Comparing long-read assemblers to explore the potential of a sustainable low-cost, low-infrastructure approach to sequence antimicrobial resistant bacteria with Oxford nanopore sequencing

Long-read sequencing (LRS) can resolve repetitive regions, a limitation of short read (SR) data. Reduced cost and instrument size has led to a steady increase in LRS across diagnostics and research. Here, we re-basecalled FAST5 data sequenced between 2018 and 2021 and analyzed the data in relation to gDNA across a large dataset (n = 200) spanning a wide GC content (25–67%). We examined whether re-basecalled data would improve the hybrid assembly, and, for a smaller cohort, compared long read (LR) assemblies in the context of antimicrobial resistance (AMR) genes and mobile genetic elements. We included a cost analysis when comparing SR and LR instruments. We compared the R9 and R10 chemistries and reported not only a larger yield but increased read quality with R9 flow cells. There were often discrepancies with ARG presence/absence and/or variant detection in LR assemblies. Flye-based assemblies were generally efficient at detecting the presence of ARG on both the chromosome and plasmids. Raven performed more quickly but inconsistently recovered small plasmids, notably a ∼15-kb Col-like plasmid harboring blaKPC. Canu assemblies were the most fragmented, with genome sizes larger than expected. LR assemblies failed to consistently determine multiple copies of the same ARG as identified by the Unicycler reference. Even with improvements to ONT chemistry and basecalling, long-read assemblies can lead to misinterpretation of data. If LR data are currently being relied upon, it is necessary to perform multiple assemblies, although this is resource (computing) intensive and not yet readily available/useable

Determination of in vitro antimicrobial susceptibility for Lefamulin (Pleuromutilin) for Ureaplasma Spp. and Mycoplasma hominis

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides; while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1–2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted

Emergence of mobile colistin resistance (mcr-8) in a highly successful Klebsiella pneumoniae sequence type 15 clone from clinical infections in Bangladesh

The emergence of mobilized colistin resistance genes (mcr) has become a serious concern in clinical practice, compromising treatment options for life-threatening infections. In this study, colistin-resistant Klebsiella pneumoniae harboring mcr-8.1 was recovered from infected patients in the largest public hospital of Bangladesh, with a prevalence of 0.3% (3/1,097). We found mcr-8.1 in an identical highly stable multidrug-resistant IncFIB(pQil) plasmid of ∼113 kb, which belonged to an epidemiologically successful K. pneumoniae clone, ST15. The resistance mechanism was proven to be horizontally transferable, which incurred a fitness cost to the host. The core genome phylogeny suggested the clonal spread of mcr-8.1 in a Bangladeshi hospital. Core genome single-nucleotide polymorphisms among the mcr-8.1-positive K. pneumoniae isolates ranged from 23 to 110. It has been hypothesized that mcr-8.1 was inserted into IncFIB(pQil) with preexisting resistance loci, blaTEM-1b and blaCTX-M-15, by IS903B. Coincidentally, all resistance determinants in the plasmid [mcr-8.1, ampC, sul2, 1d-APH(6), APH(3′′)-Ib, blaTEM-1b, blaCTX-M-15] were bracketed by IS903B, demonstrating the possibility of intra- and interspecies and intra- and intergenus transposition of entire resistance loci. This is the first report of an mcr-like mechanism from human infections in Bangladesh. However, given the acquisition of mcr-8.1 by a sable conjugative plasmid in a successful high-risk clone of K. pneumoniae ST15, there is a serious risk of dissemination of mcr-8.1 in Bangladesh from 2017 onwards

Defining fluoroquinolone resistance-mediating mutations from non-resistance polymorphisms in Mycoplasma hominis Topoisomerases

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance

Clinical and molecular description of a high-copy IncQ1 KPC-2 plasmid harbored by the international ST15 Klebsiella pneumoniae clone

This study provides the genomic characterization and clinical description of bloodstream infections (BSI) cases due to ST15 KPC-2 producer Klebsiella pneumoniae. Six KPC-K. pneumoniae isolates were recovered in 2015 in a tertiary Brazilian hospital and were analyzed by whole-genome sequencing (WGS) (Illumina MiSeq short reads). Of these, two isolates were further analyzed by Nanopore MinION sequencing, allowing complete chromosome and plasmid circularization (hybrid assembly), using Unicycler software. The clinical analysis showed that the 30-day overall mortality for these BSI cases was high (83%). The isolates exhibited meropenem resistance (MICs, 32 to 128 mg/liter), with 3/6 isolates resistant to polymyxin B. The conjugative properties of the blaKPC-2 plasmid and its copy number were assessed by standard conjugation experiments and sequence copy number analysis. We identified in all six isolates a small (8.3-kb), high-copy-number (20 copies/cell) non-self-conjugative IncQ plasmid harboring blaKPC-2 in a non-Tn4401 transposon. This plasmid backbone was previously reported to harbor blaKPC-2 only in Brazil, and it could be comobilized at a high frequency (10−4) into Escherichia coli J53 and into several high-risk K. pneumoniae clones (ST258, ST15, and ST101) by a common IncL/M helper plasmid, suggesting the potential of international spread. This study thus identified the international K. pneumoniae ST15 clone as a carrier of blaKPC-2 in a high-copy-number IncQ1 plasmid that is easily transmissible among other common Klebsiella strains. This finding is of concern since IncQ1 plasmids are efficient antimicrobial resistance determinant carriers across Gram-negative species. The spread of such carbapenemase-encoding IncQ1 plasmids should therefore be closely monitored. IMPORTANCE In many parts of the world, carbapenem resistance is a serious public health concern. In Brazil, carbapenem resistance in Enterobacterales is mostly driven by the dissemination of KPC-2-producing K. pneumoniae clones. Despite being endemic in this country, only a few reports providing both clinical and genomic data are available in Brazil, which limit the understanding of the real clinical impact caused by the dissemination of different clones carrying blaKPC-2 in Brazilian hospitals. Although several of these KPC-2-producer K. pneumoniae isolates belong to the clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number blaKPC-2 plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15

Environmental surveillance of ESBL and carbapenemase-producing gram-negative bacteria in a Ghanaian Tertiary Hospital

Background The burden of antibiotic resistant infection is mainly felt in low-to-middle income countries, where the rate of antimicrobial resistance is largely under-surveyed and under huge pressure from unregulated, disparate and often self-guided access to antimicrobials. Nosocomial infections from hospital environments have been shown to be a particularly prevalent source of multi-drug resistant strains, yet surveillance of hospital environmental contamination is often not investigated. Methods The study was prospective, observational and cross-sectional, sampling 231 high and low touch surfaces from 15th March to 13th April 2021, from five wards in the Cape Coast Teaching Hospital, Ghana. Microbial growth in the presence of vancomycin and either meropenem or cefotaxime was examined and bacterial species were identified by MALDI-TOF. The presence of common extended-spectrum β-lactamases (ESBL) and carbapenemase antimicrobial resistance genes (ARG) were identified through PCR screening, which were confirmed by phenotypic antimicrobial susceptibility determination. Isolates positive for carbapenem resistance genes were sequenced using a multi-platform approach. Results We recovered microbial growth from 99% of swabs (n = 229/231) plated on agar in the absence of antimicrobials. Multiple sites were found to be colonised with resistant bacteria throughout the hospital setting. Bacteria with multi-drug resistance and ARG of concern were isolated from high and low touch points with evidence of strain dissemination throughout the environment. A total of 21 differing species of bacteria carrying ARG were isolated. The high prevalence of Acinetobacter baumannii carrying blaNDM-1 observed was further characterised by whole genome sequencing and phylogenetic analysis to determine the relationship between resistant strains found in different wards. Conclusion Evidence of multiple clonal incursions of MDR bacteria of high sepsis risk were found in two separate wards for a regional hospital in Ghana. The prevalence of multiple blaNDM carrying species in combination with combinations of ESBLs was particularly concerning and unexpected in Africa. We also identify strains carrying tet(X3), blaVIM-5 or blaDIM-1 showing a high diversity of carbapenamases present as a reservoir in a hospital setting. Findings of multi-drug resistant bacteria from multiple environmental sites throughout the hospital will inform future IPC practices and aid research prioritisation for AMR in Ghana

Emergence of mcr-1 mediated colistin resistant Escherichia coli from a hospitalized patient in Bangladesh

Introduction: The emergence of plasmid mediated mcr in bacteria has become global public health threat. Herein, we report a mcr-1 positive E. coli in normal human flora from a patient admitted in Dhaka Medical College Hospital (DMCH). Methodology: In total, 700 non-duplicate rectal swabs were collected from DMCH during 13th May to 12th June 2018. E. coli from rectal swabs were isolated on chromogenic UTI media containing vancomycin 10mg/l (Liofilchem, Italy) and confirmed by MALDI-TOF. Minimum inhibitory concentrations (MIC) were determined by agar dilution and interpreted according to EUCAST breakpoints. Genomic analysis of mcr positive E. coli (MCRPEC) was performed by Illumina MiSeq sequencing and pulsed field gel electrophoresis (PFGE) using S1 nuclease DNA digests and blamcr-1 probing. Transferability of blamcr-1 were determined by conjugation assays. Results: We found one MCRPEC from 700 rectal swab screening which was isolated from the rectal swab culture of a 17-year boy who was admitted to the burns ICU, DMCH with 53% flame burn involving much of the trunk and face. Genome sequencing revealed that mcr-1 was present on an IncH12 plasmid of 257,243 bp and flanked by ISApaI1. The colistin resistance can be transferred to the recipient Klebsiella varricola with a frequency of 8.3 × 10-5. Transconjugants were more resistant to colistin than donor (MIC 32 µg/mL). Conclusions: This is the first human associated mcr in Bangladesh. These data indicate the need for a systematic “one health” surveillance in the country

An emerging clone, KPC-2-producing Klebsiella pneumoniae ST16, associated with high mortality rates in a CC258 endemic setting

Background Carbapenemase-producing K. pneumoniae have become a global priority, not least in low-middle income countries. Here, we report the emergence and clinical impact of a novel KPC-K. pneumoniae ST16 clone in a Clonal Complex (CC)258 endemic setting. Methods In a teaching Brazilian hospital, a retrospective cohort of adult KPC-KP bloodstream infections (BSI) cases (January 2014 to December 2016) was established to study the molecular epidemiology and its impact on outcome (30-day all-cause mortality). KPC-KP isolates were MLST-typed. Survival analysis between ST/CC groups and risk factors for fatal outcome (logistic regression) were evaluated. Representative isolates underwent whole genome sequencing (WGS), and had their virulence tested in a Galleria larvae model. Results One hundred sixty-five unique KPC-KP BSI cases were identified. CC258 was predominant (66%), followed by ST16 (12%). The overall 30-day mortality rate was 60%; in contrast, 95% of ST16 cases were fatal. Patient’s severity scores were high and baseline clinical variables were not statistically different across ST’s. In multivariate analysis, ST16 (OR 21.4; CI95% 2.3-202.8; p=0,008) and septic shock (OR 11.9; CI95% 4.2-34.1; p<0,001) were independent risk factors for fatal outcome. ST16 clone carried up to 14 resistance genes, including blaKPC-2 in an IncFIBpQIL plasmid, KL51 capsule and Yersiniabactin virulence determinants. ST16 clone was highly pathogenic in the larvae model. Conclusions Mortality rates were high in this KPC-KP BSI cohort, where CC258 is endemic. An emerging ST16 clone was associated with high mortality. Our results suggest that even in endemic settings, highly virulent clones can rapidly emerge demanding constant monitoring

Colonisation of hospital surfaces from low- and middle-income countries by extended spectrum β-lactamase- and carbapenemase-producing bacteria

Hospital surfaces can harbour bacterial pathogens, which may disseminate and cause nosocomial infections, contributing towards mortality in low- and middle-income countries (LMICs). During the BARNARDS study, hospital surfaces from neonatal wards were sampled to assess the degree of environmental surface and patient care equipment colonisation by Gram-negative bacteria (GNB) carrying antibiotic resistance genes (ARGs). Here, we perform PCR screening for extended-spectrum β-lactamases (blaCTX-M-15) and carbapenemases (blaNDM, blaOXA-48-like and blaKPC), MALDI-TOF MS identification of GNB carrying ARGs, and further analysis by whole genome sequencing of bacterial isolates. We determine presence of consistently dominant clones and their relatedness to strains causing neonatal sepsis. Higher prevalence of carbapenemases is observed in Pakistan, Bangladesh, and Ethiopia, compared to other countries, and are mostly found in surfaces near the sink drain. Klebsiella pneumoniae, Enterobacter hormaechei, Acinetobacter baumannii, Serratia marcescens and Leclercia adecarboxylata are dominant; ST15 K. pneumoniae is identified from the same ward on multiple occasions suggesting clonal persistence within the same environment, and is found to be identical to isolates causing neonatal sepsis in Pakistan over similar time periods. Our data suggests persistence of dominant clones across multiple time points, highlighting the need for assessment of Infection Prevention and Control guidelines

Characterisation of Staphylococci species from neonatal blood cultures in low- and middle-income countries

Background: In low- and middle-income countries (LMIC) Staphylococcus aureus is regarded as one of the leading bacterial causes of neonatal sepsis, however there is limited knowledge on the species diversity and antimicrobial resistance caused by Gram-positive bacteria (GPB). Methods: We characterised GPB isolates from neonatal blood cultures from LMICs in Africa (Ethiopia, Nigeria, Rwanda, and South Africa) and South-Asia (Bangladesh and Pakistan) between 2015–2017. We determined minimum inhibitory concentrations and performed whole genome sequencing (WGS) on Staphylococci isolates recovered and clinical data collected related to the onset of sepsis and the outcome of the neonate up to 60 days of age. Results: From the isolates recovered from blood cultures, Staphylococci species were most frequently identified. Out of 100 S. aureus isolates sequenced, 18 different sequence types (ST) were found which unveiled two small epidemiological clusters caused by methicillin resistant S. aureus (MRSA) in Pakistan (ST8) and South Africa (ST5), both with high mortality (n = 6/17). One-third of S. aureus was MRSA, with methicillin resistance also detected in Staphylococcus epidermidis, Staphylococcus haemolyticus and Mammaliicoccus sciuri. Through additional WGS analysis we report a cluster of M. sciuri in Pakistan identified between July-November 2017. Conclusions: In total we identified 14 different GPB bacterial species, however Staphylococci was dominant. These findings highlight the need of a prospective genomic epidemiology study to comprehensively assess the true burden of GPB neonatal sepsis focusing specifically on mechanisms of resistance and virulence across species and in relation to neonatal outcome