Re-visiting humoral constitutive antibacterial heterogeneity in bloodstream infections.

Although cellular immunity has garnered much attention in the era of single-cell technologies, humoral innate immunity has receded in priority due to its presumed limited roles. Hence, despite the long-recognised bactericidal activity of serum-a functional characteristic of constitutive humoral immunity-much remains unclear regarding mechanisms underlying its inter-individual heterogeneity and clinical implications in bloodstream infections. Recent work suggests that the immediate antimicrobial effect of humoral innate immunity contributes to suppression of the excessive inflammatory responses to infection by reducing the amount of pathogen-associated molecular patterns. In this Personal View, we propose the need to re-explore factors underlying the inter-individual heterogeneity in serum antibacterial competence as a new approach to better understand humoral innate immunity and revisit the clinical use of measuring serum antibacterial activity in the management of bacterial bloodstream infections. Given the current emphasis on subtyping sepsis, a serum bactericidal assay might prove useful in defining a distinct sepsis endotype, to enable more personalised management.

Neutrophil Extracellular Traps have DNAzyme activity that drives bactericidal potential.

The mechanisms of bacterial killing by neutrophil extracellular traps (NETs) are unclear. DNA, the largest component of NETs is believed to merely be a scaffold with minimal antimicrobial activity through the charge of the backbone. Here, we report that NETs DNA is beyond a scaffold and produces hydroxyl free radicals through the spatially concentrated G-quadruplex/hemin DNAzyme complexes, driving bactericidal effects. Immunofluorescence staining showed colocalization of G-quadruplex and hemin in extruded NETs DNA, and Amplex UltraRed assay portrayed its peroxidase activity. Proximity labeling of bacteria revealed localized concentration of radicals resulting from NETs bacterial trapping. Ex vivo bactericidal assays revealed that G-quadruplex/hemin DNAzyme is the primary driver of bactericidal activity in NETs. NETs are DNAzymes that may have important biological consequences.

Carbapenem triggers dissemination of chromosomally integrated carbapenemase genes via conjugative plasmids in Escherichia coli.

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 blaIMP-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 blaIMP-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 blaIMP-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.

Rapid Molecular Phenotypic Antimicrobial Susceptibility Test for Neisseria gonorrhoeae Based on Propidium Monoazide Viability PCR.

Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments, leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof-of-concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for the susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90 and 75% susceptible and resistant strains, respectively, to ceftriaxone and 66.7 and 83.3% susceptible and resistant strains, respectively, to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy within 30 min, a significant improvement compared to the conventional method which could take multiple days.

Rapid molecular phenotypic antimicrobial susceptibility test for Neisseria gonorrhoeae based on propidium monoazide viability PCR.

Neisseria gonorrhoeae (NG) is an urgent threat to antimicrobial resistance (AMR) worldwide. NG has acquired rapid resistance to all previously recommended treatments leaving ceftriaxone monotherapy as the first and last line of therapy for uncomplicated NG. The ability to rapidly determine susceptibility, which is currently nonexistent for NG, has been proposed as a strategy to preserve ceftriaxone by using alternative treatments. Herein, we used a DNA-intercalating dye in combination with NG-specific primers/probes to generate qPCR cycle threshold (Ct) values at different concentrations of 2 NG-relevant antimicrobials. Our proof of concept dual-antimicrobial logistic regression model based on the differential Ct measurements achieved an AUC of 0.93 with a categorical agreement for susceptibility of 84.6%. When surveying the performance against each antimicrobial separately, the model predicted 90% and 75% susceptible and resistant strains respectively to ceftriaxone and 66.7% and 83.3% susceptible and resistant strains respectively to ciprofloxacin. We further validated the model against the individual replicates and determined the accuracy of the model in classifying susceptibility agnostic of the inoculum size. We demonstrated a novel PCR-based approach to determine phenotypic ciprofloxacin and ceftriaxone susceptibility information for NG with reasonable accuracy in under 30 min, a significant improvement compared to the conventional method which takes 3 days.

A Nationwide Plasmidome Surveillance in Thailand Reveals a Limited Variety of New Delhi Metallo-ß-Lactamase-Producing Carbapenem-Resistant Enterobacteriaceae Clones and Spreading Plasmids.

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. We analyzed 371 Enterobacteriaceae isolates carrying blaNDM-1 and 114 Enterobacteriaceae isolates carrying blaNDM-5 obtained from clinical samples of 473 patients in 11 representative hospitals located in six provinces in Thailand between 2012 and 2017. The complete structures of plasmids carrying blaNDM and chromosomal phylogeny were determined by combining Southern blotting hybridization analysis and our previously performed whole-genome short-read sequencing data. Dissemination of the blaNDM-5 gene among the Enterobacteriaceae isolates in Thailand was mainly owing to the nationwide clonal spread of Escherichia coli ST410 and regional clonal spreads of Escherichia coli ST361 and ST405. Analysis of blaNDM-1-carrying isolates revealed nationwide dissemination of two specific plasmids and nationwide clonal dissemination of Klebsiella pneumoniae ST16 accompanied with regional disseminations of three distinctive K. pneumoniae clones (ST231, ST14, and ST147) with different plasmids. Dissemination of CRE carrying blaNDM in Thailand is mainly based on nationwide clonal expansions of E. coli ST410 carrying blaNDM-5 and K. pneumoniae ST16 carrying blaNDM-1, nationwide dissemination of two distinctive plasmids carrying blaNDM-1, and accumulation of clonal expansions in regional areas. Although the overuse of antibiotics can promote CRE dissemination, the limited variety of transmitters highlights the importance of preventing horizontal dissemination among patients.

[Regional dissemination of carbapenem-resistant Enterobacteriaceae accompanying with enhanced resistance in Northern Osaka, Japan].

With the rapid spread of multidrug-resistant bacteria, carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide as a major concern because of limited treatment options. Carbapenem resistance is mainly due to carbapenem-ase, a carbapenem-degrading enzyme, which is mainly encoded on a plasmid to spread across bacterial species. However, there have been only small-scale attempts to determine the similarities or accommodations of the plasmids disseminating regionwide. We analysed the 230 CRE isolates carrying blaIMP from 43 medical facilities in the northern Osaka area focusing on the plasmids, the main carriers of the drug resistance genes. Combination of whole genome sequencing and Southern blotting revealed the predominant dissemination of blaIMP-6 by the pKPI-6 plasmid among genetically distinct isolates, as well as the emergences of derivatives that acquired various advantages. We iden-tified heteroresistance likely causing stealth transmissions, which was generated by the transcriptional regu-lation of blaIMP-6, stabilization of blaIMP-6 through chromosomal integration, enhanced carbapenem resistance through plasmid multimerization, or broadened antimicrobial resistance due to a single point mutation in blaIMP-6. In this article, I dis-cussed the mechanisms of regional spread of CRE and enhancement of carbapenem resistance providing the insights to prevent their disseminations.

Clonal dissemination of carbapenem-resistant Klebsiella pneumoniae ST16 co-producing NDM-1 and OXA-232 in Thailand.

Background: Klebsiella pneumoniae ST258 and ST11 carrying bla KPC are among the most widespread carbapenem-resistant K. pneumoniae strains worldwide. Our carbapenem-resistant Enterobacteriaceae surveillance in Thailand revealed a nationwide dissemination of K. pneumoniae ST16 isolates carrying bla NDM-1 and bla OXA-232. Objectives: To analyse the genomic details of this nationwide dissemination by focusing on plasmids and virulence factors. Methods: Using WGS data of 119 K. pneumoniae ST16 isolates carrying bla NDM-1 obtained in our previous surveillance study, clonality of chromosomes and plasmids of the isolates with carriage of virulence factors was evaluated. Results: Of the 119 isolates, 111 carried plasmid pKP151_NDM1, and all 104 isolates harbouring bla OXA-232 carried plasmid pKP151_OXA232. These 104 K. pneumoniae ST16 isolates showing chromosomal clonality possessed both pKP151_NDM1 and pKP151_OXA232, demonstrating clonal dissemination of K. pneumoniae ST16 with these plasmids. The isolates had essentially similar virulence factors as those of K. pneumoniae ST16 clones carrying bla KPC, which were recently reported as highly invasive clones in Brazil. Conclusions: The potential global dissemination of these invasive clones with resistance to several antibiotics highlights the importance of appropriate monitoring and strict standard precautions.

Population analysis profiling: is it still the gold standard for the determination of heteroresistance in carbapenemase-producing Enterobacteriaceae?

Heteroresistance is the phenomenon wherein subpopulations of presumed isogenic bacteria show varied antibiotic susceptibilities, and the current gold standard for the determination of heteroresistance is population analysis profiling (PAP). However, when conducting PAP to confirm carbapenem heteroresistance in Enterobacteriaceae, the authors found some isolates that did not seem to be heteroresistant, despite meeting PAP criteria. This article elaborates on the validity of PAP for the determination of heteroresistance, especially among carbapenemase-producing Enterobacteriaceae (CPE). Bacterial cells that were originally non-viable on selective agar supplemented with a high concentration of meropenem were found to be occasionally viable, likely due to the hydrolysis of carbapenems by carbapenemases produced by dying cells, mimicking the emergence of subpopulations with enhanced resistance. As such, PAP for CPE is highly affected by carbapenemases produced by dying populations, and may not detect heterogeneity in carbapenem resistance appropriately among seemingly isogenic clones.

Diagnosis of Bloodstream Infections: An Evolution of Technologies towards Accurate and Rapid Identification and Antibiotic Susceptibility Testing.

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.

Enhanced Carbapenem Resistance through Multimerization of Plasmids Carrying Carbapenemase Genes.

The worldwide dissemination of carbapenem-resistant Enterobacteriaceae (CRE) poses a critical human health issue by limiting the range of antibiotics that are usable in the treatment of common bacterial infections. Along with CRE, carbapenem heteroresistance has disseminated worldwide, which is described as different levels of carbapenem resistance within a seemingly isogenic bacterial population. Unstable carbapenem resistance will likely lead to unexpected treatment failure due to the enhanced resistance after initiation of treatment, contradicting antimicrobial susceptibility test results. Porin mutation and tandem amplification of the carbapenemase gene have been reported as mechanisms underlying enhanced carbapenem resistance. In this study, we identified multimerization of plasmids carrying carbapenemase genes, by using Southern blotting, whole-genome sequencing, and quantitative PCR (qPCR) analysis for the CRE isolates obtained in our previous surveillance in Osaka, Japan. Plasmids harboring a carbapenemase gene were multimerized by recA, likely through recombination at two consecutive sets of transposase genes of the IS91 family, thereby producing various plasmids of discrete sizes in a single bacterial cell of an Escherichia coli isolate. This multimerization resulted in increased copy numbers of carbapenemase genes, leading to enhanced gene transcription as well as carbapenem resistance. Prior exposure to meropenem further increased the copy number of carbapenemase genes, readily resulting in enhancement of carbapenem resistance. This mechanism may lead to clinical treatment failure by sifting antimicrobial resistance after the treatment initiation. IMPORTANCE We demonstrated the multimerization of plasmids harboring carbapenemase genes, and multimeric plasmids of various discrete sizes existed in a host bacterial cell of Escherichia coli. Plasmid multimerization along with increased copy numbers of carbapenemase genes resulted in enhanced carbapenemase resistance, which was readily accelerated by an overnight preexposure to meropenem. This mechanism may lead to treatment failure in clinical settings after the initiation of antimicrobial therapy.

Effects of Ventilatory Settings on Pendelluft Phenomenon During Mechanical Ventilation.

BACKGROUND: Pendelluft phenomenon is defined as the displacement of gas from a more recruited nondependent (ND) lung region to a less recruited dependent (D) lung region. This phenomenon may cause lung injury. Thus, a lung model for pendelluft was established, and the effects of ventilatory settings on pendelluft were examined. METHODS: Two sets of the twin-bellows-type training test lung (TTL) model were utilized. One set of bellows simulated the diaphragm, and the other simulated the lung. One TTL model represented the ND region, and the other represented the D region. The lung bellows were connected to each other and were ventilated with 1 ventilator. The diaphragm bellows were ventilated with 2 synchronized ventilators that regulated pleural pressure levels. We simulated pendelluft by applying different pleural pressure levels to the D and ND bellows. The increment of the tidal volume in the D region from the "no breathing effort" condition was defined as the pendelluft volume. The effects of ventilator settings, such as ventilatory modes, triggering sensitivity, inspiratory pressurization, and inspiratory cycling-off, were examined. The changes in tidal volumes in the D region based on the control settings were compared to assess the severity of pendelluft. RESULTS: The gas flow from the D region to the ND region was found to be essential in pendelluft, but the severity of this phenomenon was not always proportional to gas flows. The severity increased with the increase in the differences in pleural pressure levels between the ND and D regions, and it was amplified by the difference in lung mechanics between the ND and D regions. However, the ventilator settings had minimal effect on the severity of pendelluft. CONCLUSIONS: The pendelluft was affected by the heterogeneity of lung mechanics and pleural pressure. Furthermore, a minimal association was observed between the ventilator settings and the severity of pendelluft.

Characterization of the Plasmidome Encoding Carbapenemase and Mechanisms for Dissemination of Carbapenem-Resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) infections, high in morbidity and mortality, pose serious clinical challenges due to limited treatment options. A previous CRE surveillance study on 1,507 patients from 43 hospitals in Osaka, Japan, revealed that 12% of patients carried CRE and that 95% of the CRE isolates were IMP-type carbapenemase producers. Here, the mechanisms for this regional dissemination of a single carbapenemase gene were investigated. Since the dissemination of CRE is primarily due to the transmission of carbapenemase genes located on plasmids, we analyzed the plasmidome of 230 CRE isolates carrying bla IMP by whole-genome sequencing and Southern blotting. bla IMP-6 was found to be predominantly disseminated among chromosomally distinct isolates through the pKPI-6 plasmid. Underlying the vast clonal dissemination of pKPI-6, various subpopulations deriving from pKPI-6 were identified, which had acquired advantages for the dissemination of CRE isolates. A cluster exhibiting heteroresistance against meropenem by the transcriptional regulation of bla IMP-6 caused an outbreak likely through covert transmission of bla IMP-6 For stable carriage of bla IMP-6, they occasionally integrated bla IMP-6 on their chromosomes. In addition, we detected one isolate that broadened the range of antimicrobial resistance through a single point mutation in bla IMP-6 on pKPI-6. Multifaceted analysis of the plasmidome granted us more accurate perspectives on the horizontal spread of CRE isolates, which is difficult to trace only by comparing the whole genomes. This study revealed the predominant spread of a specific carbapenemase-encoding plasmid accompanying the emergence of phenotypically diverse derivatives, which may facilitate further dissemination of CRE in various environments.IMPORTANCE Global dissemination of carbapenem-resistant Enterobacteriaceae (CRE) threatens human health by limiting the efficacy of antibiotics even against common bacterial infections. Carbapenem resistance, mainly due to carbapenemase, is generally encoded on plasmids and is spread across bacterial species by conjugation. Most CRE epidemiological studies have analyzed whole genomes or only contigs of CRE isolates. Here, plasmidome analysis on 230 CRE isolates carrying bla IMP was performed to shed light into the dissemination of a single carbapenemase gene in Osaka, Japan. The predominant dissemination of bla IMP-6 by the pKPI-6 plasmid among genetically distinct isolates was revealed, as well as the emergences of pKPI-6 derivatives that acquired advantages for further disseminations. Underlying vast clonal dissemination of a carbapenemase-encoding plasmid, heteroresistance was found in CRE offspring, which was generated by the transcriptional regulation of bla IMP-6, stabilization of bla IMP-6 through chromosomal integration, or broadened antimicrobial resistance due to a single point mutation in bla IMP-6.

In Vitro Efficacy of Meropenem-Cefmetazole Combination Therapy against New Delhi Metallo-ß-lactamase-producing Enterobacteriaceae.

BACKGROUND: Limited treatment options complicate management of infections with New Delhi metallo-ß-lactamase (NDM)-producing organisms. The efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) was assessed against NDM-producing Enterobacteriaceae. MATERIALS AND METHODS: Twelve Escherichia coli clinical isolates harbouring blaNDM-1 and a positive control E. coli BAA-2469 harbouring blaNDM-1 were studied. Minimum inhibitory concentrations (MICs) of MEM, ertapenem (ERT) and CMZ were determined by broth microdilution. Checkerboard and time-kill assays were performed to confirm the in vitro efficacy of the MEM/CMZ combination. Scanning electron microscopy, kinetic studies and whole-genome sequence analysis were used to determine the antimicrobial resistance mechanisms. RESULTS: MICs of MEM, ERT and CMZ in monotherapy ranged from 8 to 32, 16 to 128, and 32 to 512 µg/mL, respectively. In the checkerboard assay, MEM/ERT resulted in no synergy, whereas MEM/CMZ showed a synergistic effect in all the tested isolates. Furthermore, the MIC of MEM in combination decreased by 2- to 8-fold compared with that of MEM alone. The time-kill study revealed a bactericidal effect in 4 of 13 isolates at 24 h. Scanning electron microscopy showed spheroidisation of the bacterial cell in the MEM/CMZ combination; this was not observed in single antibiotic conditions. Kinetic studies indicated CMZ was a better antagonist for NDM-1 than ERT. Whole-genome sequence analysis did not reveal any explainable differences between isolates susceptible and those non-susceptible to combination therapy. CONCLUSION: In vitro studies showed the potential effectiveness of MEM/CMZ combination therapy against NDM-producing organisms.

Genomic characterisation of a novel plasmid carrying blaIMP-6 of carbapenem-resistant Klebsiella pneumoniae isolated in Osaka, Japan.

OBJECTIVES: To analyse plasmids carrying blaIMP-6 in Klebsiella pneumoniae isolates obtained from multicentre carbapenem-resistant Enterobacteriaceae surveillance. METHODS: Plasmids harbouring blaIMP-6 were characterised by the whole-genome sequencing of four Klebsiella pneumoniae isolates carrying blaIMP-6, and compared with the pKPI-6 plasmid, which is widespread in western Japan, through pulsed-field gel electrophoresis, Southern blotting, bacterial conjugation, and qPCR. RESULTS: 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. It was 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. CONCLUSIONS: This study 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.

Bactericidal efficacy of meropenem in combination with cefmetazole against IMP-producing carbapenem-resistant Enterobacteriaceae.

OBJECTIVE: Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to public and clinical health because of their high levels of resistance to various antibiotics. We assessed the efficacy of combination therapy with meropenem (MEM) and cefmetazole (CMZ) against Imipenemase (IMP)-producing CRE, using the checkerboard method and time-killing assay on 13 Enterobacteriaceae isolates harboring blaIMP-1 (4 Enterobacter hormaechei, 5 Escherichia coli, and 4 Klebsiella pneumoniae isolates) and 13 isolates harboring blaIMP-6 (8 E. coli and 5 K. pneumoniae isolates). RESULTS: Minimum inhibitory concentrations (MICs) of MEM and CMZ ranged from 2 to 64 and 64 to 2048 µg/mL, respectively. Checkerboard method demonstrated the synergy of the MEM/CMZ combination in all the tested IMP-producing CRE isolates, and the time-kill assay indicated a bactericidal effect for both blaIMP-1 and blaIMP-6 positive CRE when MEM/CMZ combination was used. In vitro, the MEM/CMZ combination was potentially effective against IMP-1- or IMP-6-producing CRE. Further investigations including in vivo animal studies and clinical studies are warranted to corroborate the clinical utility of the novel combination therapy.

First Identification and genomic characterization of multidrug-resistant carbapenemase-producing Enterobacteriaceae clinical isolates in Malawi, Africa.

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 ß-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.

Perioperative Enteral Nutrition After Left Ventricular Assist Device Implantation.

OBJECTIVE: We assessed what predicts nutritional adequacy at day 14 following implantation of left ventricular assist device (LVAD). METHOD: We retrospectively reviewed the cases of 97 adult patients who underwent LVAD implantation at our institution from June 2011 to June 2016. We divided the patients into two groups based on the administered enteral nutrition (EN) calories on post-operative day (POD) 14: the EN calories of group SEN (sufficient enteral nutrition) were >80% of their total target calories, or the EN calories of group IEN (insufficient enteral nutrition) were <80% of their total target calories. We compared the two groups in terms of the perioperative factors within 1 week after surgery. RESULTS: Groups SEN and IEN consisted of 53 and 44 patients, respectively. The mean doses of adrenaline and noradrenaline, mean central venous pressure (CVP), duration of nitric oxide administration, and mean residual gastric volume during 1 week after surgery in group SEN were significantly lower than those in group IEN (P < .05). In multivariate analysis, higher CVP during 1 week after surgery was identified as an independent risk factor for delayed EN on POD14 (odds ratio, 1.40; 95% confidence interval, 1.11-1.66; P = .0037). Total bilirubin, occurrence of acute kidney injury, and mixed venous blood saturation during 1 week after surgery were not significant predictors for EN on POD14. CONCLUSIONS: Increased CVP within 1 week after LVAD implantation was an independent factor for reduced EN feeding.

Factors Related to the Severity of Early Postoperative Infection After Heart Transplantation in Patients Surviving Prolonged Mechanical Support Periods: Experience at a Single University.

OBJECTIVE: The authors examined the effect of prolonged support with continuous-flow ventricular assist devices (CF-VADs) and other related factors on the severity of infections within 30 days of heart transplantation (HTx). DESIGN: A retrospective analysis of consecutive HTx procedures. SETTING: University hospital, between 2010 and 2016. PARTICIPANTS: A cohort of 53 heart transplantation recipients (median age, 38.5 yr; interquartile range [IQR], 30.3-49.2 yr; women, 34%). INTERVENTIONS: Forty-nine patients required CF-VAD support (median duration, 946 d; IQR, 600-1,132 d). MEASUREMENTS AND MAIN RESULTS: Severity of postoperative infections was categorized as follows: no infection, minor infection (resolved within 14 days), major infection (resolved after >14 days), and severe infection (septic shock). Results were expressed as number (frequency) and median with IQR. Potential risk factors for increased infection severity were expressed as odds ratio (OR) with 95% confidence interval (CI). Postoperatively, no infection, minor infection, major infection, and severe infection occurred in 32 (60.4%), 8 (15.1%), 8 (15.1%), and 5 patients (9.4%), respectively. Active ventricular assist device (VAD)-specific infections at the time of HTx occurred in 37.7% of patients. Moderate-to-severe primary graft dysfunction occurred in 26.4% of the patients. Multivariable analysis indicated that risk factors for increased infection severity included active VAD-specific infection (OR 4.8; 95% CI 2.3-11.2) and moderate-to-severe primary graft dysfunction (OR 8.8; 95% CI 2.1-42.5) but not duration of CF-VAD support (OR 1.0; 95% CI 1.0-1.0). CONCLUSION: Active VAD-specific infection and poor graft function likely contribute to the severity of early postoperative infections after HTx.