Molecular analysis of metallo-beta-lactamase-producing Pseudomonas aeruginosa in Switzerland 2022-2023.

OBJECTIVES: The occurrence of metallo-beta-lactamase-producing Pseudomonas aeruginosa (MBL-PA) isolates is increasing globally, including in Switzerland. The aim of this study was to characterise, phenotypically and genotypically, the MBL-PA isolates submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) reference laboratory over a 12-month period from July 2022 to July 2023. METHODS: Thirty-nine non-duplicate MBL-PA Isolates were submitted to NARA over the study period from across Switzerland. Susceptibility was determined by broth microdilution according to EUCAST methodology. Whole-genome sequencing was performed on 34 isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. MBL genes, blaNDM-1, blaIMP-1, and blaVIM-2, were cloned into vector pUCP24 and transformed into P. aeruginosa PA14. RESULTS: The most prevalent MBL types identified in this study were VIM (21/39; 53.8%) followed by NDM (11/39; 28.2%), IMP (6/39; 15.4%), and a single isolate produced both VIM and NDM enzymes. WGS identified 13 different STs types among the 39 isolates. They all exhibited resistance to cephalosporins, carbapenems, and the beta-lactam-beta-lactamase inhibitor combinations, ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, and 8 isolates were cefiderocol (FDC) resistant. Recombinant P. aeruginosa strains producing blaNDM-1, blaIMP-1, and blaVIM-2 exhibited FDC MICs of 16, 8, and 1 mg/L, respectively. CONCLUSIONS: This study showed that the MBL-PA in Switzerland could be attributed to the wide dissemination of high-risk clones that accounted for most isolates in this study. Although FDC resistance was only found in 8 isolates, MBL carriage was shown to be a major contributor to this phenotype.

Rapid meropenem/vaborbactam NP test for detecting susceptibility/resistance in Enterobacterales.

BACKGROUND: The treatment options for infections caused by carbapenem-resistant Enterobacterales (CRE) are extremely scarce nowadays and the development of new antibiotics does not follow the exponential increase in the dissemination of carbapenem resistance determinants worldwide. Meropenem/vaborbactam was recently approved for clinical use and it has been indicated for treating several infections. Although relatively rare, meropenem/vaborbactam resistance has already been reported in Enterobacterales and its early detection could be a valuable tool for faster clinical decision-making. OBJECTIVES: To develop a rapid test, namely the Rapid MEV NP, for the identification of meropenem/vaborbactam resistance in Enterobacterales. METHODS: The Rapid MEV NP test is based on detection of glucose metabolization occurring upon bacterial growth in the presence of meropenem/vaborbactam at a concentration of 16/8 mg/L. Bacterial growth is detectable by a colour change of phenol red (from red to yellow) subsequent of the acidification of the medium upon bacterial growth. A total of 75 Enterobacterales isolates were randomly selected for evaluating the performance of the Rapid MEV NP test. RESULTS: The test showed 97.2% sensitivity and 93.8% specificity when compared with the reference method. The results are obtained after 3 h of incubation at 35°C ±â€Š2°C, which is a gain of time of at least 15 h (one day in practice) compared with currently used antimicrobial susceptibility testing including broth microdilution methods. CONCLUSIONS: The Rapid MEV NP test, easy to perform and to interpret, showed remarkable performance while providing fast results, and is therefore suitable for implementation in routine clinical microbiology laboratories.

Rapid detection of cefiderocol susceptibility/resistance in Acinetobacter baumannii.

PURPOSE: Due to its ability to disseminate worldwide and its multiple resistance trait, Acinetobacter baumannii is becoming a threat for public health worldwide. Cefiderocol (FDC) is a promising broad-spectrum cephalosporin recently approved for treating Gram-negative infection. The aim of this study was to develop a rapid test, namely the rapid FDC Acinetobacter baumannii NP test, for the detection of FDC susceptibility/resistance in A. baumannii since the current FDC susceptibility tests are rather time-consuming (at least 24 h). MATERIALS AND METHODS: The rapid test is based on the reduction of resazurin to resorufin product by bacterial viable cells, thus detecting bacterial growth in the presence of FDC (38.4 mg/L). A color change from blue (resazurin) to violet or pink (resorufin) represents visual detection of bacterial growth. 95 randomly selected A. baumannii isolates were used to evaluate the performance of the rapid FDC Acinetobacter baumannii NP test. RESULTS: The test showed 95.5% (95% CI 78.2-99.2%) and 100.0% (95% CI 95.0-100.0%) of sensitivity and specificity, respectively. All the results were obtained within 4 h30-4 h45 incubation time at 35 °C ± 2 °C, saving virtually one day when compared with currently-used antimicrobial susceptibility tests. The test showed only a single very major error, an isolate with a MIC of 8 mg/L. CONCLUSIONS: The rapid FDC Acinetobacter baumannii NP test can be a valuable method which is easier and faster to interpret when compared with the gold standard broth microdilution method. The test showed remarkable performances; hence, it may be suitable for implementation in clinical microbiology routine laboratories.

Detection of polymyxins resistance among Enterobacterales: evaluation of available methods and proposal of a new rapid and feasible methodology.

BACKGROUND: Fast and accurate detection of polymyxins resistance is necessary as they remain the last resources to treat infections caused by Carbapenem-resistant Enterobacterales in many regions. We evaluated the rapid colorimetric polymyxin B elution (RCPE) and developed its miniaturized version, RCPE microelution (RCPEm), aiming to detect polymyxins resistance among Enterobacterales. METHODS: The methodologies consist of exposing the bacterial population in a solution (NP solution) where polymyxin B disks were previously eluted to obtain a concentration of 2 µg/mL for RCPE and 3 µg/mL for RCPEm. RESULTS: Two hundred sixty-seven Enterobacterales were evaluated, 90 (33.7%) resistant to polymyxin B by broth microdilution. It was observed 0.6% of major error (ME) by RCPE, with a specificity of 99.4%. The miniaturized version (RCPEm) presented the same ME and specificity values, but slightly higher sensitivity (97.8% vs. 95.6%) with 2.2% of very major error (VME). CONCLUSIONS: RCPE and RCPEm proved to be useful alternatives to determine polymyxin B susceptibility in clinical microbiology laboratories, presenting low cost, being easy to perform, and demanding short incubation time.

Impact of extended-spectrum chromosomal AmpC (ESAC) of Escherichia coli on susceptibility to cefiderocol.

The impact of chromosomally encoded wild-type or extended-spectrum (ESAC) AmpC ß-lactamases of Escherichia coli on susceptibility to ceftazidime, cefepime, and cefiderocol was evaluated in different genetic backgrounds, including wild-type, PBP3-modified, and porin-deficient E. coli strains. Recombinant E. coli strains possessing the different backgrounds and producing variable ESACs were evaluated. Although ESAC enzymes conferred resistance to ceftazidime and decreased susceptibility to cefepime as expected, we showed here that cefiderocol was also a substrate of ESAC enzymes. IMPORTANCE: We showed here that chromosomally encoded intrinsic extended-spectrum cephalosporinases of Escherichia coli may impact susceptibility not only to ceftazidime and cefepime but also to cefiderocol.

MultiRapid ATB NP test for detecting concomitant susceptibility and resistance of last-resort novel antibiotics available to treat multidrug-resistant Enterobacterales infections.

BACKGROUND: Recently developed therapeutics against Gram-negative bacteria include the ß-lactam-ß-lactamase inhibitor combinations ceftazidime-avibactam (CZA), meropenem-vaborbactam (MEV), and imipenem-relebatam (IPR), and the siderophore cephalosporin cefiderocol (FDC). The aim of this study was to develop a test for rapid identification of susceptibility/resistance to CZA, MEV, IPR, and FDC for Enterobacterales in a single test for rapid clinical decision making. METHODS: The MultiRapid ATB NP test is based on the detection of glucose metabolism occurring after bacterial growth in the presence of defined concentrations of CZA, MEV, IPR, and FDC, followed by visual detection of colour change of the pH indicator red phenol (red to yellow) generated by the acidification of the medium upon bacterial growth. This test is performed in 96-well microplates. The MultiRapid ATB NP test was evaluated using 78 Enterobacterales isolates and compared to the reference method broth microdilution. RESULTS: The MultiRapid ATB NP test displayed 97.0% (confidence interval [CI] 92.6-98.8) sensitivity, 97.7% (CI 94.3-99.1) specificity, and 97.4% (CI 95.0-98.7) accuracy. The results were obtained after 3 h of incubation at 35 °C ± 2 °C, representing at least a 15-h gain-of-time compared with currently used antimicrobial susceptibility testing methods. CONCLUSION: The MultiRapid ATB NP test provided accurate results for the concomitant detection of susceptibility/resistance to CZA, MEV, IPR, and FDC in Enterobacterales, independent of the resistance mechanism. This test may be suitable for implementation in any microbiology routine laboratory.

Effect of Zinc Oxide and Copper Sulfate on Antibiotic Resistance Plasmid Transfer in Escherichia coli.

Heavy metals such as zinc (Zn) and copper (Cu) may be associated with antibiotic resistance dissemination. Our aim was to investigate whether sub-lethal dosage of Zn and Cu may enhance plasmid transfer and subsequently resistance genes dissemination. Plasmid conjugation frequencies (PCF) were performed with Escherichia coli strains bearing IncL-blaOXA-48, IncA/C-blaCMY-2, IncI1-blaCTX-M-1, IncF-blaCTX-M-1, and IncX3-blaNDM-5 as donors. Mating-out assays were performed with sub-dosages of zinc oxide (ZnO) and Cu sulfate (CuSO4). Quantification of the SOS response-associated gene expression levels and of the production of reactive oxygen species were determined. Increased PCF was observed for IncL, IncA/C, and IncX3 when treated with ZnO. PCF was only increased for IncL when treated with CuSO4. The ROS production presented an overall positive correlation with PCF after treatment with ZnO for IncL, IncA/C, and IncX3. For CuSO4 treatment, the same was observed only for IncL. No increase was observed for expression of SOS response-associated genes under CuSO4 treatment, and under ZnO treatment, we observed an increase in SOS response-associated genes only for IncX3. Our data showed that sub-dosages of ZnO and CuSO4 could significantly enhance PCF in E. coli, with a more marked effect observed with IncL, IncA/C, and IncX3 scaffolds. Our study suggested that use of certain heavy metals is not the panacea for avoiding use of antibiotics in order to prevent the dissemination of antibiotic resistance.

Rapid detection of imipenem/relebactam susceptibility/resistance in Enterobacterales.

OBJECTIVES: The treatment options for infections caused by carbapenem-resistant Enterobacterales are scarce and the development of new antibiotics is an urgent necessity. Imipenem/relebactam (IPR) has been recently introduced for treating severe infections related to multidrug-resistant bacteria. However, IPR resistance has already been reported in Enterobacterales, thus its rapid detection may be interesting for clinical decision-making. The aim of the study was to develop a rapid and accurate test, namely the Rapid IPR Nordmann Poirel (NP) test, for the identification of IPR resistance among multidrug-resistant Enterobacterales. METHODS: The Rapid IPR NP test is based on the detection of glucose metabolization because of bacterial growth in the presence of IPR. Bacterial growth is visually detectable by a colour change of the red phenol pH indicator, turning from red to yellow subsequent to the acidification of the medium upon bacterial growth. Cultures of a total of 94 Enterobacterales isolates were selected for evaluating the performance of the Rapid IPR NP test. RESULTS: The sensitivity and specificity of the test were found to be 95.2% (95.2%, CI 84.2-98.7%) and 100% (100%, CI 93.1-100%), respectively. All the results were obtained within 3 hours incubation time at 35°C ± 2°C, which is a gain of time of at least 15 hours when compared with currently used antimicrobial susceptibility. The test showed two very major errors corresponding to OXA-48-producing Klebsiella pneumoniae isolates with MICs of IPR at 8 mg/L. DISCUSSION: The Rapid IPR NP test is simple to perform and interpret, and shows excellent performances. Thus, it may suitable for implementation in clinical microbiology routine laboratories.

Plasmid-mediated fosfomycin resistance in Escherichia coli isolates of worldwide origin.

OBJECTIVES: Fosfomycin is a first-line treatment for uncomplicated urinary tract infections (UTIs) in several European countries, and it is increasingly becoming the treatment of choice globally. Resistance to fosfomycin in Escherichia coli can be exerted through several mechanisms, including the acquisition of fosfomycin-modifying enzymes, of which the FosA-type enzymes are the most common. This study analysed, both phenotypically and genotypically, an international collection of E. coli strains harbouring acquired fosA genes. METHODS: Thirty-one fosA-positive E. coli isolates were obtained from both clinical and environmental sources, from seven countries (Portugal (n = 12), Switzerland (n = 9), China (n = 3), France (n = 2), Nepal (n = 2), South Africa (n = 2), Kuwait (n = 1)). MICs were determined according to EUCAST guidelines. Whole genome sequencing (WGS) was performed on 23 isolates, and complete fosA plasmid sequences were determined for 12. Conjugation assays were performed on seven isolates. RESULTS: All isolates exhibited high-level resistance to fosfomycin (64 to >256 mg/L). WGS of 23 isolates identified 17 sequence types (STs), and 16 harboured fosA3, four fosA4, two fosA8, and one fosA10. ESBLs, pAmpC, or carbapenemase genes were present in 15, four, and three isolates, respectively. The fosA plasmids of 12 isolates were determined and were diverse in size (∼67 kb to ∼235 kb), resistance gene carriage, and replicon types. Six fosA plasmids additionally carried ESBL or carbapenemase genes. Conjugation assays, performed on seven isolates harbouring diverse plasmids, identified that all were capable of being transmitted. CONCLUSION: This study highlights the necessity of the surveillance and close monitoring of fosfomycin resistance in E. coli, essential to maintain the optimal use of this treatment option.

Carbapenemase-Producing Klebsiella pneumoniae From Transplanted Patients in Brazil: Phylogeny, Resistome, Virulome and Mobile Genetic Elements Harboring bla KPC- 2 or bla NDM- 1.

OBJECTIVES: Carbapenemase-producing Klebsiella pneumoniae (CP-Kp) is a major cause of infections in transplanted patients and has been associated with high mortality rates in this group. There is a lack of information about the Brazilian structure population of CP-Kp isolated from transplanted patients. By whole-genome sequencing (WGS), we analyzed phylogeny, resistome, virulome of CP-Kp isolates, and the structure of plasmids encoding bla KPC- 2 and bla NDM- 1 genes. METHODS: One K. pneumoniae isolated from each selected transplanted patient colonized or infected by CP-Kp over a 16-month period in a hospital complex in Porto Alegre (Brazil) was submitted for WGS. The total number of strains sequenced was 80. The hospital complex in Porto Alegre comprised seven different hospitals. High-resolution SNP typing, core genome multilocus sequence typing (cgMLST), resistance and virulence genes inference, and plasmid reconstruction were performed in 80 CP-Kp. RESULTS: The mortality rate of CP-Kp colonized or infected transplanted inpatients was 21.3% (17/80). Four CP-Kp epidemic clones were described: ST11/KPC-2, ST16/KPC-2, and ST15/NDM-1, all responsible for interhospital outbreaks; and ST437/KPC-2 affecting a single hospital. The average number of acquired resistance and virulence genes was 9 (range = 2-14) and 27 (range = 6-36), respectively. Two plasmids carrying the bla KPC - 2 were constructed and belonged to IncN and IncM types. Additionally, an IncFIB plasmid carrying the bla NDM- 1 was described. CONCLUSION: We detected intrahospital and interhospital spread of mobile structures and international K. pneumoniae clones as ST11, ST16, and ST15 among transplanted patients, which carry a significant range of acquired resistance and virulence genes and keep spreading across the world.

Carbapenem-resistant Acinetobacter baumannii contamination in an intensive care unit.

INTRODUCTION:: Acinetobacter baumannii is a major pathogen causing infections in intensive care units (ICUs). In this study, we aimed to evaluate the presence of A. baumannii in an ICU environment and gloves from ICU workers and to characterize the antimicrobial resistance of the isolates in comparison with those isolated from ICU patients at the same hospital. METHODS:: ICU samples were collected from March to November 2010. Isolates biochemically characterized as Acinetobacter calcoaceticus-Acinetobacter baumannii complex were evaluated by PCR targeting the 16S rDNA and bla OXA-51 genes. Antimicrobial susceptibility was determined using the disk diffusion method, and carbapenem-resistant isolates were also evaluated for the minimum inhibitory concentration of imipenem using broth microdilution. The presence of the bla OXA-23 gene was evaluated in isolates with reduced susceptibility to carbapenems. RESULTS:: A. baumannii was detected in 9.5% (84) of the 886 samples collected from the ICU environment, including from furniture, medical devices, and gloves, with bed rails being the most contaminated location (23.8%; 20/84). Multidrug-resistant (MDR) A. baumannii was found in 98.8% (83/84) of non-clinical and 97.8% (45/46) of clinical isolates. Reduced susceptibility to carbapenems was detected in 83.3% (70/84) of non-clinical and 80.4% (37/46) of clinical isolates. All isolates resistant to carbapenems harbored bla OXA-23. CONCLUSIONS:: We found a strong similarity between the antimicrobial susceptibility profiles of non-clinical and clinical A. baumannii isolates. Such data highlight the ICU environment as a potential origin for the persistence of MDR A. baumannii, and hence the ICU may be a source of hospital-acquired infections caused by this microorganism.

Carbapenem-resistant Acinetobacter baumannii contamination in an intensive care unit

Abstract INTRODUCTION: Acinetobacter baumannii is a major pathogen causing infections in intensive care units (ICUs). In this study, we aimed to evaluate the presence of A. baumannii in an ICU environment and gloves from ICU workers and to characterize the antimicrobial resistance of the isolates in comparison with those isolated from ICU patients at the same hospital. METHODS: ICU samples were collected from March to November 2010. Isolates biochemically characterized as Acinetobacter calcoaceticus-Acinetobacter baumannii complex were evaluated by PCR targeting the 16S rDNA and bla OXA-51 genes. Antimicrobial susceptibility was determined using the disk diffusion method, and carbapenem-resistant isolates were also evaluated for the minimum inhibitory concentration of imipenem using broth microdilution. The presence of the bla OXA-23 gene was evaluated in isolates with reduced susceptibility to carbapenems. RESULTS: A. baumannii was detected in 9.5% (84) of the 886 samples collected from the ICU environment, including from furniture, medical devices, and gloves, with bed rails being the most contaminated location (23.8%; 20/84). Multidrug-resistant (MDR) A. baumannii was found in 98.8% (83/84) of non-clinical and 97.8% (45/46) of clinical isolates. Reduced susceptibility to carbapenems was detected in 83.3% (70/84) of non-clinical and 80.4% (37/46) of clinical isolates. All isolates resistant to carbapenems harbored bla OXA-23. CONCLUSIONS: We found a strong similarity between the antimicrobial susceptibility profiles of non-clinical and clinical A. baumannii isolates. Such data highlight the ICU environment as a potential origin for the persistence of MDR A. baumannii, and hence the ICU may be a source of hospital-acquired infections caused by this microorganism.