannot be overlooked.