Rapid Detection of Ceftazidime/Avibactam Susceptibility/Resistance in Enterobacterales by Rapid CAZ/AVI NP Test.

We developed a novel culture-based test, the Rapid CAZ/AVI NP test, for rapid identification of ceftazidime/avibactam susceptibility/resistance in Enterobacterales. This test is based on glucose metabolization upon bacterial growth in the presence of a defined concentration of ceftazidime/avibactam (128/53 µg/mL). Bacterial growth is visually detectable by a red to yellow color change of red phenol, a pH indicator. A total of 101 well characterized enterobacterial isolates were used to evaluate the test performance. This test showed positive percent agreement of 100% and negative percent agreement of 98.5% with overall percent agreement of 99%, by comparison with the MIC gradient strip test (Etest) taken as the reference standard method. The Rapid CAZ/AVI NP test had only 1.5% major errors and 0% extremely major errors. This test is rapid (result within 2 hours 45 minutes), reliable, affordable, easily interpretable, and easy to implement in clinical microbiology laboratories without requiring any specific equipment.

Effect of modification of penicillin-binding protein 3 on susceptibility to ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and cefiderocol of Escherichia coli strains producing broad-spectrum ß-lactamases.

The impact of penicillin-binding protein 3 (PBP3) modifications that may be identified in Escherichia coli was evaluated with respect to susceptibility to ß-lactam/ß-lactamase inhibitor combinations including ceftazidime-avibactam, imipenem-relebactam, meropenem-vaborbactam, aztreonam-avibactam, cefepime-taniborbactam, and to cefiderocol. A large series of E. coli recombinant strains producing broad-spectrum ß-lactamases was evaluated. While imipenem-relebactam showed a similar activity regardless of the PBP3 background, susceptibility to other molecules tested was affected at various levels. This was particularly the case for ceftazidime-avibactam, aztreonam-avibactam, and cefepime-taniborbactam.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor taniborbactam against metallo-ß-lactamases.

Taniborbactam (TAN) is a novel broad-spectrum ß-lactamase inhibitor with significant activity against subclass B1 metallo-ß-lactamases (MBLs). Here, we showed that TAN exhibited an overall excellent activity against B1 MBLs including most NDM- and VIM-like as well as SPM-1, GIM-1, and DIM-1 enzymes, but not against SIM-1. Noteworthy, VIM-1-like enzymes (particularly VIM-83) were less inhibited by TAN than VIM-2-like. Like NDM-9, NDM-30 (also differing from NDM-1 by a single amino acid substitution) was resistant to TAN.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor xeruborbactam in comparison with taniborbactam against metallo-ß-lactamases produced in Escherichia coli and Pseudomonas aeruginosa.

Xeruborbactam is a newly developed ß-lactamase inhibitor designed for metallo-ß-lactamases (MBLs). This study assessed the relative inhibitory properties of this novel inhibitor in comparison with another MBL inhibitor, namely taniborbactam (TAN), against a wide range of acquired MBL produced either in Escherichia coli or Pseudomonas aeruginosa. As observed with taniborbactam, the combination of xeruborbactam (XER) with ß-lactams, namely, ceftazidime, cefepime and meropenem, led to significantly decreased MIC values for a wide range of B1-type MBL-producing E. coli, including most recombinant strains producing NDM, VIM, IMP, GIM-1, and DIM-1 enzymes. Noteworthily, while TAN-based combinations significantly reduced MIC values of ß-lactams for MBL-producing P. aeruginosa recombinant strains, those with XER were much less effective. We showed that this latter feature was related to the MexAB-OprM efflux pump significantly impacting MIC values when testing XER-based combinations in P. aeruginosa. The relative inhibitory concentrations (IC50 values) were similar for XER and TAN against NDM and VIM enzymes. Noteworthily, XER was effective against NDM-9, NDM-30, VIM-83, and most of IMP enzymes, although those latter enzymes were considered resistant to TAN. However, no significant inhibition was observed with XER against IMP-10, SPM-1, and SIM-1 as well as the representative subclass B2 and B3 enzymes, PFM-1 and AIM-1. The determination of the constant inhibition (Ki) of XER revealed a much higher value against IMP-10 than against NDM-1, VIM-2, and IMP-1. Hence, IMP-10 that differs from IMP-1 by a single amino-acid substitution (Val67Phe) can, therefore, be considered resistant to XER.

Inter-phylum circulation of a beta-lactamase-encoding gene: a rare but observable event.

Beta-lactamase-mediated degradation of beta-lactams is the most common mechanism of beta-lactam resistance in Gram-negative bacteria. Beta-lactamase-encoding genes can be transferred between closely related bacteria, but spontaneous inter-phylum transfers (between distantly related bacteria) have never been reported. Here, we describe an extended-spectrum beta-lactamase (ESBL)-encoding gene (blaMUN-1) shared between the Pseudomonadota and Bacteroidota phyla. An Escherichia coli strain was isolated from a patient in Münster (Germany). Its genome was sequenced. The ESBL-encoding gene (named blaMUN-1) was cloned, and the corresponding enzyme was characterized. The distribution of the gene among bacteria was investigated using the RefSeq Genomes database. The frequency and relative abundance of its closest homolog in the global microbial gene catalog (GMGC) were analyzed. The E. coli strain exhibited two distinct morphotypes. Each morphotype possessed two chromosomal copies of the blaMUN-1 gene, with one morphotype having two additional copies located on a phage-plasmid p0111. Each copy was located within a 7.6-kb genomic island associated with mobility. blaMUN-1 encoded for an extended-spectrum Ambler subclass A2 beta-lactamase with 43.0% amino acid identity to TLA-1. blaMUN-1 was found in species among the Bacteroidales order and in Sutterella wadsworthensis (Pseudomonadota). Its closest homolog in GMGC was detected frequently in human fecal samples. This is, to our knowledge, the first reported instance of inter-phylum transfer of an ESBL-encoding gene, between the Bacteroidota and Pseudomonadota phyla. Although the gene was frequently detected in the human gut, inter-phylum transfer was rare, indicating that inter-phylum barriers are effective in impeding the spread of ESBL-encoding genes, but not entirely impenetrable.

Detection of volatile organic compounds as new paradigm to accelerate antimicrobial susceptibility testing: performance evaluation of VITEK® REVEAL™.

OBJECTIVES: The measurement of VOCs release in the headspace of a bacterial culture represents a new approach to rapidly assess antimicrobial susceptibility. Herein, we evaluated the diagnostic performance of the VITEK® REVEAL™ system directly from a collection of Gram-negative positive blood cultures. MATERIALS AND METHODS: One hundred and twenty-eight positive blood cultures were included in the analysis (Enterobacterales, n = 95; Pseudomonas aeruginosa, n = 21; Acinetobacter baumannii complex, n = 12). Samples were processed using VITEK® REVEAL™ according to the manufacturer's recommendations, and MICs of 22 antimicrobials were compared with those obtained using reference methods. Categorical agreement (CA), essential agreement (EA) and categorical errors were calculated. RESULTS: Overall, 2220 strain/antibiotic pair combinations were analysed. Of these, most were classified as resistant by reference antimicrobial susceptibility testing (1091/2220; 48.7%). The overall CA and EA were 97.6% and 97.7%, respectively. CA ranged from 97.5% in Enterobacterales to 97.9% in both P. aeruginosa and A. baumannii complex. The overall number of categorical discrepancies were: 18 very major errors (1.6%), 13 major errors (1.2%) and 22 minor errors (2.4%). EA ranged from 95.2% in P. aeruginosa to 98.1% in Enterobacterales. Screening test for ESBL phenotype was positive, indeterminate and negative in 13.7%, 32.6% and 27.4% of Enterobacterales isolates tested by both VITEK® REVEAL™ and the reference method, showing 100% CA. CONCLUSIONS: VITEK® REVEAL™ represents a reliable tool to obtain antimicrobial susceptibility results of the main Gram-negative species directly from positive blood cultures with time to results of less than 8 h.

Multidrug-resistant Gram-negative clinical isolates with reduced susceptibility/resistance to cefiderocol: which are the best present and future therapeutic alternatives?

PURPOSE: To evaluate the different present and future therapeutic ß-lactam/ß-lactamase inhibitor (BL/BLI) alternatives, namely aztreonam-avibactam, imipenem-relebactam, meropenem-vaborbactam, cefepime-zidebactam, cefepime-taniborbactam, meropenem-nacubactam, and sulbactam-durlobactam against clinical isolates showing reduced susceptibility or resistance to cefiderocol in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa. METHODS: MIC values of aztreonam, aztreonam-avibactam, cefepime, cefepime-taniborbactam, cefepime-zidebactam, imipenem, imipenem-relebactam, meropenem, meropenem-vaborbactam, meropenem-nacubactam, sulbactam-durlobactam, and cefiderocol combined with a BLI were determined for 67, 9, and 11 clinical Enterobacterales, P. aeruginosa or A. baumannii isolates, respectively, showing MIC values of cefiderocol being ≥1 mg/L. If unavailable, the respective ß-lactam breakpoints according to EUCAST were used for BL/BLI combinations. RESULTS: For Enterobacterales, the susceptibility rates for aztreonam, cefepime, imipenem, and meropenem were 7.5%, 0%, 10.4%, and 10.4%, respectively, while they were much higher for cefepime-zidebactam (91%), cefiderocol-zidebactam (91%), meropenem-nacubactam (71.6%), cefiderocol-nacubactam (74.6%), and cefiderocol-taniborbactam (76.1%), as expected. For P. aeruginosa isolates, the higher susceptibility rates were observed for imipenem-relebactam, cefiderocol-zidebactam, and meropenem-vaborbactam (56% for all combinations). For A. baumannii isolates, lower susceptibility rates were observed with commercially or under development BL/BLI combos; however, a high susceptibility rate (70%) was found for sulbactam-durlobactam and when cefiderocol was associated to some BLIs. CONCLUSIONS: Zidebactam- and nacubactam-containing combinations showed a significant in vitro activity against multidrug-resistant Enterobacterales clinical isolates with reduced susceptibility to cefiderocol. On the other hand, imipenem-relebactam and meropenem-vaborbactam showed the highest susceptibility rates against P. aeruginosa isolates. Finally, sulbactam-durlobactam and cefiderocol combined with a BLI were the only effective options against A. baumannii tested isolates.

Reduced susceptibility to aztreonam-avibactam conferred by acquired AmpC-type ß-lactamases in PBP3-modified Escherichia coli.

PURPOSE: Carbapenemase-producing Enterobacterales are a growing threat, and very few therapeutic options remain active against those multidrug resistant bacteria. Aztreonam is the molecule of choice against metallo-beta-lactamases (MBL) producers since it is not hydrolyzed by those enzymes, but the co-production of acquired plasmidic cephalosporinases or extended-spectrum ß-lactamases leading to aztreonam resistance may reduce the efficacy of this molecule. Hence, the development of the aztreonam-avibactam (AZA) combination provides an interesting therapeutic alternative since avibactam inhibits the activity of both cephalosporinases and extended-spectrum ß-lactamases. However, structural modifications of penicillin binding protein PBP3, the target of aztreonam, may lead to reduced susceptibility to aztreonam-avibactam. METHODS: Here the impact of various plasmid-encoded AmpC-type ß-lactamases (ACC-1, ACT-7, ACT-17, CMY-2, CMY-42, DHA-1, FOX-1, and FOX-5) on susceptibility to aztreonam-avibactam was evaluated using isogenic E. coli MG1655 strains harboring insertions in PBP3 (YRIN and YRIK). The inhibitory activity of various ß-lactamase inhibitors (clavulanic acid, tazobactam, avibactam, relebactam, and vaborbactam) were also compared against these enzymes. RESULTS: Hence, we showed that reduced susceptibility to AZA was due to the combined effect of both AmpC production and amino acid insertions in PBP3. The highest resistance level was achieved in strains possessing the insertions in PBP3 in association with the production of ACT-7, ACC-1, or CMY-42. CONCLUSION: Although none of the recombinant strains tested displayed clinical resistance to aztreonam-avibactam, our data emphasize that the occurrence of such profile might be of clinical relevance for MBL-producing strains.

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.

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.

Impact of Acquired Broad Spectrum ß-Lactamases on Susceptibility to Novel Combinations Made of ß-Lactams (Aztreonam, Cefepime, Meropenem, and Imipenem) and Novel ß-Lactamase Inhibitors in Escherichia coli and Pseudomonas aeruginosa.

The impact of broad-spectrum ß-lactamases on the susceptibility to novel ß-lactamase/ß-lactamase inhibitor combinations was evaluated both in Pseudomonas aeruginosa and Escherichia coli using isogenic backgrounds. Cefepime-zidebactam displayed low MICs, mainly due to the significant intrinsic antibacterial activity of zidebactam. Cefepime-taniborbactam showed excellent activity against recombinant E. coli strains, including metallo-ß-lactamase producers, whereas aztreonam-avibactam remained the best therapeutic option against class B ß-lactamase-producing P. aeruginosa.

Impact of Minor Carbapenemases on Susceptibility to Novel ß-Lactam/ß-Lactamase Inhibitor Combinations and Cefiderocol in Enterobacterales.

The in vitro activity of imipenem-relebactam, meropenem-vaborbactam, ceftazidime-avibactam, and cefiderocol was evaluated against both clinical and isogenic enterobacterial isolates producing carbapenemases of the SME, NmcA, FRI, and IMI types. Ceftazidime-avibactam and meropenem-vaborbactam showed the highest activity against all tested isolates; imipenem-relebactam showed only moderate activity. All isolates remained susceptible to cefiderocol. Furthermore, avibactam and vaborbactam have greater inhibitory activity than relebactam against the tested carbapenemases. Overall, ceftazidime-avibactam, meropenem-vaborbactam, and cefiderocol were the most effective therapeutic options for treating infections caused by the tested minor carbapenemase producers.

Impact of acquired broad-spectrum ß-lactamases on susceptibility to oral penems/carbapenems (tebipenem, sulopenem, and faropenem) alone or in combination with avibactam and taniborbactam ß-lactamase inhibitors in Escherichia coli.

The impact of ß-lactamases on susceptibility to oral penems/carbapenems (tebipenem, sulopenem, and faropenem) and other carbapenem molecules was evaluated in Escherichia coli, alone and in combination with avibactam or taniborbactam ß-lactamase inhibitors. Tebipenem and sulopenem exhibited a similar spectrum of activity compared to the intravenous carbapenems and displayed lower MIC values than ceftibuten-avibactam against E. coli producing extended-spectrum ß-lactamases or AmpC enzymes. Combined with taniborbactam, tebipenem and sulopenem exhibited low MIC values against almost all tested recombinant E. coli, including metallo-ß-lactamase producers.

An improved PKPD modeling approach to characterize the pharmacodynamic interaction over time between ceftazidime/avibactam and colistin from in vitro time-kill experiments against multidrug-resistant Klebsiella pneumoniae isolates.

In contrast to the checkerboard method, bactericidal experiments [time-kill curves (TKCs)] allow an assessment of pharmacodynamic (PD) interactions over time. However, TKCs in combination pose interpretation problems. The objective of this study was to characterize the PD interaction over time between ceftazidime/avibactam (CZA) and colistin (CST) using TKC against four multidrug-resistant Klebsiella pneumoniae susceptible to both antibiotics and expressing a widespread carbapenemase determinant KPC-3. In vitro TKCs were performed and analyzed using pharmacokinetic/pharmacodynamic (PKPD) modeling. The general pharmacodynamic interaction model was used to characterize PD interactions between drugs. The 95% confidence intervals (95%CIs) of the expected additivity and of the observed interaction were built using parametric bootstraps and compared to evaluate the in vitro PD interaction over time. Further simulations were conducted to investigate the effect of the combination at varying concentrations typically observed in patients. Regrowth was observed in TKCs at high concentrations of drugs alone [from 4 to 32× minimum inhibitory concentrations (MIC)], while the combination systematically prevented the regrowth at concentrations close to the MIC. Significant synergy or antagonism were observed under specific conditions but overall 95%CIs overlapped widely over time indicating an additive interaction between antibiotics. Moreover, simulations of typical PK profile at standard dosages indicated that the interaction should be additive in clinical conditions. The nature of the PD interaction varied with time and concentration in TKC. Against the four K. pneumoniae isolates, the bactericidal effect of CZA + CST combination was predicted to be additive and to prevent the emergence of resistance at clinical concentrations.

A Selective Culture Medium for Screening Cefiderocol Resistance in Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii.

Cefiderocol (FDC) is a siderophore cephalosporin with a broad spectrum of activity against many multidrug-resistant Gram-negative bacteria. Acquired resistance to FDC has been already reported among Gram-negative isolates, thus highlighting the need for rapid and accurate identification of such resistant pathogens, in order to control their spread. Therefore, the SuperFDC medium was developed to screen FDC-resistant Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. After testing several culture conditions, a selective medium was set up by supplementing an iron-depleted agar medium with 8 µg/mL of FDC and evaluated with a collection of 68 FDC-susceptible and 33 FDC-resistant Gram-negative isolates exhibiting a variety of ß-lactam resistance mechanisms. The sensitivity and specificity of detection of this medium were evaluated at 97% and 100%, respectively. In comparison with the reference broth microdilution method, only 3% very major errors were found. In addition, excellent detection performances were obtained by testing spiked stools with a lower limit of detection ranging between 100 and 103 CFU/mL. The SuperFDC medium allows detection of FDC-resistant Gram-negative isolates regardless of their corresponding resistance mechanisms.

Rapid Aztreonam/Avibactam NP test for detection of aztreonam/avibactam susceptibility/resistance in Enterobacterales.

Aztreonam-avibactam (AZA), a newly developed ß-lactam/ß-lactamase inhibitor combination, is a treatment option for infections due to carbapenem-resistant Enterobacterales (CRE), including metallo-ß-lactamase producers, regardless of additional production of broad-spectrum serine-ß-lactamases. However, AZA-resistance has already been reported in Enterobacterales and its early detection could be a valuable tool for faster and more accurate clinical decision-making. We therefore developed a rapid culture-based test for the identification of AZA resistance among multidrug-resistant Enterobacterales. The Rapid Aztreonam/Avibactam NP test is based on resazurin reduction when bacterial growth occurs in the presence of AZA at 8/4 µg/mL (protocol 1) or 12/4 µg/mL (protocol 2). Given the absence of guidelines on AZA susceptibility testing, two tentative breakpoints were indeed used to categorize AZA-susceptible isolates: ≤4 µg/mL in protocol 1 and ≤ 8 µg/mL in protocol 2. Bacterial growth was visually detectable by a blue-to-purple or blue-to-pink color change of the medium. A total of 78 enterobacterial isolates (among which 34 AZA-resistant and 13 AZA-resistant according to protocols 1 and 2, respectively) were used to evaluate the test performance using protocol 1 or protocol 2. The sensitivity and specificity of the test were found to be 100% and 97.7%, respectively, following protocol 1 and 100% and 100%, respectively, following protocol 2, in comparison with broth microdilution. All results were obtained within 4.5 hours corresponding to a time saving of ca. 14 hours compared with currently available methods for AZA susceptibility testing. The Rapid Aztreonam/Avibactam NP test is rapid, highly sensitive, specific, easily interpretable, and easy to implement in routine.

Evaluation of Phenotypic Tests to Detect Extended-Spectrum ß-Lactamase (ESBL)-Producing Klebsiella oxytoca Complex Strains.

Klebsiella oxytoca complex (KoC) species may overproduce their chromosomal class A OXY ß-lactamases, conferring reduced susceptibility to piperacillin-tazobactam, expanded-spectrum cephalosporins and aztreonam. Moreover, since clavulanate maintains its ability to inhibit these enzymes, the resulting resistance phenotype may falsely resemble the production of acquired extended-spectrum ß-lactamases (ESBLs). In this work, a collection of 44 KoC strains of human and animal origin was characterized with whole-genome sequencing (WGS) and broth microdilution (BMD) susceptibility testing. Comparison of ESBL producers (n = 11; including CTX-M-15 [n = 6] and CTX-M-1 [n = 5] producers) and hyperproducers of OXYs (n = 21) showed certain phenotypic differences: piperacillin-tazobactam (MIC90s: 16 versus >64 µg/mL), cefotaxime (MIC90s: 64 versus 4 µg/mL), ceftazidime (MIC90s: 32 versus 4 µg/mL), cefepime (MIC90s: 8 versus 4 µg/mL) and associated resistance to non-ß-lactams (e.g., trimethoprim-sulfamethoxazole: 90.9% versus 14.3%, respectively). However, a clear phenotype-based distinction between the two groups was difficult. Therefore, we evaluated 10 different inhibitor-based confirmatory tests to allow such categorization. All tests showed a sensitivity of 100%. However, only combination disk tests (CDTs) with cefepime/cefepime-clavulanate and ceftazidime/ceftazidime-clavulanate or the double-disk synergy test (DDST) showed high specificity (100%, 95.5%, and 100%, respectively). All confirmatory tests in BMD or using the MIC gradient strip did not perform well (specificity, ≤87.5%). Of note, ceftazidime/ceftazidime-avibactam tests also exhibited low specificity (CDT, 87.5%; MIC gradient strip, 77.8%). Our results indicate that standard antimicrobial susceptibility profiles can raise some suspicion, but only the use of cefepime/cefepime-clavulanate CDT or DDST can guarantee distinction between ESBL-producing KoC strains and those hyperproducing OXY enzymes.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

In vitro activity of cefepime/zidebactam and cefepime/taniborbactam against aztreonam/avibactam-resistant NDM-like-producing Escherichia coli clinical isolates.

BACKGROUND: Aztreonam/avibactam is one of the last therapeutic options for treating infections caused by NDM-like-producing Enterobacterales. However, PBP3-modified and NDM-producing Escherichia coli strains that co-produce CMY-42 have been shown to be resistant to this drug combination. The aim of our study was to assess the in vitro activity of cefepime/taniborbactam and cefepime/zidebactam against such aztreonam/avibactam-resistant E. coli strains. METHODS: MIC values of aztreonam, aztreonam/avibactam, cefepime, cefepime/taniborbactam, cefepime/zidebactam and zidebactam alone were determined for 28 clinical aztreonam/avibactam-resistant E. coli isolates. Those isolates produced either NDM-5 (n = 24), NDM-4 (n = 2) or NDM-1 (n = 2), and they all co-produced CMY-42 (n = 28). They all harboured a four amino acid insertion in PBP-3 (Tyr-Arg-Ile-Asn or Tyr-Arg-Ile-Lys). RESULTS: All strains were resistant to aztreonam/avibactam and cefepime, as expected. The resistance rate to cefepime/taniborbactam was 100%, with MIC50 and MIC90 being at 16 mg/L and 64 mg/L, respectively. Conversely, all strains were susceptible to cefepime/zidebactam, with both MIC50 and MIC90 at 0.25 mg/L. Notably, all strains showed low MICs for zidebactam alone, with MIC50 and MIC90 at 0.5 mg/L and 1 mg/L. CONCLUSIONS: Our data highlighted the excellent in vitro performance of the newly developed ß-lactam/ß-lactamase inhibitor combination cefepime/zidebactam against aztreonam/avibactam-resistant E. coli strains, suggesting that this combination could be considered as an efficient therapeutic option in this context. Our study also highlights the cross-resistance between acquired resistance to aztreonam/avibactam and the cefepime/taniborbactam combination.

Evaluation of in vitro pharmacodynamic drug interactions of ceftazidime/avibactam and fosfomycin in Escherichia coli.

BACKGROUND: Combination therapy can increase efficacy of antibiotics and prevent emergence of resistance. Ceftazidime/avibactam and fosfomycin may be empirically combined for this purpose, but a systematic and quantitative evaluation of this combination is needed. OBJECTIVES: In this study, a systematic analysis of the pharmacodynamic interactions of ceftazidime/avibactam and fosfomycin in clinical and isogenic Escherichia coli strains carrying genes coding for several carbapenemases or ESBLs was performed and pharmacodynamic interactions were quantified by modelling and simulations. METHODS: Pharmacodynamic interactions were evaluated in 'dynamic' chequerboard experiments with quantification of viable bacteria in eight isogenic and six clinical E. coli strains. Additionally, supplemental time-kill experiments were performed and genomic analyses were conducted on representative fosfomycin-resistant subpopulations. Models were fitted to all data using R and NONMEM®. RESULTS: Synergistic drug interactions were identified for 67% of the clinical and 75% of the isogenic isolates with a mean EC50 reduction of >50%. Time-kill experiments confirmed the interactions and modelling quantified EC50 reductions up to 97% in combination and synergy prevented regrowth of bacteria by enhanced killing effects. In 9 out of 12 fosfomycin-resistant mutants, genomic analyses identified previously reported mutations. CONCLUSIONS: The broad synergistic in vitro activity of ceftazidime/avibactam and fosfomycin confirms the potential of the application of this drug combination in clinics. The substantial reduction of the EC50 in combination may allow use of lower doses or treatment of organisms with higher MIC values and encourage further research translating these findings into the clinical setting.