Bloodstream infections (BSI) are a leading cause of death worldwide. The lack of timely and reliable diagnostic practices is an ongoing issue for managing BSI. The current gold standard blood culture practice for pathogen identification and antibiotic susceptibility testing is time-consuming. Delayed diagnosis warrants the use of empirical antibiotics, which could lead to poor patient outcomes, and risks the development of antibiotic resistance. Hence, novel techniques that could offer accurate and timely diagnosis and susceptibility testing are urgently needed. This review focuses on BSI and highlights both the progress and shortcomings of its current diagnosis. We surveyed clinical workflows that employ recently approved technologies and showed that, while offering improved sensitivity and selectivity, these techniques are still unable to deliver a timely result. We then discuss a number of emerging technologies that have the potential to shorten the overall turnaround time of BSI diagnosis through direct testing from whole blood—while maintaining, if not improving—the current assay’s sensitivity and pathogen coverage. We concluded by providing our assessment of potential future directions for accelerating BSI pathogen identification and the antibiotic susceptibility test. While engineering solutions have enabled faster assay turnaround, further progress is still needed to supplant blood culture practice and guide appropriate antibiotic administration for BSI patients.

Purpose. Carbapenemase-producing Enterobacteriaceae (CPE) have become a global concern and a serious threat to human health due to their resistance to multiple antibiotics. In this study, we identified and characterized CPE for the first time in Malawi, southeastern Africa. Methodology. We investigated the possible presence of carbapenemases among a collection of 200 ceftriaxone-nonsusceptible Gram-negative clinical isolates obtained from five Malawian hospitals between January 2016 and December 2017, using both phenotypic and genotypic tests. Molecular typing of CPE was done by PFGE, multilocus sequence typing (ST) or phylogenetic grouping. Resistant plasmids were characterized by S1 PFGE, Southern blotting and conjugation assays. Results. Out of 200 isolates, we detected 16 (8 %) CPE of which all originated from one referral hospital, Kamuzu Central Hospital, in the Central part of Malawi. Of 16 isolates, seven Klebsiella pneumoniae ST340/CC258 carried bla KPC-2, two Escherichia coli ST636 (phylogroup B2) carried bla NDM-5, six E. coli ST617 (phylogroup A) and one Klebsiella variicola carried bla OXA-48. All carbapenemases were plasmid-encoded, but only bla NDM-5-carrying plasmids could be conjugated. Most isolates co-harboured other β-lactamases and consequently exhibited a wider spectrum of resistance to commonly used antibiotics. We observed indistinguishable genetic profiles between strain types, despite originating from different wards, suggesting acquisition during admission and intra-hospital spread. Conclusion. This report strongly suggests a probable existence of highly resistant various types of CPE organisms in Malawi including KPC-2-producing K. pneumoniae ST340/CC258, a known high-risk epidemic lineage.

Despite frequent identification of plasmids carrying carbapenemase genes, the transfer of plasmids carrying carbapenemase genes is not well recognized in clinical settings because of technical limitations. To investigate the detailed mechanisms of the spread of carbapenem-resistant Enterobacteriaceae (CRE), we performed multifaceted genomic surveillance of CRE isolates in Thailand and analyzed their plasmidome.

To analyse plasmids carrying blaIMP-6 in isolates of Klebsiella pneumoniae obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. We characterised plasmids harbouring blaIMP-6 by the whole-genome sequencing of four K. pneumoniae isolates carrying blaIMP-6, and compared them with the pKPI-6 plasmid widespread in western Japan through PFGE, Southern blotting, bacterial conjugation, and qPCR. Whole-genome sequencing analysis revealed that three of the four isolates carried approximately 50 kbp plasmids similar to the pKPI-6 plasmid, however, one isolate carried a 250 kbp plasmid harbouring blaIMP-6 (pE196_IMP6). So far, all of the reported plasmids carrying blaIMP-6 were similar to the pKPI-6 plasmid, and this plasmid was a novel blaIMP6-carrier. The size and transferability of this plasmid was confirmed by Southern hybridisation and conjugation experiments. We demonstrated that the generation of plasmid pE196_IMP6 was due to an intramolecular transposition mediated by IS26, and a homologous recombination between plasmids pKPI-6 and pE013 that was obtained from another carbapenem-resistant Enterobacteriaceae isolate in this analysis. As a result of co-integration with pE013, pE196_IMP6 acquired six additional pairs of type II toxin-antitoxin systems that pKPI-6 does not carry. Transcription of all of the toxin-antitoxin systems were confirmed in an isolate carrying pE196_IMP6 by qPCR. We detected a novel plasmid carrying blaIMP-6, and revealed the origin of this plasmid. Toxin-antitoxin system acquisition could enable pE196_IMP6 maintenance persistently through successions, even without selection pressure by the clinical usage of antimicrobials, generating broad dissemination and longer carbapenem-resistant Enterobacteriaceae colonisation duration in patients.

Epidemiological surveys have shown that carbapenem resistance is mainly transmitted across species by carbapenemase genes located on conjugative plasmids. As chromosomal integration of carbapenemase genes has rarely been identified, only a few studies have investigated their advantages to the carbapenem-resistant bacterial community. Here, we confirmed the increased stability of bla _IMP-6 on a chromosome-integrated plasmid in an Escherichia coli isolate compared with that on original plasmids in the absence of antibiotic pressure. Although plasmids carrying carbapenemase genes are supposedly lost in successive generations, we found that the complete plasmid backbone was retained in bacterial cells even after the occasional loss of their antibiotic-resistance cassettes. This backbone structure has been observed worldwide to carry various antimicrobial resistance genes. Although the chromosomally integrated plasmid carrying bla _IMP-6 could not be transmitted by conjugation, we found that meropenem treatment for 1 wk allowed the plasmid to be released from the chromosome and spread among E. coli strains that were susceptible to meropenem. The copy number of bla _IMP-6 on the plasmid was amplified eight times, resulting in enhanced resistance. Although the carbapenemase producers that carry chromosomal carbapenemase genes comprised of small subpopulations, they functioned as stable, long-term reservoirs of carbapenem resistance that could be disseminated via plasmids with amplified resistance upon meropenem stimulation. Although plasmids occasionally lose their resistance cassettes as a scaffold for the acquisition of another resistance gene, chromosomal integration may contribute to the effective sharing of carbapenem resistance within a population, complicating the development of a strategy to avoid the dissemination of antimicrobial resistance. IMPORTANCE: Although carbapenem antibiotics are the last resort for combating multidrug-resistant organisms, global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens public health. Carbapenemases, which are enzymes responsible for carbapenem resistance, are mainly encoded by genes on plasmids that can be transmitted across bacterial species. Owing to the rarity of chromosomally encoded carbapenemase genes, studies investigating their properties in bacterial communities are lacking. In our study, we revealed the stability of carbapenemase genes on chromosomes compared with those on plasmids, which can be lost through the loss of antimicrobial resistance cassettes despite robust retention of plasmid backbones. Following exposure to meropenem, the carbapenemase gene integrated into the chromosome was released as a plasmid, restarting the dissemination of enhanced carbapenem resistance through amplified copy numbers of carbapenemase genes. Chromosomally encoded carbapenemase genes may function as a reservoir of resistance genes within the bacterial community and challenge infection control against CRE dissemination.