The combination of manogepix and itraconazole is synergistic and inhibits the growth of Madurella mycetomatis in vitro but not in vivo.

Mycetoma is a neglected tropical disease commonly caused by the fungus Madurella mycetomatis. Standard treatment consists of extensive treatment with itraconazole in combination with surgical excision of the infected tissue, but has a low success rate. To improve treatment outcomes, novel treatment strategies are needed. Here, we determined the potential of manogepix, a novel antifungal agent that targets the GPI-anchor biosynthesis pathway by inhibition of the GWT1 enzyme. Manogepix was evaluated by determining the minimal inhibitory concentrations (MICs) according to the CLSI-based in vitro susceptibility assay for 22 M. mycetomatis strains and by in silico protein comparison of the target protein. The synergy between manogepix and itraconazole was determined using a checkerboard assay. The efficacy of clinically relevant dosages was assessed in an in vivo grain model in Galleria mellonella larvae. MICs for manogepix ranged from <0.008 to >8 mg/l and 16/22 M. mycetomatis strains had an MIC ≥4 mg/ml. Differences in MICs were not related to differences observed in the GWT1 protein sequence. For 70% of the tested isolates, synergism was found between manogepix and itraconazole in vitro. In vivo, enhanced survival was not observed upon admission of 8.6 mg/kg manogepix, nor in combination treatment with 5.7 mg/kg itraconazole. MICs of manogepix were high, but the in vitro antifungal activity of itraconazole was enhanced in combination therapy. However, no efficacy of manogepix was found in an in vivo grain model using clinically relevant dosages. Therefore, the therapeutic potential of manogepix in mycetoma caused by M. mycetomatis seems limited.

Treatment of Madurella mycetomatis-caused mycetoma consists of extensive exposure to antifungals and surgery. To improve therapy, we evaluated manogepix, a novel antifungal agent, as a therapeutic option against M. mycetomatis. Our findings suggest limited therapeutic potential for manogepix.

Inhibiting DHN- and DOPA-melanin biosynthesis pathway increased the therapeutic value of itraconazole in Madurella mycetomatis infected Galleria mellonella.

Eumycetoma is a neglected tropical disease, and Madurella mycetomatis, the most common causative agent of this disease forms black grains in hosts. Melanin was discovered to be one of the constituents in grains. Melanins are hydrophobic, macromolecular pigments formed by oxidative polymerisation of phenolic or indolic compounds. M. mycetomatis was previously known to produce DHN-melanin and pyomelanin in vitro. These melanin was also discovered to decrease M. mycetomatis's susceptibility to antifungals itraconazole and ketoconazole in vitro. These findings, however, have not been confirmed in vivo. To discover the melanin biosynthesis pathways used by M. mycetomatis in vivo and to determine if inhibiting melanin production would increase M. mycetomatis's susceptibility to itraconazole, inhibitors targeting DHN-, DOPA- and pyomelanin were used. Treatment with DHN-melanin inhibitors tricyclazole, carpropamid, fenoxanil and DOPA-melanin inhibitor glyphosate in M. mycetomatis infected Galleria mellonella larvae resulted in presence of non-melanized grains. Our finding suggested that M. mycetomatis is able to produce DOPA-melanin in vivo. Inhibiting DHN-melanin with carpropamid in combination with the antifungal itraconazole also significantly increased larvae survival. Our results suggested that combination treatment of antifungals and melanin inhibitors can be an alternative treatment strategy that can be further explored. Since the common black-grain eumycetoma causing agents uses similar melanin biosynthesis pathways, this strategy may be applied to them and other eumycetoma causative agents. LAY SUMMARY: Melanin protects fungi from environmental stress and antifungals. We have discovered that Madurella mycetomatis produces DHN-, pyomelanin and DOPA-melanin in vivo. Inhibiting M. mycetomatis DHN-melanin biosynthesis increases therapeutic value of the antifungal itraconazole in vivo.

Rapid and Accurate Detection of Aminoglycoside-Modifying Enzymes and 16S rRNA Methyltransferases by Targeted Liquid Chromatography-Tandem Mass Spectrometry.

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in curbing drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside-modifying enzymes and 16S rRNA methyltransferases in Escherichia coli and Klebsiella pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6')-Ib, AAC(6')-Ib-cr, ANT(2″)-I, APH(3')-VI, ArmA, RmtB, RmtC, and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole-genome sequencing results. Of the 2,460 isolate and resistance mechanism combinations tested, 2,416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin, and amikacin in only the E. coli and K. pneumoniae isolates (n = 191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

Characterization of Staphylococcus roterodami sp. nov., a new species within the Staphylococcus aureus complex isolated from a human foot infection.

This article introduces a new Staphylococcus species cultivated from a human foot wound infection in a Dutch traveller returning from the island of Bali, Indonesia: Staphylococcus roterodami sp. nov. Based on the genomic sequence, there is strong molecular evidence for assigning the strain to a novel species within the S. aureus complex. Differences in cellular fatty acid spectrum and biochemical tests underline these findings. Its ecological niche and pathogenicity require further study. The type strain is DSM111914T (JCM34415T).

Genome-guided analysis allows the identification of novel physiological traits in Trichococcus species.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 2094T), is able to grow on glycerol with the production of 1,3-propanediol.

Trichococcus shcherbakoviae sp. nov., isolated from a laboratory-scale anaerobic EGSB bioreactor operated at low temperature.

A new species of the genus Trichococcus, strain Art1T, was isolated from a psychrotolerant syntrophic propionate-oxidizing consortium, obtained before from a low-temperature EGSB reactor fed with a mixture of VFAs (acetate, propionate and butyrate). The 16S rRNA gene sequence of strain Art1T was highly similar to those of other Trichococcus species (99.7-99.9 %) but digital DNA-DNA hybridization values were lower than those recommended for the delineation of a novel species, indicating that strain Art1T is a novel species of the genus Trichococcus. Cells of strain Art1T are non-motile cocci with a diameter of 0.5-2.0 µm and were observed singularly, in pairs, short chains and irregular conglomerates. Cells of Art1T stained Gram-positive and produced extracellular polymeric substances . Growth was optimal at pH 6-7.5 and cells could grow in a temperature range of from -2 to 30 °C (optimum 25-30 °C). Strain Art1T can degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate and ethanol. The genomic DNA G+C content of strain Art1T is 46.7 %. The major components of the cellular fatty acids are C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c. Based on genomic and physiological characteristics of strain Art1T, a new species of the genus Trichococcus, Trichococcusshcherbakoviae, is proposed. The type strain of Trichococcusshcherbakoviae is Art1T (=DSM 107162T = VKM B-3260T).

Description of Trichococcus ilyis sp. nov. by combined physiological and in silico genome hybridization analyses.

Species of the genus Trichococcus share high similarity of their 16S rRNA gene sequences (>99 %). Digital DNA-DNA hybridization values (dDDH) among type strains of all described species of the genus Trichococcus (T. flocculiformis DSM 2094T, T. pasteurii DSM 2381T, T. collinsii DSM 14526T, T. palustris DSM 9172T, and T. patagoniensisDSM 18806T) indicated that Trichococcus sp. strain R210T represents a novel species of the genus Trichococcus. The dDDH values showed a low DNA relatedness between strain R210T and all other species of the genus Trichococcus (23-32%). Cells of strain R210T were motile, slightly curved rods, 0.63-1.40×0.48-0.90 µm and stained Gram-positive. Growth was optimal at pH 7.8 and at temperature of 30 °C. Strain R210T could utilize several carbohydrates, and the main products from glucose fermentation were lactate, acetate, formate and ethanol. The genomic DNA G+C content of strain R210T was 47.9 mol%. Based on morphological, physiological and biochemical characteristics along with measured dDDH values for all species of the genus Trichococcus, it is suggested that strain R210T represents a novel species within the genus Trichococcus, for which the name Trichococcus ilyis sp. nov. is proposed. The type strain is R210T (=DSM 22150T=JCM 31247T).

First Report of Colistin-Resistant Escherichia coli Carrying mcr-1 IncI2(delta) and IncX4 Plasmids from Camels (Camelus dromedarius) in the Gulf Region.

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 (mcr) in Escherichia coli 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 mcr-genes, plasmid profiling, and whole-genome sequencing (WGS) were conducted. Eleven of fifty camel fecal samples (22%) yielded colonies positive for E. coli isolates carrying the mcr-1 gene on mobile genetic elements. No other mcr-gene variants and no chromosomally located colistin resistance genes were detected. Following plasmid profiling and WGS, nine E. coli isolates from eight camels were selected for in-depth analysis. E. coli 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 mcr-1 carriage in these isolates. These findings represent the first identification of mcr-1-carrying plasmids associated with camels in the Gulf region. The presence of mcr-1 in camels from this region was previously unreported and serves as a novel finding in the field of AMR surveillance.

High Prevalence of ST502 Carrying an OXA-24 Carbapenemase gene in Carbapenem-Nonsusceptible Acinetobacter baumannii-calcoaceticus Isolates in Romania.

Background: Acinetobacter baumannii can cause difficult-to-treat infections because it can acquire extensive antimicrobial resistance mechanisms. We aim to describe the antimicrobial resistance pattern and the genetic basis of carbapenem-nonsusceptible A. baumannii isolates in a University Hospital in Romania, a country where multidrug-resistant A. baumannii is widespread. Methods: We collected 104 consecutive meropenem-nonsusceptible A. baumannii isolates from 104 patients (36% female, mean age [SD] of 63 [16] years) between May 2015 and August 2017 from a large tertiary center in Romania. Whole-genome sequencing of representative isolates from amplified fragment length polymorphism clusters was used to determine clonality and resistance patterns. Results: All isolates were resistant to piperacillin/tazobactam, ceftazidime, and ciprofloxacin; 88.5% to gentamicin; and 90.4% to trimethoprim/sulfamethoxazole. In contrast, 79.8% and 99.0% were susceptible to tobramycin and colistin, respectively. The only isolate resistant to colistin had an minimum inhibitory concentration (MIC) of ≥16 mg/L. The blaOXA-24 gene was detected in 79.1% and blaOXA-23 in 20.9% of the isolates. In one isolate, blaOXA-23 was copresent with blaOXA-24. ST502 (Oxford scheme) was the most prevalent sequence type and was exclusively associated with blaOXA-24. Conclusions: ST502 associated with blaOXA-24 was frequently observed in the region where carbapenem-nonsusceptible A. baumannii was found to be endemic. In these isolates, tobramycin and colistin might be the remaining therapeutic options. Due to differences in gentamicin and tobramycin resistance in these isolates, surveillance data should not group gentamicin, tobramycin, and amikacin together as aminoglycosides.

Pre-COVID-19 international travel and admission to hospital when back home: travel behavior, carriage of highly resistant microorganisms, and risk perception of patients admitted to a large tertiary care hospital.

BACKGROUND: When people who recently travelled abroad are admitted to a hospital back home, there is a risk of introducing highly resistant microorganisms (HRMO) into the hospital. To minimize this risk, a feasible infection prevention strategy should be developed. In this study, we investigated patients' travel history and behavior during travel and analyzed whether this was correlated to HRMO carriage at admission. METHODS: From May 2018 until August 2019, adult patients admitted to a large tertiary care center in the Netherlands were asked upon hospital admission to participate in the study. Included patients received a questionnaire about risk perception, travel history in the last year, and behavior during travel, and were screened for HRMO carriage at admission using a perianal swab. RESULTS: Six hundred and eight questionnaires were handed out, of which 247 were returned (40.6%). One hundred and thirty (52.6%) patients did not travel abroad in the last year, of whom eight (6.2%) were HRMO carrier at admission. One hundred seventeen (47.4%) patients travelled in the preceding year, of whom seven patients (6.0%) were HRMO carrier at admission. Thirty patients (12%) travelled outside of Europe; in this group HRMO prevalence was 13.3% (4 out of 30). The majority of patients (71.3%) were aware that international travel could lead to carriage of HRMO, and an even larger majority (89.5%) would support a screening strategy upon hospital admission in case of a travel history, to minimize the risk of introducing HRMO. CONCLUSIONS: We identified that half of admitted patients to a large tertiary care hospital travelled abroad in the last year, with only a small percentage outside Europe. We discuss several screening strategies and propose a strategy of screening and preemptive isolation of patients who travelled to Asia or Africa in the 2 months before their hospital admission; a strategy that patients would support.

Using Targeted Liquid Chromatography-Tandem Mass Spectrometry to Rapidly Detect ß-Lactam, Aminoglycoside, and Fluoroquinolone Resistance Mechanisms in Blood Cultures Growing E. coli or K. pneumoniae.

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 ß-lactam, aminoglycoside, and fluoroquinolone resistance mechanisms in blood cultures growing Escherichia coli or Klebsiella pneumoniae complex. Selected targets were the ß-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.

The effect of 100% single-occupancy rooms on acquisition of extended-spectrum beta-lactamase-producing Enterobacterales and intra-hospital patient transfers: a prospective before-and-after study.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. TRIAL REGISTRATION: This study was retrospectively registered in the Dutch National Trial Register on 24-02-2020, with registration number NL8406.

Liquid Chromatography-Tandem Mass Spectrometry Analysis Demonstrates a Decrease in Porins and Increase in CMY-2 ß-Lactamases in Escherichia coli Exposed to Increasing Concentrations of Meropenem.

While Extended-Spectrum ß-Lactamases (ESBL) and AmpC ß-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 Enterobacterales can become resistant to carbapenems when high concentrations of ESBL and AmpC ß-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 Escherichia coli porins OmpC and OmpF, its chromosomal AmpC ß-lactamase, and the plasmid-mediated CMY-2 ß-lactamase. Bla CMY-2-like positive E. coli 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 ß-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 E. coli.

CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics.

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.

Genetic Analysis of mcr-1-Carrying Plasmids From Gram-Negative Bacteria in a Dutch Tertiary Care Hospital: Evidence for Intrapatient and Interspecies Transmission Events.

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.

Cefpirome Treatment Results in Limited Selection of Stable Derepressed Enterobacter cloacae Mutants in the Intestinal Flora of Rats Treated for an Experimental Klebsiella pneumoniae Pulmonary Infection.

Background: Fourth-generation cephalosporins have been developed to improve their potency, that is, low minimal inhibitory concentrations (MICs) and to prevent resistance selection of derepressed AmpC-producing mutants in comparison to third-generation cephalosporins as ceftazidime. Objectives: We investigated the role of the administered cefpirome dose on the efficacy of treatment of a Klebsiella pneumoniae lung infection as well as in the selection of resistant Enterobacter cloacae isolates in the intestines of rats treated for a K. pneumoniae lung infection. Materials and Methods: Rats with K. pneumoniae lung infection received therapy with cefpirome doses of 0.4 to 50 mg/kg/day b.i.d. for 18 days. Resistance selection in intestinal E. cloacae was monitored during 43 days. Mutants were checked for ß-lactamase activity, mutations in their structural ampC gene, ampD gene, and omp39-40 gene. Results: A 45% and 100% rat survival rate was obtained by administration of 3.1 and 12.5 mg/kg b.i.d. of cefpirome. A significant correlation was demonstrated in the reduction of the susceptible E. cloacae isolates with %fT>MIC at days 7, 14, 22, and 29. Cefpirome E. cloacae mutants, with increased cefpirome MICs, were obtained in only four rats. Conclusions: The treatment with cefpirome resulted in less selection of derepressed mutants in comparison to ceftazidime as shown by their low number per gram of feces and in a limited number of animals.

Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry.

BACKGROUND: 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. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: 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. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.

Co-culture of a Novel Fermentative Bacterium, Lucifera butyrica gen. nov. sp. nov., With the Sulfur Reducer Desulfurella amilsii for Enhanced Sulfidogenesis.

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 ALET) that is able to use a great variety of substrates including glycerol. Strain ALET 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 Propionispora hippei (91.4% 16S rRNA gene identity) from the Sporomusaceae family (Selenomonadales order, Negativicutes class, Firmicutes phylum). Based on the distinctive physiological and phylogenetic characteristics of strain ALET, a new genus and species Lucifera butyrica gen. nov., sp. nov., is proposed. The type strain is ALET (=JCM 19373T = DSM 27520T). Strain ALET is an incomplete oxidizer and acetate, among other products, accumulates during glycerol conversion. Strain ALET was used to extend the substrate range for sulfur reduction by constructing co-cultures with the acetate oxidizer and sulfur reducer Desulfurella amilsii. The co-culture was tested with glycerol as substrate in batch and chemostat experiments. Acetate formed by fermentation of glycerol by strain ALET resulted in sulfur reduction by D. amilsii. 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. Note: The locus tag for the genes encoded in Lucifera butyrica is LUCI_∗. To avoid repetition of the prefix along the text, the locus tags are represented by the specific identifier.

Draft Genome Sequence of Actinomyces glycerinitolerans Strain G10T, Isolated from Sheep Rumen Fluid.

Actinomyces glycerinitolerans strain G10T, which was isolated from sheep rumen fluid, can metabolize a range of substrates, including complex carbohydrates to organic acids (OAs). Here, we report a 3.69-Mbp draft genome of Actinomyces glycerinitolerans.