Alexander M. Wailan

The spread and acquisition of NDM-1: a multifactorial problem

bla NDM is a major mechanism of resistance of Gram-negative bacteria to β-lactam antibiotics including the carbapenems. bla NDM has been acquired by a large range of Gram-negative bacilli, especially by the Enterobacteriaceae and Acinetobacter spp. The combination of human factors (suboptimal antibiotic stewardship and infection control, movement of people between countries) plus bacterial factors (hospital adapted clones, environmental persistence and prolific horizontal gene transfer) have led to global spread of bla NDM at a rapid pace. Treatment options for New Delhi metallo-β-lactamase (NDM) producers are very limited. For serious infections, combination therapy including a polymyxin is preferred. However, resistance to polymyxins is emerging. Clearly, substantial international efforts must be made to control the spread of NDM producers or else many of the advances of modern medicine may be undermined by untreatable infections.

Genomic Characteristics of NDM-Producing Enterobacteriaceae Isolates in Australia and Their blaNDM Genetic Contexts

ABSTRACT blaNDM has been reported in different Enterobacteriaceae species and on numerous plasmid replicon types (Inc). Plasmid replicon typing, in combination with genomic characteristics of the bacterial host (e.g., sequence typing), is used to infer the spread of antimicrobial resistance determinants between genetically unrelated bacterial hosts. The genetic context of blaNDM is heterogeneous. In this study, we genomically characterized 12 NDM-producing Enterobacteriaceae isolated in Australia between 2012 and 2014: Escherichia coli (n = 6), Klebsiella pneumoniae (n = 3), Enterobacter cloacae (n = 2) and Providencia rettgeri (n = 1). We describe their blaNDM genetic contexts within Tn125, providing insights into the acquisition of blaNDM into Enterobacteriaceae. IncFII-type (n = 7) and IncX3 (n = 4) plasmids were the most common plasmid types found. The IncHI1B (n = 1) plasmid was also identified. Five different blaNDM genetic contexts were identified, indicating four particular plasmids with specific blaNDM genetic contexts (NGCs), three of which were IncFII plasmids (FII-A to -C). Of note, the blaNDM genetic context of P. rettgeri was not conjugative. Epidemiological links between our NDM-producing Enterobacteriaceae were established by their acquisition of these five particular plasmid types. The combination of different molecular and genetic characterization methods allowed us to provide insight into the spread of plasmids transmitting blaNDM.

Draft Genome Sequence of NDM-5-Producing Escherichia coli Sequence Type 648 and Genetic Context of blaNDM-5 in Australia

ABSTRACT We report here the draft genome sequence of uropathogenic Escherichia coli sequence type 648 (ST648) possessing blaNDM-5 from a 55-year-old female in Australia with a history of travel to India. The plasmid-mediated blaNDM-5 was in a genetic context nearly identical to that of the GenBank entry of an IncX3 blaNDM-5 plasmid previously reported from India (Klebsiella pneumoniae MGR-K194).

Mechanisms Involved in Acquisition of blaNDM Genes by IncA/C2 and IncFIIY Plasmids

ABSTRACT blaNDM genes confer carbapenem resistance and have been identified on transferable plasmids belonging to different incompatibility (Inc) groups. Here we present the complete sequences of four plasmids carrying a blaNDM gene, pKP1-NDM-1, pEC2-NDM-3, pECL3-NDM-1, and pEC4-NDM-6, from four clinical samples originating from four different patients. Different plasmids carry segments that align to different parts of the blaNDM region found on Acinetobacter plasmids. pKP1-NDM-1 and pEC2-NDM-3, from Klebsiella pneumoniae and Escherichia coli, respectively, were identified as type 1 IncA/C2 plasmids with almost identical backbones. Different regions carrying blaNDM are inserted in different locations in the antibiotic resistance island known as ARI-A, and ISCR1 may have been involved in the acquisition of blaNDM-3 by pEC2-NDM-3. pECL3-NDM-1 and pEC4-NDM-6, from Enterobacter cloacae and E. coli, respectively, have similar IncFIIY backbones, but different regions carrying blaNDM are found in different locations. Tn3-derived inverted-repeat transposable elements (TIME) appear to have been involved in the acquisition of blaNDM-6 by pEC4-NDM-6 and the rmtC 16S rRNA methylase gene by IncFIIY plasmids. Characterization of these plasmids further demonstrates that even very closely related plasmids may have acquired blaNDM genes by different mechanisms. These findings also illustrate the complex relationships between antimicrobial resistance genes, transposable elements, and plasmids and provide insights into the possible routes for transmission of blaNDM genes among species of the Enterobacteriaceae family.

Genetic Contexts of blaNDM-1 in Patients Carrying Multiple NDM-Producing Strains

ABSTRACT The carbapenem resistance determinant blaNDM-1 has been found in various Gram-negative bacteria and upon different plasmid replicon types (Inc). Here, we present four patients within two hospitals in Pakistan harboring between two and four NDM-1-producing Gram-negative bacilli of different species coresident in their stool samples. We characterize the blaNDM-1 genetic contexts of these 11 NDM-1-producing Gram-negative bacilli in addition to other antimicrobial resistance mechanisms, plasmid replicon profiles, and sequence types (STs) in order to understand the underlying acquisition mechanisms of carbapenem resistance within these bacteria. Two common plasmid types (IncN2 and IncA/C) were identified to carry blaNDM-1 among the six different bacterial species isolated from the four patients. Two of these strains were novel Citrobacter freundii ST 20 and ST 21. The same IncN2-type blaNDM-1 genetic context was found in all four patients and within four different species. The IncA/C-type blaNDM-1 genetic context was found in two different species and in two of the four patients. Combining genetic context characterization with other molecular epidemiology methods, we were able to establish the molecular epidemiological links between genetically unrelated bacterial species by linking their acquisition of an IncN2 or IncA/C plasmid carrying blaNDM-1 for carbapenem resistance. By combining plasmid characterization and in-depth genetic context assessment, this analysis highlights the importance of plasmids in antimicrobial resistance. It also provides a novel approach for investigating the underlying mechanisms of blaNDM-1-related spread between bacterial species and genera via plasmids.

Species identification within Acinetobacter calcoaceticus–baumannii complex using MALDI-TOF MS

Acinetobacter baumannii, one of the more clinically relevant species in the Acinetobacter genus is well known to be multi-drug resistant and associated with bacteremia, urinary tract infection, pneumonia, wound infection and meningitis. However, it cannot be differentiated from closely related species such as Acinetobacter calcoaceticus, Acinetobacter pittii and Acinetobacter nosocomialis by most phenotypic tests and can only be differentiated by specific, time consuming genotypic tests with very limited use in clinical microbiological laboratories. As a result, these species are grouped into the A. calcoaceticus-A. baumannii (Acb) complex. Herein we investigated the mass spectra of 73 Acinetobacter spp., representing ten different species, using an AB SCIEX 5800 MALDI-TOF MS to differentiate members of the Acinetobacter genus, including the species of the Acb complex. RpoB gene sequencing, 16S rRNA sequencing, and gyrB multiplex PCR were also evaluated as orthogonal methods to identify the organisms used in this study. We found that whilst 16S rRNA and rpoB gene sequencing could not differentiate A. pittii or A. calcoaceticus, they can be differentiated using gyrB multiplex PCR and MALDI-TOF MS. All ten Acinetobacter species investigated could be differentiated by their MALDI-TOF mass spectra.

The Murray collection of pre-antibiotic era Enterobacteriacae: a unique research resource.

Studies of historical isolates inform on the evolution and emergence of important pathogens and phenotypes, including antimicrobial resistance. Crucial to studying antimicrobial resistance are isolates that predate the widespread clinical use of antimicrobials. The Murray collection of several hundred bacterial strains of pre-antibiotic era Enterobacteriaceae is an invaluable resource of historical strains from important pathogen groups. Studies performed on the Collection to date merely exemplify its potential, which will only be realised through the continued effort of many scientific groups. To enable that aim, we announce the public availability of the Murray collection through the National Collection of Type Cultures, and present associated metadata with whole genome sequence data for over half of the strains. Using this information we verify the metadata for the collection with regard to subgroup designations, equivalence groupings and plasmid content. We also present genomic analyses of population structure and determinants of mobilisable antimicrobial resistance to aid strain selection in future studies. This represents an invaluable public resource for the study of these important pathogen groups and the emergence and evolution of antimicrobial resistance.

Global prevalence of carbapenem resistance in neutropenic patients and association with mortality and carbapenem use: systematic review and meta-analysis.

Background: Carbapenem-resistant Gram-negative bacteria are recognized as a cause of difficult-to-treat infections associated with high mortality. Objectives: To perform a systematic review of currently available data on distribution, characteristics and outcome associated with carbapenem-resistant bloodstream infections in adult neutropenic patients. Methods: Included studies were identified through Medline, Embase and Cochrane databases between January 1995 and April 2016. Random effect meta-analysis was used to quantify the association between carbapenem resistance and mortality and between carbapenem exposure and resistance. Results: A total of 30 studies from 21 countries were included. Overall carbapenem resistance varied from 2% to 53% (median 9%) among studies. Infections due to carbapenem-resistant Pseudomonas spp. were reported in 18 (60%) studies showing high median resistance rates (44% of all carbapenem-resistant Gram-negatives and 19% of Pseudomonas isolates). Resistance of Enterobacteriaceae was less commonly reported and bloodstream infections due to carbapenem-resistant Klebsiella spp. were mainly documented from endemic areas (Greece, Italy, Israel). Carbapenem resistance in Acinetobacter spp. was reported in 9 (30%) studies (median resistance 58% of Acinetobacter isolates). Mortality rates ranged from 33% to 71% (median 50%) in patients with carbapenem-resistant infections. Carbapenem resistance appeared to correlate with mortality (OR 4.89, 95% CI 3.30–7.26) and previous exposure to carbapenems (OR 4.63, 95% CI 3.08–6.96). Conclusions: Carbapenem resistance represents a threat to neutropenic patients. In this group, resistance is likely promoted by previous carbapenem use and leads to high mortality rates. The knowledge of resistance patterns is crucial and can direct clinicians in the use of alternatives to carbapenem-based regimens.

PME-1 Producing Pseudomonas aeruginosa in Qatar

The novel extended-spectrum β-lactamase (ESBL) PME-1 was first described in 2010 in an isolate from a Pseudomonas aeruginosa strain obtained from multiple clinical specimens from a single patient admitted to the University of Pittsburgh Medical Center in 2008. The patient had had a prolonged period

Whole genome analysis of cephalosporin-resistant Escherichia coli from bloodstream infections in Australia, New Zealand and Singapore: high prevalence of CMY-2 producers and ST131 carrying blaCTX-M-15 and blaCTX-M-27

Objectives To characterize MDR Escherichia coli from bloodstream infections (BSIs) in Australia, New Zealand and Singapore. Methods We collected third-generation cephalosporin-resistant (3GC-R) E. coli from blood cultures in patients enrolled in a randomized controlled trial from February 2014 to August 2015. WGS was used to characterize antibiotic resistance genes, MLST, plasmids and phylogenetic relationships. Antibiotic susceptibility was determined using disc diffusion and Etest. Results A total of 70 3GC-R E. coli were included, of which the majority were ST131 (61.4%). BSI was most frequently from a urinary source (69.6%), community associated (62.9%) and in older patients (median age 71 years). The median Pitt score was 1 and ICU admission was infrequent (3.1%). ST131 possessed more acquired resistance genes than non-ST131 (P = 0.003). Clade C1/C2 ST131 predominated (30.2% and 53.5% of ST131, respectively) and these were all ciprofloxacin resistant. All clade A ST131 (n = 6) were community associated. The predominant ESBL types were blaCTX-M (80.0%) and were strongly associated with ST131 (95% carried blaCTX-M), with the majority blaCTX-M-15. Clade C1 was associated with blaCTX-M-14 and blaCTX-M-27, whereas blaCTX-M-15 predominated in clade C2. Plasmid-mediated AmpC genes (mainly blaCMY-2) were frequent (17.1%) but were more common in non-ST131 (P < 0.001) isolates from Singapore and Brisbane. Two strains carried both blaCMY-2 and blaCTX-M. The majority of plasmid replicon types were IncF. Conclusions In a prospective collection of 3GC-R E. coli causing BSI, community-associated Clade C1/C2 ST131 predominate in association with blaCTX-M ESBLs, although a significant proportion of non-ST131 strains carried blaCMY-2.