Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome ...sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.
The genus Trichococcus currently contains nine species: T. flocculiformis, T. pasteurii, T. palustris, T. collinsii, T. patagoniensis, T. ilyis, T. paludicola, T. alkaliphilus, and T. shcherbakoviae. ...In general, Trichococcus species can degrade a wide range of carbohydrates. However, only T. pasteurii and a non-characterized strain of Trichococcus, strain ES5, have the capacity of converting glycerol to mainly 1,3-propanediol. Comparative genomic analysis of Trichococcus species provides the opportunity to further explore the physiological potential and uncover novel properties of this genus.
In this study, a genotype-phenotype comparative analysis of Trichococcus strains was performed. The genome of Trichococcus strain ES5 was sequenced and included in the comparison with the other nine type strains. Genes encoding functions related to e.g. the utilization of different carbon sources (glycerol, arabinan and alginate), antibiotic resistance, tolerance to low temperature and osmoregulation could be identified in all the sequences analysed. T. pasteurii and Trichococcus strain ES5 contain a operon with genes encoding necessary enzymes for 1,3-PDO production from glycerol. All the analysed genomes comprise genes encoding for cold shock domains, but only five of the Trichococcus species can grow at 0 °C. Protein domains associated to osmoregulation mechanisms are encoded in the genomes of all Trichococcus species, except in T. palustris, which had a lower resistance to salinity than the other nine studied Trichococcus strains.
Genome analysis and comparison of ten Trichococcus strains allowed the identification of physiological traits related to substrate utilization and environmental stress resistance (e.g. to cold and salinity). Some substrates were used by single species, e.g. alginate by T. collinsii and arabinan by T. alkaliphilus. Strain ES5 may represent a subspecies of Trichococcus flocculiformis and contrary to the type strain (DSM 2094
), is able to grow on glycerol with the production of 1,3-propanediol.
New and rapid antimicrobial susceptibility/resistance testing methods are required for bacteria from positive blood cultures. In this study, a multiplex-targeted liquid chromatography-tandem mass ...spectrometry (LC-MS/MS) assay was developed and validated for the detection of β-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing
Escherichia coli
or
Klebsiella pneumoniae
complex. Selected targets were the β-lactamases SHV, TEM, OXA-1-like, CTX-M-1-like, CMY-2-like, chromosomal
E. coli
AmpC (cAmpC), OXA-48-like, NDM, VIM, and KPC; the aminoglycoside-modifying enzymes AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6′)-Ib, ANT(2
′
′
)-I, and APH(3′)-VI; the 16S-RMTases ArmA, RmtB, RmtC, and RmtF; the quinolone resistance mechanisms QnrA, QnrB, AAC(6′)-Ib-cr; the wildtype quinolone resistance determining region of GyrA; and the
E. coli
porins OmpC and OmpF. The developed assay was evaluated using 100 prospectively collected positive blood cultures, and 148 negative blood culture samples spiked with isolates previously collected from blood cultures or isolates carrying less prevalent resistance mechanisms. The time to result was approximately 3 h. LC-MS/MS results were compared with whole-genome sequencing and antimicrobial susceptibility testing results. Overall, there was a high agreement between LC-MS/MS results and whole-genome sequencing results. In addition, the majority of susceptible and non-susceptible phenotypes were correctly predicted based on LC-MS/MS results. Exceptions were the predictions for ciprofloxacin and amoxicillin/clavulanic acid that matched with the phenotype in 85.9 and 63.7% of the isolates, respectively. Targeted LC-MS/MS based on parallel reaction monitoring can be applied for the rapid and accurate detection of various resistance mechanisms in blood cultures growing
E. coli
or
K. pneumoniae
complex.
At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for ...confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases.
Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62
and
isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates.
For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected.
The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.
The role of plasmids in the complex pandemic of antimicrobial resistance is increasingly being recognized. In this respect, multiple mobile colistin resistance (
mcr
) gene-carrying plasmids have ...been described. However, the characteristics and epidemiology of these plasmids within local healthcare settings are largely unknown. We retrospectively characterized the genetic composition and epidemiology of plasmids from
mcr-1-
positive bacterial isolates identified from patients from a large academic hospital in the Netherlands. Clinical Gram-negative bacteria with an MIC > 2 μg/mL for colistin, obtained from patients hospitalized at the Erasmus MC University Medical Center Rotterdam during the years 2010–2018, were screened for presence of the
mcr-1
gene. Extracted plasmids from
mcr-1
-positive isolates were sequenced using a combination of short- and long-read sequencing platforms, characterized by incompatibility type and genetic composition and compared to publicly available
mcr-1-
carrying plasmid sequences. In 21 isolates from 14 patients,
mcr-1
was located on a plasmid. These plasmids were of diverse genetic background involving Inc types IncX4, IncI2(delta), IncHI2, as well as double Inc types IncHI2/IncN and IncHI2/IncQ.
mcr-1
-carrying plasmids were found in
Escherichia coli, Klebsiella pneumoniae, and Kluyvera georgiana
, and within the chromosome of an ST147
K. pneumoniae
isolate. In depth analysis indicated intrapatient, interpatient, and interspecies transmission events of
mcr-1-
carrying plasmids. In addition, our results show that the
mcr-1
gene resides in a rich environment full of other (
mcr-1
negative) plasmids and of many different Inc types, enabling interplasmidal transfer events and facilitating widespread dissemination of the
mcr-1
gene. Multiple
mcr-1
-carrying plasmid transmission events had likely occurred among isolates from hospitalized patients. Recognition and identification of plasmid transmission events within hospitals is necessary in order to design and implement effective infection control measures.
Enterobacter hormaechei has emerged as a significant pathogen within healthcare settings due to its ability to develop multidrug resistance (MDR) and survive in hospital environments. This study ...presents a genome-based analysis of carbapenem-resistant Enterobacter hormaechei isolates from two major hospitals in the United Arab Emirates. Eight isolates were subjected to whole-genome sequencing (WGS), revealing extensive resistance profiles including the blaNDM-1, blaOXA-48, and blaVIM-4 genes. Notably, one isolate belonging to ST171 harbored dual carbapenemase genes, while five isolates exhibited colistin resistance without mcr genes. The presence of the type VI secretion system (T6SS), various adhesins, and virulence genes contributes to the virulence and competitive advantage of the pathogen. Additionally, our isolates (87.5%) possessed ampC β-lactamase genes, predominantly blaACT genes. The genomic context of blaNDM-1, surrounded by other resistance genes and mobile genetic elements, highlights the role of horizontal gene transfer (HGT) in the spread of resistance. Our findings highlight the need for rigorous surveillance, strategic antibiotic stewardship, and hospital-based WGS to manage and mitigate the spread of these highly resistant and virulent pathogens. Accurate identification and monitoring of Enterobacter cloacae complex (ECC) species and their resistance mechanisms are crucial for effective infection control and treatment strategies.
Biosulfidogenesis can be used to remediate low pH and high metal content waters such as acid mine drainage and recover the present metals. The selection of a cheap electron donor for the process is ...important for the economic viability. In this work we isolated a novel versatile acidotolerant fermentative bacterium (strain ALE
) that is able to use a great variety of substrates including glycerol. Strain ALE
is an obligate anaerobe, and cells are motile, rod-shaped, spore-forming, and stain Gram-positive. Growth occurred in a pH range from 3.5 to 7 (optimum 5.5), and temperature range from 25 to 40°C (optimum 37°C). It grows by fermentation of sugars, organic acids and glycerol. It has the ability to use thiosulfate, iron and DMSO as electron acceptors. Its genome is 4.7 Mb with 5122 protein-coding sequences, and a G+C content of 46.9 mol%. Based on 16S rRNA gene sequence analysis, the closest cultured species is
(91.4% 16S rRNA gene identity) from the
family (
order,
class,
phylum). Based on the distinctive physiological and phylogenetic characteristics of strain ALE
, a new genus and species
gen. nov., sp. nov., is proposed. The type strain is ALE
(=JCM 19373
= DSM 27520
). Strain ALE
is an incomplete oxidizer and acetate, among other products, accumulates during glycerol conversion. Strain ALE
was used to extend the substrate range for sulfur reduction by constructing co-cultures with the acetate oxidizer and sulfur reducer
The co-culture was tested with glycerol as substrate in batch and chemostat experiments. Acetate formed by fermentation of glycerol by strain ALE
resulted in sulfur reduction by
. The co-culture strategy offers good perspectives to use a wide range of cost-efficient substrates, including glycerol, to produce sulfide by specialized sulfur reducers. The recovery of heavy metals from metalliferous streams may become economically feasible by this approach.
The locus tag for the genes encoded in
is LUCI_
. To avoid repetition of the prefix along the text, the locus tags are represented by the specific identifier.
Addressing the emergence of antimicrobial resistance (AMR) poses a significant challenge in veterinary and public health. In this study, we focused on determining the presence, phenotypic background, ...and genetic epidemiology of plasmid-mediated colistin resistance (
) in
bacteria isolated from camels farmed in the United Arab Emirates (UAE). Fecal samples were collected from 50 camels at a Dubai-based farm in the UAE and colistin-resistant Gram-negative bacilli were isolated using selective culture. Subsequently, a multiplex PCR targeting a range of
-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for
isolates carrying the
gene on mobile genetic elements. No other
-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine
isolates from eight camels were selected for in-depth analysis.
sequence types (STs) identified included ST7, ST21, ST24, ST399, ST649, ST999, and STdaa2. Seven IncI2(delta) and two IncX4 plasmids were found to be associated with
carriage in these isolates. These findings represent the first identification of
-1-carrying plasmids associated with camels in the Gulf region. The presence of
in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance.
While Extended-Spectrum β-Lactamases (ESBL) and AmpC β-lactamases barely degrade carbapenem antibiotics, they are able to bind carbapenems and prevent them from interacting with penicillin-binding ...proteins, thereby inhibiting their activity. Further, it has been shown that
can become resistant to carbapenems when high concentrations of ESBL and AmpC β-lactamases are present in the bacterial cell in combination with a decreased influx of antibiotics (due to a decrease in porins and outer-membrane permeability). In this study, a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed for the detection of the
porins OmpC and OmpF, its chromosomal AmpC β-lactamase, and the plasmid-mediated CMY-2 β-lactamase.
positive
isolates were cultured in the presence of increasing concentrations of meropenem, and resistant mutants were analyzed using the developed LC-MS/MS assay, Western blotting, and whole genome sequencing. In five strains that became meropenem resistant, a decrease in OmpC and/or OmpF (caused by premature stop codons or gene interruptions) was the first event toward meropenem resistance. In four of these strains, an additional increase in MICs was caused by an increase in CMY-2 production, and in one strain this was most likely caused by an increase in CTX-M-15 production. The LC-MS/MS assay developed proved to be suitable for the (semi-)quantitative analysis of CMY-2-like β-lactamases and porins within 4 h. Targeted LC-MS/MS could have additional clinical value in the early detection of non-carbapenemase-producing carbapenem-resistant
.
Extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) are a well-known cause of healthcare-associated infections. The implementation of single-occupancy rooms is believed to decrease ...the spread of ESBL-E. Additionally, implementation of single-occupancy rooms is expected to reduce the need for intra-hospital patient transfers. We studied the impact of a new hospital with 100% single-occupancy rooms on the acquisition of ESBL-E and on intra-hospital patient transfers.
In 2018, the Erasmus MC University Medical Center moved from an old, 1200-bed hospital with mainly multiple-occupancy rooms, to a newly constructed 522-bed hospital with 100% single-occupancy rooms. Adult patients admitted between January 2018 and September 2019 with an expected hospitalization of ≥ 48 h were asked to participate in this study. Perianal samples were taken at admission and discharge. Patient characteristics and clinical information, including number of intra-hospital patient transfers, were collected from the patients' electronic health records.
Five hundred and ninety-seven patients were included, 225 in the old and 372 in the new hospital building. Fifty-one (8.5%) ESBL-E carriers were identified. Thirty-four (66.7%) patients were already positive at admission, of which 23 without recent hospitalization. Twenty patients acquired an ESBL-E, seven (3.1%) in the old and 13 (3.5%) in the new hospital building (P = 0.801). Forty-one (80.4%) carriers were only detected by the active screening performed during this study. Only 10 (19.6%) patients, six before and four during hospitalization, showed ESBL-E in a clinical sample taken on medical indication. Fifty-six (24.9%) patients were transferred to other rooms in the old hospital, compared to 53 (14.2%) in the new hospital building (P = 0.001). Intra-hospital patient transfers were associated with ESBL-E acquisition (OR 3.18, 95%CI 1.27-7.98), with increasing odds when transferred twice or more.
Transitioning to 100% single-occupancy rooms did not decrease ESBL-E acquisition, but did significantly decrease the number of intra-hospital patient transfers. The latter was associated with lower odds on ESBL-E acquisition. ESBL-E carriers remained largely unidentified through clinical samples.
This study was retrospectively registered in the Dutch National Trial Register on 24-02-2020, with registration number NL8406.
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