Dissemination Of Carbapenem-resistant Enterobacteriaceae Research Articles

Genomically diverse carbapenem resistant Enterobacteriaceae from wild birds provide insight into global patterns of spatiotemporal dissemination

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenically-associated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

Clonal and plasmid-mediated dissemination of environmental carbapenem-resistant Enterobacteriaceae in large animal breeding areas in northern China

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) constitute a major global health problem. The environment plays an important role in the dissemination of CRE, but large-scale studies on CRE in groundwater environments in animal breeding areas are scarce. The aim of this study was to investigate CRE occurrence and environmental transmission of carbapenem resistance genes in large animal breeding areas in northern China. In total, 280 well water and 102 animal feces samples in large animal breeding areas in six counties from the two provinces Inner Mongolia and Shandong in China, were screened for CRE. A total of 39 CRE were isolated and characterized with next-generation sequencing. 5.3% of well water samples were contaminated with CRE. The well water in chicken farms had the highest number of detections of CRE (15.9%). More than half of the isolates carried closely related, conjugative IncX3 plasmids with blaNDM-genes from multiple geographic areas, indicating that this kind of plasmid plays an important role in dissemination of carbapenem resistance determinants. The clonal expansion of various CRE isolates in well water and animal feces were demonstrated; clonally related CRE were isolated from different wells within the same county, from different counties in the same province, and even from different provinces. In addition to harboring various ARGs, two closely related K. pneumoniae belonging to ST11 isolated from well water carried genetic hypervirulence determinants on a virulence plasmid, highlighting the potential health risk posed by further dissemination of this strain. These findings suggest that groundwater may be an underappreciated reservoir and source of dissemination of CRE, from which resistance genes may disseminate among different bacterial strains and over large geographic distances. Further research and multi-sectorial monitoring, with a “One health” perspective, is urgently needed to investigate the need for interventions aimed at preventing CRE dissemination.

Characterization of Carbapenem-Resistant Enterobacteriaceae Cultured From Retail Meat Products, Patients, and Porcine Excrement in China.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern to animal and public health. However, little is known about the spread of CRE in food and livestock and its potential transmission to humans. To identify CRE strains from different origins and sources, 53 isolates were cultured from 760 samples including retail meat products, patients, and porcine excrement. Antimicrobial susceptibility testing was carried out, followed by phylogenetic typing, whole-genome sequencing, broth mating assays, and plasmids analyses. Forty-three Escherichia coli, nine Klebsiella pneumoniae, and one Enterobacter cloacae isolates were identified, each exhibiting multidrug-resistant phenotypes. Genetically, the main sequence types (STs) of E. coli were ST156 (n = 7), ST354 (n = 7), and ST48 (n = 7), and the dominant ST of K. pneumoniae is ST11 (n = 5). blaNDM–5 (n = 40) of E. coli and blaKPC–2 (n = 5) were the key genes that conferred carbapenem resistance phenotypes in these CRE strains. Additionally, the mcr-1 gene was identified in 17 blaNDM-producing isolates. The blaNDM–5 gene from eight strains could be transferred to the recipients via conjugation assays. Two mcr-1 genes in the E. coli isolates could be co-transferred along with the blaNDM–5 genes. IncF and IncX3 plasmids have been found to be predominantly associated with blaNDM gene in these strains. Strains isolated in our study from different sources and regions tend to be concordant and overlap. CRE strains from retail meat products are a reservoir for transition of CRE strains between animals and humans. These data also provide evidence of the dissemination of CRE strains and carbapenem-resistant genes between animal and human sources.

In vitro and in silico screening of Klebsiella pneumoniae new Delhi metallo-β-lactamase-1 inhibitors from endophytic Streptomyces spp.

The increase in drug resistance over the last two decades is a big threat in health care settings. More importantly, the dissemination of carbapenem-resistant Enterobacteriaceae is the major threat to public health with an increase in morbidity and mortality. β-lactamase is known to confer enteric bacteria with nearly complete resistance to all β-lactam antibiotics including the late-generation carbapenems. The commercially available β-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam are being met with an increasing number of resistant phenotypes and are ineffective against pathogens harbouring New Delhi metallo-β-lactamase (NDM-1). Inhibition of New Delhi metallo-β-lactamase-1 activity is one potential way to treat metallo β-lactamase (MBL) producing multi drug resistant (MDR) pathogen. The present study focused on screening of Klebsiella pneumoniae New Delhi metallo-β-lactamase-1 (BLIs) from endophytic Streptomyces spp. using in vitro and in silico methods. The study identified three potential inhibitors of New Delhi metallo-β-lactamase-1, namely dodecanoic acid, dl-alanyl- l-leucine and phenyl propanedioic acid. These molecules were found to bind to other MBLs namely, IMP-1 and VIM-2. To the best of our knowledge, this is the first kind of study reporting the binding mode of these molecules with New Delhi metallo-β-lactamase-1. Communicated by Ramaswamy H. Sarma

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.

<p>Microbiological Characteristics of Carbapenem-Resistant <em>Enterobacteriaceae</em> Clinical Isolates Collected from County Hospitals</p>

ObjectiveTo investigate the molecular characteristics of carbapenem-resistant Enterobacteriaceae (CRE) from county hospitals in China.Materials and MethodsForty-three sequential non-duplicate CRE strains (including 33 Klebsiella pneumoniae isolates, 4 Enterobacter cloacae isolates, 3 Escherichia coli isolates, 1 Serratia marcescens, 1 Morganella morganii and 1 Citrobacter freundii) were collected from 4 county hospitals and 2 municipal hospitals. Antimicrobial susceptibility testing was conducted by broth microdilution method, using 3-aminophenylboronic acid and EDTA and the modified carbapenem inactivation method (mCIM) to screen phenotype of carbapenemase. β-Lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of blaNDM-5 was investigated by transformation experiment. Clonal relatedness was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing .ResultsThe results of antimicrobial susceptibility testing indicated that 43 CRE strains were resistant to most of the antimicrobial agents, except tigecycline and colistin. Overall, 93%, 93%, and 97.7% of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. PCR and DNA sequencing indicated that 67.4% (29/43) were blaKPC-2 positive isolates, in which 3.4% (1/29) was coproduced with blaNDM-1. In addition, 7.0% (3/43), 4.7% (2/43), 4.7% (2/43), 2.3% (1/43), 2.3% (1/43) were blaNDM-1, blaNDM-16, blaNDM-4, blaNDM-5, blaIMP-4 positive isolates, respectively. The 29 blaKPC-2-positive isolates belonged to 12 different PFGE type and designated as ST11 (n=20) and ST15, ST39, ST116, ST667, ST2245, ST2338. The plasmid bearing blaNDM-5 could be transferred into recipient E. coli J53 through transformation.ConclusionOur study indicated the dissemination of CRE between the tertiary hospitals and secondary hospitals.

Demonstration of bactericidal and synergistic activity of quercetin with meropenem among pathogenic carbapenem resistant Escherichia coli and Klebsiella pneumoniae

Rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE) is considered to be a global issue. Quercetin is a well known antimicrobial agent. Hence, it would be important to investigate bactericidal and synergistic interaction of quercetin with meropenem and elucidate effects of quercetin alone and quercetin-meropenem combination on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions, cellular morphology and cell-wall/membrane integrity. MIC, Time-kill and Baclight assays were performed to determine antibacterial/bactericidal activity of quercetin. Synergism with meropenem was evaluated by checkerboard assay followed by dose-response, isobologram analysis and FIC index, combination index calculation. Effects of meropenem, quercetin and their combinations on blaNDM, blaVIM,acrB, ompC, ompF, ompk35 and ompk36 expressions were evaluated by qRT-PCR. SEM was performed to evaluate effects of aforesaid combinations on cellular morphology. Quercetin alone exhibited at least four-fold reduced MIC value (16–256 μg/mL) than that of meropenem against CRE. It exhibited synergism with meropenem against 89.25% CRE. Again, only 128 μg/mL quercetin killed upto 99.95% bacteria within 4–6 h of dosing, which increased further to 99.99% in MIC combination of meropenem-quercetin. Thus, effective bactericidal activity of quercetin-meropenem combination might have been achieved through alteration of blaVIM, ompC expression and cellular morphology of bacteria. Quercetin exhibited bactericidal and synergistic activity with meropenem.

Emergence of New Delhi metallo beta lactamase blaNDM-1 and Oxacillinases blaOXA-48 producing Klebsiella pneumoniae in an Egyptian hospital

The rapid dissemination of carbapenem resistant Enterobacteriaceae (CRE) all over the world represents a matter of concern. This study aims to investigate the prevalence of CRE in clinical isolates recovered from Al Kasr Al-Ainy hospital in the period between August 2015 and February 2017. The isolates were identified by conventional methods and Maldi-TOF spectroscopy. Phenotypic identification of CRE was carried out by Modified Hodge test and genotypic characterization for Extended-Spectrum Beta-Lactamase (ESBL) and CRE genes was also performed. Carbapenemase activity of CRE isolates was confirmed in 46% of the isolates of which, blaNDM-1 and blaOXA-48 were detected in 75%, 59% of the isolates respectively, while blaVIM was detected in 2.3% only. However, blaIMP and blaKPC were not detected in any isolate. All CRE isolates carried at least one ESBL gene, 95.4% of CRE isolates had blaCTX-M-15, 88.6% blaTEM-1, and 68.2% blaSHV. The blaSHV gene showed different alleles in the CRE isolates. All the isolates were sensitive to polymyxin B and colistin while only 36.4% were sensitive to tigecycline. Consequently, microbiologists and clinicians should implement the necessary control measures to prevent the spreading of these resistant bacteria.

New Delhi Metallo-β-lactamase and other mechanisms of carbapenemases among Enterobacteriaceae in rural South India.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is an important public health problem. This study aimed to understand the prevalence and mechanisms of carbapenem resistance in clinically important members of Enterobacteriaceae in rural South India. Routine clinical isolates of Escherichia coli and Klebsiella spp. were tested for Ertapenem (ETP) non-susceptibility by the disk diffusion method over a 3-year period (2012-2014). The ETP non-susceptible isolates were preserved, and tested for the MIC of carbapenems and the carriage of major carbapenemase-encoding genes. Representative genes were sequenced and selective isolates were tested for the production of carbapenemase by carbapenem inactivation method. A total of 444 ETP non-susceptible isolates were identified in increasing incidence over the study period. Among them, MIC50 and MIC90 of carbapenems (excluding ETP) were 0.25-0.5μg/mL and 8-16μg/mL, respectively, and the prevalence of non-ETP carbapenem resistance was estimated as 3%. Among the 177 tested isolates, 65 (37%) had one or more carbapenemase-encoding genes with a predominance of New Delhi Metallo-β-lactamase (NDM; 32 of 65; 49.2%). This study documented the MIC range for carbapenems, prevalence and mechanisms of carbapenem resistance among Enterobacteriaceae in rural South India. It substantiated NDM as a leading mechanism of carbapenem resistance and highlighted the importance of MIC testing in patient management.

Carbapenemase Of Intestinal Rods – The Beginning Of Post-Antibiotic Era?

Abstract In recent years in Poland as well as globally at an alarming rate, the number of bacteria producing mechanisms of antibiotic resistance has been increased. The major source of concern is the emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Carbapenems are considered as last resort drugs for the treatment of multidrug-resistant (MDR) bacterial infections. At the present time the greatest menaces to public health are strains producing KPC (Klebsiella pneumoniae carbapenemases), NDM (New Delhi Metallo-β-lactamase) and OXA-48 (Oxacillinase-48). Carbapenemase-producing Enterobacterales have been resistant to most and sometimes even to all drugs that would be considered for treatment. Therefore, the accurate therapeutic options for the treatment of infections due to CRE strains are limited to the following antibiotics: colistin, tigecycline, fosfomycin, and aminoglycosides. Moreover, combination therapy containing two or more antibiotics has been recommended for the treatment of severe infections caused by carbapenemase-producing Enterobacterales. Due to the rapid spread of carbapenem-resistant strains and the lack of new antibiotic drug development, there is an urgent need to broaden our knowledge regarding antibiotic resistance. 1. Introduction. 2. Carbapenemases. 2.1. Metallo-β-lactamases. 2.2. Class A Carbapenemases. 2.3. Class D Carbapenemases (OXA). 3. Review of antibiotic treatment options of infections due to carbapenem-resistant strains. 3.1. Colistin. 3.2. Fosfomycin. 3.3. Tigecycline. 3.4. Aminoglycosides. 3.5. Carbapenems. 3.6. Mechanism of NDM – likely antibiotic/ chemotherapeutics could be used in the therapy. 3.7. Mechanism of KPC – likely antibiotic/ chemotherapeutics could be used in the therapy. 3.8. Mechanism of OXA-48 – likely antibiotic/ chemotherapeutics could be used in the therapy. 4. Summary

English

Dissemination of carbapenem resistant Enterobacteriaceae (CRE) poses a considerable threat to public health. The aim of the present work was to estimate the prevalence of metallo beta lactamases (MBL) among CRE isolated from Alexandria Main University Hospital, Egypt, to evaluate the performance of different phenotypic methods for the detection of MBL, and to investigate the local antimicrobial sensitivity profile of these isolates. Eighty CRE were tested for MBL production by Etest® MBL MP/MPI, EDTA double disc synergy test, and EDTA combined disc test. All isolates were confirmed as MBL producers by polymerase chain reaction (PCR). Antibiotic sensitivity testing was performed using disc diffusion method. Among the 80 CRE, 56 isolates (70%) were MBL by PCR. Fifty-four isolates were positive for NDM (96.4%). Meropenem EDTA CDT was the most sensitive test (94.6%). Blood was the most frequent sample from which MBL were isolated (51.7%). Majority of the isolates were isolated from intensive care units (82.1%). All MBL were multidrug resistant; Colistin and polymyxin B showed the lowest resistance rate (26.8 and 19.6%, respectively). The EDTA-CDT will provide a reliable, convenient, and cost-effective approach for detection of MBL in laboratories, which cannot afford to perform molecular tests. Key words: Metallo beta lactamases, carbapenem resistant Enterobacteriaceae, New Delhi lactamase.

The role of surveillance systems in confronting the global crisis of antibiotic-resistant bacteria.

It is widely accepted that infection control, advanced diagnostics, and novel therapeutics are crucial to mitigate the impact of antibiotic-resistant bacteria. The role of global, national, and regional surveillance systems as part of the response to the challenge posed by antibiotic resistance is not sufficiently highlighted. We provide an overview of contemporary surveillance programs, with emphasis on gram-negative bacteria. The WHO and public health agencies in Europe and the United States recently published comprehensive surveillance reports. These highlight the emergence and dissemination of carbapenem-resistant Enterobacteriaceae and other multidrug-resistant gram-negative bacteria. In Israel, public health action to control carbapenem-resistant Enterobacteriaceae, especially Klebsiella pneumoniae carbapenemase producing K. pneumoniae, has advanced together with a better understanding of its epidemiology. Surveillance models adapted to the requirements and capacities of each country are in development. Robust surveillance systems are essential to combat antibiotic resistance, and need to emphasize a 'one health' approach. Refinements in surveillance will come from advances in bioinformatics and genomics that permit the integration of global and local information about antibiotic consumption in humans and animals, molecular mechanisms of resistance, and bacterial genotyping.

Community acquisition of β-lactamase producing Enterobacteriaceae in neonatal gut

BackgroundCommensal flora constitutes a reservoir of antibiotic resistance. The increasing variety of β-lactamases and the emergence of Carbapenem resistant Enterobacteriaceae (CRE) in community, raise concerns regarding efficacy of β-lactams. It is important to know the exact load of antibiotic resistance in the absence of any antibiotic selection pressure including via food and water.In the present study gut colonization in neonates with no direct antibiotic pressure was used as a model to evaluate β-lactam resistance in the community.ResultsIn this prospective study, 75 healthy, vaginally delivered, antibiotic naive, breast fed neonates were studied for gut colonization by Extended spectrum β-lactamases (ESBL), AmpC β-lactamases hyperproducing Enterobacteriaceae and CRE on day 0, 21 and 60. Total 267 Enterobacteriaceae were isolated and E.coli was the predominant flora. ESBL, AmpC and coproduction was seen in 20.6%, 19.9% and 11.2% isolates respectively. ESBL carriage increased threefold from day 1 to 60 showing predominance of CTX-M group 15 (82.5%), ampC genes were heterogeneous. Colonization with CRE was rare, only one baby harboured Enterobacter sp positive for kpc-2. The reservoirs for these genes are likely to be mother and the environment.ConclusionsData strongly suggests that in absence of any antibiotic pressure there is tremendous load of antibiotic resistance to β-lactam drugs. Wide spread presence of ESBL and AmpC can drive rapid emergence and dissemination of CRE. This is the first report from India which depicts the smaller picture of true antibiotic pressure present in the Indian community.

Environmental contamination by carbapenem-resistant Enterobacteriaceae.

In the last decade, the global emergence of carbapenem resistance in Enterobacteriaceae has posed great concern to public health. Data concerning the role of environmental contamination in the dissemination of carbapenem-resistant Enterobacteriaceae (CRE) are currently lacking. Here, we aimed to examine the extent of CRE contamination in various sites in the immediate surroundings of CRE carriers and to assess the effects of sampling time and cleaning regimens on the recovery rate. We evaluated the performance of two sampling methods, CHROMAgar KPC contact plate and eSwab, for the detection of environmental CRE. eSwab was followed either by direct plating or by broth enrichment. First, 14 sites in the close vicinity of the carrier were evaluated for environmental contamination, and 5, which were found to be contaminated, were further studied. The environmental contamination decreased with distance from the patient; the bed area was the most contaminated site. Additionally, we found that the sampling time and the cleaning regimen were critical factors affecting the prevalence of environmental CRE contamination. We found that the CHROMAgar KPC contact plate method was a more effective technique for detecting environmental CRE than were eSwab-based methods. In summary, our study demonstrated that the vicinity of patients colonized with CRE is often contaminated by these organisms. Using selective contact plates to detect environmental contamination may guide cleaning efficacy and assist with outbreak investigation in an effort to limit the spread of CRE.