scholarly journals Meropenem-Vaborbactam Resistance Selection, Resistance Prevention, and Molecular Mechanisms in Mutants of KPC-Producing Klebsiella pneumoniae

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Dongxu Sun ◽  
Debora Rubio-Aparicio ◽  
Kirk Nelson ◽  
Michael N. Dudley ◽  
Olga Lomovskaya
Keyword(s):  
Klebsiella Pneumoniae ◽  
Copy Number ◽  
Molecular Mechanisms ◽  
Resistance Mutation ◽  
Gene Copy Number ◽  
Resistance Mechanisms ◽  
Gene Copy ◽  
Coding Region ◽  
Content Type ◽  
Drug Concentrations

ABSTRACT Vaborbactam (formerly RPX7009) is a new β-lactamase inhibitor based on a cyclic boronic acid pharmacophore with potent inhibitory activity against Klebsiella pneumoniae carbapenemases (KPC). It has been developed in combination with meropenem. The objective of these studies was to identify the concentrations of both agents associated with the selection or prevention of single-step mutations leading to reduced sensitivity to the combination and to characterize the selected mutations. Eighteen strains of KPC-producing Klebsiella pneumoniae with various degrees of sensitivity to meropenem (MICs, 8 to 512 μg/ml) and meropenem-vaborbactam (MICs, ≤0.06 to 32 μg/ml) and preexisting resistance mechanisms were selected from a worldwide collection of isolates recovered from surveillance studies, emphasizing strains for which MICs were in the upper range of the meropenem-vaborbactam MIC distribution. Meropenem and vaborbactam at 8 μg/ml each suppressed the drug resistance mutation frequency to <1 × 10−8 in 77.8% (14/18) of strains, and all strains were inhibited when the meropenem concentration was increased to 16 μg/ml. Mutants selected at lower drug concentrations showed phenotypes associated with previously described carbapenem resistance mechanisms, including ompK36 inactivation in mutants selected from OmpK36-proficient strains and an increased bla KPC gene copy number in strains with partially functional ompK36. No mutations in the coding region of bla KPC were identified. These data indicate that the selection of mutants with reduced sensitivity to meropenem-vaborbactam from KPC-producing Klebsiella pneumoniae strains is associated with previously described mechanisms involving porin mutations and the increase in the bla KPC gene copy number and not changes in the KPC enzyme and can be prevented by the drug concentrations achieved with optimal dosing of the combination.

Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates

2021 ◽  
Author(s):  
Alessandra Carattoli ◽  
Gabriele Arcari ◽  
Giulia Bibbolino ◽  
Federica Sacco ◽  
Dario Tomolillo ◽  
...  
Keyword(s):  
High Risk ◽  
Klebsiella Pneumoniae ◽  
Copy Number ◽  
Residual Activity ◽  
Gene Copy Number ◽  
Resistance Mechanisms ◽  
University Hospital ◽  
Gene Copy ◽  
Carbapenemase Gene ◽  
Evolutionary Trajectories

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA) resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the ST512, ST101 and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among them, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69 and KPC-70 showed amino acid substitutions, insertions and deletions in the Ω loop of the protein. KPC-29 has the duplication, while the novel KPC-67 has the triplication of the KDD triplet in the 270-loop of the protein. Genomics performed on contemporary resistant and susceptible clones underlined that those novel mutations emerged in bla KPC-3 genes located on conserved plasmids: in ST512, all bla KPC-3 mutant genes were located in pKpQIL plasmids, while the three novel bla KPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy number pTOPO vector, the Ω loop mutated variants showed CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA-resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

scholarly journals Ceftazidime-Avibactam Resistance Associated with Increased blaKPC-3 Gene Copy Number Mediated by pKpQIL Plasmid Derivatives in Sequence Type 258 Klebsiella pneumoniae

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Marco Coppi ◽  
Vincenzo Di Pilato ◽  
Francesco Monaco ◽  
Tommaso Giani ◽  
Pier Giulio Conaldi ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Copy Number ◽  
Acquired Resistance ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Content Type ◽  
Outer Membrane Porins ◽  
Long Read ◽  
Multiple Copies

ABSTRACT This study reports on the characterization of two ceftazidime-avibactam (CZA)-resistant KPC-producing Klebsiella pneumoniae strains (KP-14159 and KP-8788) sequentially isolated from infections occurred in a patient never treated with CZA. Whole-genome sequencing characterization using a combined short- and long-read sequencing approach showed that both isolates belonged to the same ST258 strain, had altered outer membrane porins (a truncated OmpK35 and an Asp137Thr138 duplication in the L3 loop of OmpK36), and carried novel pKpQIL plasmid derivatives (pIT-14159 and pIT-8788, respectively) harboring two copies of the Tn4401a KPC-3-encoding transposon. Plasmid pIT-8788 was a cointegrate of pIT-14159 with a ColE replicon (that was also present in KP-14159) apparently evolved in vivo during infection. pIT-8788 was maintained at a higher copy number than pIT-14159 and, upon transfer to Escherichia coli DH10B, was able to increase the CZA MIC by 32-fold. The present findings provide novel insights about the mechanisms of acquired resistance to CZA, underscoring the role that the evolution of broadly disseminated pKpQIL plasmid derivatives may have in increasing the blaKPC gene copy number and KPC-3 expression in bacterial hosts. Although not self-transferable, similar elements, with multiple copies of Tn4401 and maintained at a high copy number, could mediate transferable CZA resistance upon mobilization.

scholarly journals The Copy Number of the spoVA2mob Operon Determines Pressure Resistance of Bacillus Endospores

2019 ◽  
Vol 85 (19) ◽  
Author(s):  
Zhen Li ◽  
Felix Schottroff ◽  
David J. Simpson ◽  
Michael G. Gänzle
Keyword(s):  
High Pressure ◽  
Genome Sequencing ◽  
Copy Number ◽  
Digital Pcr ◽  
Resistance Mechanisms ◽  
Droplet Digital Pcr ◽  
Food Microbiology ◽  
Gene Copy ◽  
Content Type ◽  
Pressure Resistance

ABSTRACT The spoVA2mob operon confers heat resistance to Bacillus spp., and the resistance correlates to the copy number of the operon. Bacillus endospores also exhibit a strong variation in resistance to pressure, but the underlying mechanisms of endospore resistance to pressure are not fully understood. We determined the effects of multiple spoVA2mob operons on high-pressure resistance in Bacillus endospores. The copy numbers of the spoVA2mob operon in 17 strains of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus velezensis, and Bacillus pumilus were determined via droplet digital PCR (ddPCR) and genome sequencing. These strains contained between 0 and 3 copies of the spoVA2mob operon; the quantification of the gene copy number by ddPCR was as accurate as whole-genome sequencing. We further tested the pressure resistance of 17 Bacillus endospores at 600 MPa and 80°C. Strains with one or no spoVA2mob operon had significantly lower pressure resistance than strains with two or three copies of the operons (P < 0.001), indicating that redundant spoVA2mob operons in Bacillus contributed to higher pressure resistance of endospores. The copy number of the spoVA2mob operon was not related to the dipicolinic acid (DPA) content of endospores. Overall, the copy number of the spoVA2mob operon contributes to pressure resistance of Bacillus endospores. This improves our understanding of the pressure resistance mechanisms in Bacillus spp. and may inform the development of high-pressure sterilization in food processing. IMPORTANCE Bacillus spp. are considered pressure-resistant microorganisms, but the resistance mechanisms remain unknown. The spoVA2mob operon is a mobile genetic element, and it can transfer to pathogenic or spoilage organisms by horizontal gene transfer. Results in this study indicate that multiple copies of the spoVA2mob operon mediate high-pressure resistance of Bacillus endospores, and it might contribute to the identification of the source of pressure-resistant pathogens and spoilage organisms that may contaminate the food supply. The droplet digital PCR (ddPCR) system is well suited for analysis in some human diseases due to its high efficiency and capability to provide high precision; however, no relevant studies in food microbiology have been reported so far. This study demonstrates a novel application of ddPCR in food microbiology.

scholarly journals Genetic Factors Associated with Elevated Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

2010 ◽  
Vol 54 (10) ◽  
pp. 4201-4207 ◽  
Author(s):  
Brandon Kitchel ◽  
J. Kamile Rasheed ◽  
Andrea Endimiani ◽  
Andrea M. Hujer ◽  
Karen F. Anderson ◽  
...  
Keyword(s):  
United States ◽  
Klebsiella Pneumoniae ◽  
Copy Number ◽  
Gene Copy Number ◽  
Carbapenem Resistance ◽  
The United States ◽  
Gene Copy ◽  
Sequencing Analysis ◽  
Low Level ◽  
High Level

ABSTRACT In the United States, the most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the production of a Klebsiella pneumoniae carbapenemase (KPC). KPC-producing isolates often exhibit a range of carbapenem MICs. To better understand the factors that contribute to overall carbapenem resistance, we analyzed 27 KPC-producing K. pneumoniae isolates with different levels of carbapenem resistance, 11 with low-level (i.e., meropenem or imipenem MIC ≤ 4 μg/ml), 2 with intermediate-level (i.e., meropenem and imipenem MIC = 8 μg/ml), and 14 with high-level (i.e., imipenem or meropenem MIC ≥ 16 μg/ml) carbapenem resistance, that were received from throughout the United States. Among 14 isolates that exhibited high-level carbapenem resistance, Western blot analysis indicated that 10 produced an elevated amount of KPC. These isolates either contained an increased bla KPC gene copy number (n = 3) or had deletions directly upstream of the bla KPC gene (n = 7). Four additional isolates lacked elevated KPC production but had high-level carbapenem resistance. Porin sequencing analysis identified 22 isolates potentially lacking a functional OmpK35 and three isolates potentially lacking a functional OmpK36. The highest carbapenem MICs were found in two isolates that lacked both functioning porins and produced elevated amounts of KPC. The 11 isolates with low-level carbapenem resistance contained neither an upstream deletion nor increased bla KPC copy number. These results suggest that both bla KPC copy number and deletions in the upstream genetic environment affect the level of KPC production and may contribute to high-level carbapenem resistance in KPC-producing K. pneumoniae, particularly when coupled with OmpK36 porin loss.

scholarly journals blaKPCRNA Expression Correlates with Two Transcriptional Start Sites but Not Always with Gene Copy Number in Four Genera of Gram-Negative Pathogens

2011 ◽  
Vol 55 (8) ◽  
pp. 3936-3938 ◽  
Author(s):  
Amanda L. Roth ◽  
Philip M. Kurpiel ◽  
Philip D. Lister ◽  
Nancy D. Hanson
Keyword(s):  
Gene Expression ◽  
Klebsiella Pneumoniae ◽  
Copy Number ◽  
Susceptibility Testing ◽  
Gene Copy Number ◽  
Clinical Isolates ◽  
Gene Copy ◽  
Gram Negative ◽  
Transcriptional Start Sites

ABSTRACTKlebsiella pneumoniaecarbapenemase (KPC)-producing organisms are therapeutically and diagnostically challenging. It is possible thatblaKPCgene expression plays a role in the variability observed in clinical susceptibility testing.blaKPCtransformants together with 10 clinical isolates representing four genera were evaluated forblaKPCcopy number and gene expression and correlated with β-lactam MIC data. The data suggest that mechanisms other than gene copy number and expression ofblaKPCcontribute to variability in susceptibility when testing KPC-producing isolates.

scholarly journals Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Giovanni Bussotti ◽  
Evi Gouzelou ◽  
Mariana Côrtes Boité ◽  
Ihcen Kherachi ◽  
Zoubir Harrat ◽  
...  
Keyword(s):  
Environmental Change ◽  
Copy Number ◽  
Gene Copy Number ◽  
Environmental Adaptation ◽  
Gene Copy ◽  
Protozoan Parasites ◽  
Content Type ◽  
Intrinsic Factors ◽  
Genomic Adaptation

ABSTRACT Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

scholarly journals Gene Copy Number and Post-Transductional Mechanisms Regulate TRAP1 Expression in Human Colorectal Carcinomas

2019 ◽  
Vol 21 (1) ◽  
pp. 145 ◽  
Author(s):  
Michele Pietrafesa ◽  
Francesca Maddalena ◽  
Luciana Possidente ◽  
Valentina Condelli ◽  
Pietro Zoppoli ◽  
...  
Keyword(s):  
Copy Number ◽  
Molecular Mechanisms ◽  
Tumor Necrosis Factor Receptor ◽  
Gene Copy Number ◽  
Protein S ◽  
Lymph Node Involvement ◽  
The Cancer Genome Atlas ◽  
Gene Copy ◽  
Colorectal Carcinomas ◽  
Protein Levels

Tumor Necrosis Factor Receptor-Associated Protein 1 (TRAP1) is a heat shock protein 90 (HSP90) molecular chaperone overexpressed in 60–70% human colorectal carcinomas (CRCs) and the co-upregulation of TRAP1 and associated 6-related proteins identifies metastatic CRCs with poor prognosis. Since the molecular mechanisms responsible for TRAP1 regulation are still unknown, the significance of TRAP1 gene copy number (CN) and the role of post-transductional protein modifications were addressed. TRAP1 gene aneuploidy accounted for 34.5% of cases in a cohort of 58 human CRCs and TRAP1 CN correlated with its mRNA and protein expression, suggesting that transcriptional mechanisms are responsible for TRAP1 upregulation. Furthermore, the analysis of the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium/The Cancer Genome Atlas (CPTAC/TCGA) CRC database showed that TRAP1 polysomy significantly correlates with lymph node involvement. However, a subgroup of tumors showed TRAP1 protein levels independent from its CN. Of note, a direct correlation was observed between TRAP1 protein levels and the expression of S-nitrosoglutathione reductase (GSNOR), a denitrosylase involved in the regulation of protein S-nitrosylation. Furthermore, CRC cell lines exposed to hypoxia or dichloroacetate treatment showed the downregulation of TRAP1 upon GSNOR silencing and this resulted in increased TRAP1 mono/polyubiquitination. These data suggest that transcriptional and post-transductional mechanisms account for TRAP1 expression in human CRCs and GSNOR protects TRAP1 from S-nitrosylation and consequent proteasome degradation mostly in conditions of stress.

scholarly journals Coordination of Polyploid Chromosome Replication with Cell Size and Growth in a Cyanobacterium

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Ryudo Ohbayashi ◽  
Ai Nakamachi ◽  
Tetsuhiro S. Hatakeyama ◽  
Satoru Watanabe ◽  
Yu Kanesaki ◽  
...  
Keyword(s):  
Cell Growth ◽  
Chromosome Number ◽  
Atpase Activity ◽  
Cell Volume ◽  
Cell Size ◽  
Copy Number ◽  
Gene Copy Number ◽  
Model Organisms ◽  
Gene Copy ◽  
Content Type

ABSTRACTHomologous chromosome number (ploidy) has diversified among bacteria, archaea, and eukaryotes over evolution. In bacteria, model organisms such asEscherichia colipossess a single chromosome encoding the entire genome during slow growth. In contrast, other bacteria, including cyanobacteria, maintain multiple copies of individual chromosomes (polyploid). Although a correlation between ploidy level and cell size has been observed in bacteria and eukaryotes, it is poorly understood how replication of multicopy chromosomes is regulated and how ploidy level is adjusted to cell size. In addition, the advantages conferred by polyploidy are largely unknown. Here we show that only one or a few multicopy chromosomes are replicated at once in the cyanobacteriumSynechococcus elongatusand that this restriction depends on regulation of DnaA activity. Inhibiting the DnaA intrinsic ATPase activity inS. elongatusincreased the number of replicating chromosomes and chromosome number per cell but did not affect cell growth. In contrast, when cell growth rate was increased or decreased, DnaA level, DnaA activity, and the number of replicating chromosomes also increased or decreased in parallel, resulting in nearly constant chromosome copy number per unit of cell volume at constant temperature. When chromosome copy number was increased by inhibition of DnaA ATPase activity or reduced culture temperature, cells exhibited greater resistance to UV light. Thus, it is suggested that the stepwise replication of the genome enables cyanobacteria to maintain nearly constant gene copy number per unit of cell volume and that multicopy chromosomes function as backup genetic information to compensate for genomic damage.IMPORTANCEPolyploidy has evolved many times across the kingdom of life. The relationship between cell growth and chromosome replication in bacteria has been studied extensively in monoploid model organisms such asEscherichiacolibut not in polyploid organisms. Our study of the polyploid cyanobacteriumSynechococcus elongatusdemonstrates that replicating chromosome number is restricted and regulated by DnaA to maintain a relatively stable gene copy number/cell volume ratio during cell growth. In addition, our results suggest that polyploidy confers resistance to UV, which damages DNA. This compensatory polyploidy is likely necessitated by photosynthesis, which requires sunlight and generates damaging reactive oxygen species, and may also explain how polyploid bacteria can adapt to extreme environments with high risk of DNA damage.

scholarly journals The influence of antibiotics on transitory resistome during gut colonization with CTX-M-15 and OXA-162 producing Klebsiella pneumoniae ST15

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Balázs Stercz ◽  
Ferenc B. Farkas ◽  
Ákos Tóth ◽  
Márió Gajdács ◽  
Judit Domokos ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Gene ◽  
Copy Number ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Antibiotic Administration ◽  
Mice Model ◽  
Gut Colonization ◽  
Colonization Rates

AbstractGreat efforts have been made to limit the transmission of carbapenemase-producing Enterobacteriaceae (CPE), however, the intestinal reservoir of these strains and its modulation by various antibiotics remain largely unexplored. Our aim was to assess the effects of antibiotic administration (ampicillin, ceftazidime, ciprofloxacin) on the establishment and elimination of intestinal colonization with a CTX-M-15 ESBL and OXA-162 carbapenemase producing Klebsiella pneumoniae ST15 (KP5825) in a murine (C57BL/6 male mice) model. Whole genome sequencing of KP5825 strain was performed on an Illumina MiSeq platform. Conjugation assays were carried out by broth mating method. In colonization experiments, 5 × 106 CFU of KP5825 was administered to the animals by orogastric gavage, and antibiotics were administered in their drinking water for two weeks and were changed every day. The gut colonization rates with KP5825 were assessed by cultivation and qPCR. In each of the stool samples, the gene copy number of blaOXA-162 and blaCTX-M-15 were determined by qPCR. Antibiotic concentrations in the stool were determined by high pressure liquid chromatography and a bioanalytical method. The KP5825 contained four different plasmid replicon types, namely IncFII(K), IncL, IncFIB and ColpVC. IncL (containing the blaOXA-162 resistance gene within a Tn1991.2 genetic element) and IncFII(K) (containing the blaCTX-M-15 resistance gene) plasmids were successfully conjugated. During ampicillin and ceftazidime treatments, colonization rate of KP5825 increased, while, ciprofloxacin treatments in both concentrations (0.1 g/L and 0.5 g/L) led to significantly decreased colonization rates. The gene copy number blaOXA-162 correlated with K. pneumoniae in vivo, while a major elevation was observed in the copy number of blaCTX-M-15 from the first day to the fifteenth day in the 0.5 g/L dose ceftazidime treatment group. Our results demonstrate that commonly used antibiotics may have diverse impacts on the colonization rates of intestinally-carried CPE, in addition to affecting the gene copy number of their resistance genes, thus facilitating their stable persistance and dissemination.

scholarly journals Gene copy-number polymorphism in nature

2010 ◽  
Vol 277 (1698) ◽  
pp. 3213-3221 ◽  
Author(s):  
Daniel R. Schrider ◽  
Matthew W. Hahn
Keyword(s):  
Natural Selection ◽  
Copy Number Variation ◽  
Copy Number ◽  
Molecular Mechanisms ◽  
Copy Number Variants ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Phenotypic Differences ◽  
Sequencing Technologies ◽  
Number Variation

Differences between individuals in the copy-number of whole genes have been found in every multicellular species examined thus far. Such differences result in unique complements of protein-coding genes in all individuals, and have been shown to underlie adaptive phenotypic differences. Here, we review the evidence for copy-number variants (CNVs), focusing on the methods used to detect them and the molecular mechanisms responsible for generating this type of variation. Although there are multiple technical and computational challenges inherent to these experimental methods, next-generation sequencing technologies are making such experiments accessible in any system with a sequenced genome. We further discuss the connection between copy-number variation within species and copy-number divergence between species, showing that these values are exactly what one would expect from similar comparisons of nucleotide polymorphism and divergence. We conclude by reviewing the growing body of evidence for natural selection on copy-number variants. While it appears that most genic CNVs—especially deletions—are quickly eliminated by selection, there are now multiple studies demonstrating a strong link between copy-number differences at specific genes and phenotypic differences in adaptive traits. We argue that a complete understanding of the molecular basis for adaptive natural selection necessarily includes the study of copy-number variation.

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