Optical maps of plasmids as a proxy for clonal spread of MDR bacteria: A case study of an outbreak in a rural Ethiopian hospital

Objectives: MDR bacteria have become a prevailing health threat worldwide. We here aimed to use optical DNA mapping (ODM) as a rapid method to trace nosocomial spread of bacterial clones and gene elements.We believe that this method has the potential to be a tool of pivotal importance for MDR control. Methods: Twenty-four Escherichia coli samples of ST410 from three different wards were collected at an Ethiopian hospital and their plasmids were analysed by ODM. Plasmids were specifically digested with Cas9 targeting the antibiotic resistance genes, stained by competitive binding and confined in nanochannels for imaging. The resulting intensity profiles (barcodes) for each plasmid were compared to identify potential clonal spread of resistant bacteria. Results: ODM demonstrated that a large fraction of the patients carried bacteria with a plasmid of the same origin, carrying the ESBL gene blaCTX-M-15, suggesting clonal spread. The results correlate perfectly with core genome (cg)MLST data, where bacteria with the same plasmid also had very similar cgMLST profiles. Conclusions: ODM is a rapid discriminatory method for identifying plasmids and antibiotic resistance genes. Long-range deletions/insertions, which are challenging for short-read next-generation sequencing, can be easily identified and used to trace bacterial clonal spread. We propose that plasmid typing can be a useful tool to identify clonal spread of MDR bacteria. Furthermore, the simplicity of the method enables possible future application in low-and middle-income countries

Polyclonal spread of blaCTX-M-15 through high-risk clones of Escherichia coli at a tertiary hospital in Ethiopia

Objectives: The burden of antimicrobial resistance and spread of epidemic clones are rarely reported from low-income countries. We aimed to investigate the genome-based epidemiology of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) at a tertiary hospital in Jimma, Ethiopia. Methods: Bacteria were isolated from clinical specimens at Jimma Medical Center and subjected to species identification (MALDI-TOF), antimicrobial susceptibility testing (disk diffusion) and whole-genome sequencing (Illumina, HiSeq2500). Genomic data analysis was performed using EnteroBase and Center for Genomic Epidemiology bioinformatics pipelines. A maximum likelihood tree was generated using FastTree/2.1.8 based on single nucleotide polymorphisms (SNPs) in shared genomic regions to identify transmission clusters. Results: Escherichia coli isolates (n = 261) were collected from 1087 single non-duplicate clinical specimens over a 5-month period in 2016. The prevalence of ESBL-EC was 54.8% (143/261), 96% of which were resistant to multiple antibiotic classes. The blaCTX-M-15 ESBL gene was present in 88.4.% of isolates (122/138). Genes conferring resistance to aminoglycosides and ciprofloxacin [aac(6′)-Ib-cr, 62.3% (86/138)], phenicols [catB3, 56.5% (78/138)], sulfonamides [sul1, 68.1% (94/138), trimethoprim [dfrA17, 58.0% (80/138)] and macrolides [mph(A), 67.4% (93/138) were detected. The most prevalent sequence types were ST410 (23%), ST648 (17%), ST131 (10%) and ST167 (7%). Isolates of the same sequence type collected from different units of the hospital were highly similar in the SNP analysis. Conclusion: A high prevalence of ESBLs and dissemination of blaCTX-M-15 through multiple high-risk E. coli clones was detected. Nosocomial spread of multidrug-resistant ESBL-EC within the hospital puts vulnerable patients at risk of difficult-to-treat infections

Optical dna mapping of plasmids reveals clonal spread of carbapenem-resistant klebsiella pneumoniae in a large thai hospital

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1

High prevalence of bla(CTX-M-15) and nosocomial transmission of hypervirulent epidemic clones of Klebsiella pneumoniae at a tertiary hospital in Ethiopia

Background: Genomic epidemiology of antibiotic resistance is not sufficiently studied in low-income countries. Objectives: To determine prevalence of ESBL production, and resistome and virulome profiles, of Klebsiella pneumoniae isolated at Jimma Medical Center, Ethiopia. Methods: Strains isolated from patients with suspected infections between June and November 2016 were characterized by MALDI-TOF for species identification and disc diffusion for antimicrobial susceptibility testing. All K. pneumoniae isolates were characterized by double disc diffusion for ESBL production and all ESBL-producing strains (ESBL-KP) were subjected to WGS on the Illumina (HiSeq 2500) platform. DNA was extracted by automated systems (MagNA Pure 96). Genome assembly was performed using SPAdes (v. 3.9) and draft genomes were used for analysing molecular features of the strains. Maximum likelihood trees were generated using FastTree/2.1.8 based on SNPs in shared genomic regions to identify transmission clusters. Results: Of the 146 K. pneumoniae strains isolated, 76% were ESBL-KP; 93% of the ESBL-KP strains showed resistance to multiple antimicrobial classes. bla(CTX-M-15) (84.4%) was the most prevalent ESBL gene. Resistance genes for aminoglycosides and/or fluoroquinolones [aac(6)-Ib-cr (65.1%)], phenicols [catB3 (28.4%)], sulphonamides [sul1 (61.2%) and sul2 (60.5%)], trimethoprim [dfrA27 (32.1%)], macrolides [mph(A) (12.8%)] and rifampicin [arr2/arr3 (39.4%)] were prevalent. Plasmids of the IncF and IncR families were prevalent among ST218, ST147, ST15 and ST39. KL64 and KL57 capsular types and O1 and O2 LPSs were prevalent. A high-risk clone, ST218-KL57 encoding rmpA1/rmpA2 and iutA, was detected. Phylogenetic analysis showed a cluster of clonally related strains from different units of the hospital. Conclusions: Prevalence of ESBL-KP was high and bla(CTX-M-15) was the predominant ESBL gene. ESBL genes had spread through both clonal and polyclonal expansion of high-risk and hypervirulent clones. Nosocomial transmission of MDR strains between different units of the hospital was observed

Molecular and genetic characterization of emerging carbapenemase-producing Acinetobacter baumannii strains from patients and hospital environments in Bangladesh

Background Carbapenemase-producing multidrug-resistant (MDR) Acinetobacter baumannii is a global health care problem. MDR A. baumannii has emerged as an important nosocomial pathogen, costing many lives worldwide including Bangladesh. Aim To investigate the detailed molecular epidemiology of carbapenem-resistant A. baumannii (CRAB) both from patients and the hospital environment, to shed light on genetic characteristics and transmission dynamics. Methods A set of 49 clinical A. baumannii strains collected during early 2015 was received from the clinical microbiology laboratory of Dhaka Medical College Hospital (DMCH) in Bangladesh. Additionaly, 100 environmental samples were also collected from the hospital surfaces of Dhaka Medical College Hospital and analyzed for carbapenamase-producing A. baumannii. CRAB were identified by culture on selective plates, biochemical testing and MALDI-TOF. All isolates were characterized by susceptibility testing, realtime-PCRs, conventional PCR, MLST and sequencing. Findings Clinical A. baumannii were resistant to ciprofloxacin (100%), imipenem (91.8%), meropenem (91.8%), gentamicin (91.8%), amikacin (87.7%), and trimethoprim-sulfamethoxazole (61.2%). The majority (59%) of the isolates were MDR. All environmental A. baumannii (n=10) were resistant to imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin. Strains carried the following antibiotic resistant genes; blaOXA-23, blaOXA-58, blaPER-7, qnrB1, qnrC1, aac(6′)1b-cr and armA. A total of 36 different clones were identified by rep-PCR and common clonal clusters were found both in patients and hospital environments. MLST analysis revealed different sequence types (ST2, ST10, ST149, ST575, ST1063 and ST1065). In clinical and environmental settings. A. baumannii ST2 dominated in both clinical and environmental settings. Both clinical and environmental A. baumannii strains with known STs carried several biofilm-related genes; bap, csuE, and pgaB. Conclusion Widespread dissemination of MDR A. baumannii in the DMC hospital of Bangladesh is a serious problem

Antimicrobial susceptibility testing of Bacteroides species by disk diffusion: The NordicAST Bacteroides study

Objectives - Antimicrobial susceptibility testing (AST) of anaerobic bacteria has until recently been done by MIC methods. We have carried out a multi-centre evaluation of the newly validated EUCAST disk diffusion method for AST of Bacteroides spp. Methods - A panel of 30 Bacteroides strains was assembled based on reference agar dilution MICs, resistance gene detection and quantification of cfiA carbapenemase gene expression. Nordic clinical microbiology laboratories (n = 45) performed disk diffusion on Fastidious Anaerobe Agar with 5% mechanically defibrinated horse blood (FAA-HB) for piperacillin-tazobactam, meropenem and metronidazole. Results - A total of 43/45 (95.6%) laboratories carried out disk diffusion per protocol. Intraclass correlation coefficients were 0.87 (0.80–0.93) for piperacillin-tazobactam, 0.95 (0.91–0.97) for meropenem and 0.89 (0.83–0.94) for metronidazole. For metronidazole, one media lot yielded smaller zones and higher variability than another. Piperacillin-tazobactam and meropenem zone diameters correlated negatively with cfiA expression. A meropenem zone diameter of Conclusions - Inter-laboratory agreement by disk diffusion was good or very good. The main challenges were media-related variability for metronidazole and categorical disagreement with the reference method for piperacillin-tazobactam in some cfiA-positive strains. An area of technical uncertainty specific for such strains may be warranted

Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of bla NDM-1 in India, Sweden, and the United Kingdom

Clinical isolates of Klebsiella pneumoniae producing NDM-1 carbapenemase from India (n = 22), the United Kingdom (n = 13), and Sweden (n = 4) were subjected to multilocus sequence typing (MLST), automated repetitive sequence-based PCR (rep-PCR), serotyping, virulence gene screening, and plasmid replicon typing. The most frequently detected MLST sequence types (STs) were ST14 (n = 13; all serotype K2), ST11, ST149, ST231, and ST147. The correlation between MLST and automated rep-PCR was excellent. IncA/C was the most frequently detected plasmid replicon type (n = 14). ST14, ST11, and other successful clones may be important for the dissemination of bla . Copyrigh

Duration of Treatment for Pseudomonas aeruginosa Bacteremia: a Retrospective Study

Introduction: There is no consensus regarding optimal duration of antibiotic therapy for Pseudomonas aeruginosa bacteremia. We aimed to evaluate the impact of short antibiotic course. Methods: We present a retrospective multicen ter study including patients with P. aeruginosa bacteremia during 2009–2015. We evaluated outcomes of patients treated with short (6–- 10 days) versus long (11–15 days) antibiotic courses. The primary outcome was a composite of 30-day mortality or bacteremia recurrence and/or persistence. Univariate and inverse probability treatment-weighted (IPTW) adjusted multivariate analysis for the primary outcome was performed. To avoid immortal time bias, the landmark method was used. Results: We included 657 patients; 273 received a short antibiotic course and 384 a long course. There was no significant difference in baseline characteristics of patients. The com posite primary outcome occurred in 61/384 patients in the long-treatment group (16%) versus 32/273 in the short-treatment group (12%) (p = 0.131). Mortality accounted for 41/384 (11%) versus 25/273 (9%) of cases, respectively. Length of hospital stay was signif icantly shorter in the short group [median 13 days, interquartile range (IQR) 9–21 days, versus median 15 days, IQR 11–26 days, p = 0.002]. Ten patients in the long group dis continued antibiotic therapy owing to adverse events, compared with none in the short group. On univariate and multivariate analyses, dura tion of therapy was not associated with the primary outcome. Conclusions: In this retrospective study, 6–- 10 days of antibiotic course for P. aeruginosa bacteremia were as effective as longer courses in terms of survival and recurrence. Shorter ther apy was associated with reduced length of stay and less drug discontinuation

Molecular Epidemiology of OXA-48 and NDM-1 Producing Enterobacterales Species at a University Hospital in Tehran, Iran, Between 2015 and 2016

Carbapenem-resistant Enterobacterales (CRE) is an increasing problem worldwide. Here, we examined the clonal relatedness of 71 non-repetitive CRE isolates collected in a university hospital in Tehran, Iran, between February 2015 and March 2016. Pulsed-field gel electrophoresis (PFGE) and MLST were used for epidemiological analysis. Screening for antibiotic resistance genes, PCR-based replicon typing, conjugation experiments, and optical DNA mapping were also performed. Among all 71 isolates, 47 isolates of Klebsiella pneumoniae (66.2%), eight Escherichia coli (11.2%), five Serratia marcescens (7%), and two Enterobacter cloacae (2.8%) harbored blaNDM–1 and blaOXA–48 genes together or alone. PFGE analysis revealed that most of the OXA-48- and NDM-1-producing K. pneumoniae and all of OXA-48-producing S. marcescens were clonally related, while all eight E. coli and two E. cloacae isolates were clonally unrelated. The predominant clones of carbapenemase-producing K. pneumoniae associated with outbreaks within the hospital were ST147 (n = 13) and ST893 (n = 10). Plasmids carrying blaNDM–1 and blaOXA–48 were successfully transferred to an E. coli K12-recipient strain. The blaOXA–48 gene was located on an IncL/M conjugative plasmid, while the blaNDM–1 gene was located on both IncFII ∼86-kb to ∼140-kb and IncA/C conjugative plasmids. Our findings provide novel epidemiologic data on carbapenemase-producing Enterobacterales (CPE) in Iran and highlight the importance of horizontal gene transfer in the dissemination of blaNDM–1 and blaOXA–48 genes. The occurrence and transmission of distinct K. pneumoniae clones call for improved infection control to prevent further spread of these pathogens in Iran

Systematic Comparison of Epidemic and Non-Epidemic Carbapenem Resistant Klebsiella pneumoniae Strains

Over the past few decades, extensively drug resistant (XDR) resistant Klebsiella pneumoniae has become a notable burden to healthcare all over the world. Especially carbapenemase-producing strains are problematic due to their capability to withstand even last resort antibiotics. Some sequence types (STs) of K. pneumoniae are significantly more prevalent in hospital settings in comparison to other equally resistant strains. This provokes the question whether or not there are phenotypic characteristics that may render certain K. pneumoniae more suitable for epidemic dispersal between patients, hospitals, and different environments. In this study, we selected seven epidemic and non-epidemic carbapenem resistant K. pneumoniae isolates for extensive systematic characterization for phenotypic and genotypic qualities in order to identify potential factors that precede or emerge from epidemic successfulness. Studied characteristics include growth rates and densities in different conditions (media, temperature, pH, resource levels), tolerance to alcohol and drought, inhibition between strains, ability to compensate pH, as well as various genomic features. Overall, there are clear differences between isolates, yet, only drought tolerance was found to notably associate with non-epidemic K. pneumoniae strains. We further report a preliminary study on the potential to control K. pneumoniae ST11 with an antimicrobial component produced by a non-epidemic K. pneumoniae. This component initially restricts bacterial growth, but stable resistance develops rapidly in vitro