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-bla <sub>OXA-48</sub> , IncA/C-bla <sub>CMY-2</sub> , IncI1-bla <sub>CTX-M-1</sub> , IncF-bla <sub>CTX-M-1</sub> , and IncX3-bla <sub>NDM-5</sub> as donors. Mating-out assays were performed with sub-dosages of zinc oxide (ZnO) and Cu sulfate (CuSO <sub>4</sub> ). 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 CuSO <sub>4</sub> . The ROS production presented an overall positive correlation with PCF after treatment with ZnO for IncL, IncA/C, and IncX3. For CuSO <sub>4</sub> treatment, the same was observed only for IncL. No increase was observed for expression of SOS response-associated genes under CuSO <sub>4</sub> 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 CuSO <sub>4</sub> 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

Impact of veterinary antibiotics on plasmid-encoded antibiotic resistance transfer.

Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the environment, or human and animal guts (One Health concept). The aim of our study was to evaluate whether subdosages of antibiotics administered in veterinary medicine could enhance plasmid transfer and, consequently, resistance gene exchange in gut microbiota. Conjugation frequencies were determined with Escherichia coli strains carrying IncL- (blaOXA-48) or IncI1-type (blaCTX-M-1) plasmids subjected to a series of subinhibitory concentrations of antibiotics used in veterinary medicine, namely amoxicillin, ceftiofur, apramycin, neomycin, enrofloxacin, colistin, erythromycin, florfenicol, lincomycin, oxytetracycline, sulfamethazine, tiamulin and the ionophore narasin. Treatments with subinhibitory dosages were performed with and without supplementation with the antioxidant edaravone, known as a mitigator of the inducibility effect of several antibiotics on plasmid conjugation frequency (PCF). Expression of SOS-response associated genes and fluorescence-based reactive oxygen species (ROS) detection assays were performed to evaluate the stress oxidative response. Increased PCFs were observed for both strains when treating with florfenicol and oxytetracycline. Increased expression of the SOS-associated recA gene also occurred concomitantly, as well as increased ROS production. Addition of edaravone to the treatments reduced their PCF and also showed a decreasing effect on SOS and ROS responses for both plasmid scaffolds. We showed here that some antibiotics used in veterinary medicine may induce transfer of plasmid-encoded resistance and therefore may contribute to the worldwide spread of antibiotic resistance genes

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

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. 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. 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. 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

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

Among the recently developed antibacterial therapeutics against Gram-negatives, there are the β-lactam-β-lactamase inhibitor combinations ceftazidime-avibactam (CZA), meropenem-vaborbactam (MEV), and imipenem-relebatam (IPR), and there is the siderophore cephalosporin cefiderocol (FDC). This study is aimed to develop a rapid test, namely the MultiRapid ATB NP test, for rapid identification of susceptibility/resistance to CZA, MEV, IPR, and FDC for Enterobacterales.in a single test for rapid clinical decision making. The MultiRapid ATB NP test is based on the detection of glucose metabolization occurring after bacterial growth in the presence of defined concentrations of CZA, MEV, IPR, and FDC. Followed by visual detection of color change of the pH indicator red phenol (red to yellow) generated by the acidification of the medium upon bacterial growth. It is performed in 96-well microplates. The Multi Rapid ATB NP test was evaluated with 78 Enterobacterales isolates, and its performances compared to the reference method broth microdilution. The MultiRapid ATB NP test displayed 97.0% (97.0%, CI 92.6 - 98.8%) of sensitivity, 97.7% (97.7%, CI 94.3 - 99.1%) of specificity, and 97.4% (97.4%, CI 95.0% - 98.7%) of 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. The MultiRapid ATB NP test was capable of providing accurate results for a concomitant detection of susceptibility/resistance to CZA, MEV, IPR, and FDC in Enterobacterales, independent from its resistance mechanism. It may be suitable for implementation in any microbiology routine laboratory

Rapid detection of cefiderocol susceptibility/resistance in Acinetobacter baumannii.

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). 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. 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. 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

Emergence of Carbapenemase-Producing Hypervirulent Klebsiella pneumoniae in Switzerland.

Increasing occurrence of multidrug-resistant (MDR) and hypervirulent (hv) Klebsiella pneumoniae (MDR-hvKp) convergent clones is being observed. Those strains have the potential of causing difficult-to-treat infections in healthy adults with an increased capacity for mortality. It is therefore crucial to track their dissemination to prevent their further spread. The aim of our study was to investigate the occurrence of carbapenemase-producing hvKp isolates in Switzerland and to determine their genetic profile. A total of 279 MDR carbapenemase-producing K. pneumoniae from patients hospitalized all over Switzerland was investigated, and a rate of 9.0% K. pneumoniae presenting a virulence genotype was identified. Those isolates produced either KPC, NDM, or OXA-48 and had been either recovered from rectal swabs, urine, and blood. A series of previously reported K. pneumoniae clones such as ST23-K1, ST395-K2, and ST147-K20 or ST147-K64 were identified. All the isolates defined as MDR-hvKp (4.7%) possessed the aerobactin and the yersiniabactin clusters. The ST23-K1s were the only isolates presenting the colibactin cluster and achieved higher virulence scores. This study highlights the occurrence and circulation of worrisome MDR-hvKp and MDR nonhypervirulent K. pneumoniae (MDR-nhv-Kp) isolates in Switzerland. Our findings raise an alert regarding the need for active surveillance networks to track and monitor the spread of such successful hybrid clones representing a public health threat worldwide

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

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. To develop a rapid test, namely the Rapid MEV NP, for the identification of meropenem/vaborbactam resistance in Enterobacterales. 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. 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 ± 2°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. 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