Elevated MICs Research Articles

Cefepime-Taniborbactam: A Novel Cephalosporin/β-Lactamase Inhibitor Combination.

Taniborbactam (formerly known as VNRX-5133) is a novel bicyclic boronate β-lactamase inhibitor of serine β-lactamases (SBLs) [Ambler classes A, C, and D] and metallo-β-lactamases (MBLs) [Ambler class B], including NDM and VIM, but not IMP. Cefepime-taniborbactam is active in vitro against most isolates of carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA), including both carbapenemase-producing and carbapenemase-non-producing CRE and CRPA, as well as against multidrug-resistant (MDR), ceftazidime-avibactam-resistant, meropenem-vaborbactam-resistant, and ceftolozane-tazobactam-resistant Enterobacterales and P. aeruginosa. The addition of taniborbactam to cefepime resulted in a > 64-fold reduction in MIC90 compared with cefepime alone for a 2018-2021 global collection of > 13,000 clinical isolates of Enterobacterales. In the same study, against > 4600 P. aeruginosa, a fourfold MIC reduction was observed with cefepime-taniborbactam, compared with cefepime alone. Whole genome sequencing studies have shown that resistance towards cefepime-taniborbactam in Enterobacterales arises due to the presence of multiple resistance mechanisms, often in concert, including production of IMP, PBP3 alterations, permeability (porin) defects, and upregulation of efflux pumps. In P. aeruginosa, elevated cefepime-taniborbactam MICs are also associated with the presence of multiple, concurrent mechanisms, most frequently IMP, PBP3 mutations, and upregulation of efflux pumps, as well as AmpC (PDC) overexpression. The pharmacokinetics of taniborbactam are dose proportional, follow a linear model, and do not appear to be affected when combined with cefepime. Taniborbactam's approximate volume of distribution (Vd) at steady state is 20L and the approximate elimination half-life (t½) is 2.3 h, which are similar to cefepime. Furthermore, like cefepime, taniborbactam is primarily cleared renally, and clearance corresponds with renal function. Pharmacodynamic studies (in vitro and in vivo) have reported that cefepime-taniborbactam has bactericidal activity against various β-lactamase-producing Gram-negative bacilli that are not susceptible to cefepime alone. It has been reported that antimicrobial activity best correlated with taniborbactam exposure (area under the curve). A phase III clinical trial showed that cefepime-taniborbactam (2g/0.5g administered as an intravenous infusion over 2h) was superior to meropenem for the treatment of complicated urinary tract infection (cUTI), including acute pyelonephritis, caused by Enterobacterales species and P. aeruginosa while demonstrating similar safety compared with meropenem. The safety and tolerability of taniborbactam and cefepime-taniborbactam has been reported in one pharmacokinetic trial, and in two pharmacokinetic trials and one phase III clinical trial, respectively. Cefepime-taniborbactam appears to be well tolerated in both healthy subjects and patients. Headache and gastrointestinal upset are the most common drug-related adverse effects associated with cefepime-taniborbactam use. Cefepime-taniborbactam will likely have a role in the treatment of infections proven or suspected to be caused by MDR Gram-negative bacteria, including Enterobacterales and P. aeruginosa. In particular, it may be useful in the treatment of infections caused by isolates that harbor an MBL (NDM, VIM) enzyme, although further clinical data are needed. Additional safety and efficacy studies may support indications for cefepime-taniborbactam beyond cUTI.

Antimicrobial susceptibility testing reveals reduced susceptibility to azithromycin and other antibiotics in Legionella pneumophila serogroup 1 isolates from Portugal

BackgroudAlthough not fully investigated, studies show that Legionella pneumophila can develop antibiotic resistance. As there is limited data available for Portugal, we determined the antibiotic susceptibility profile of Portuguese L. pneumophila serogroup 1 (LpnSg1) isolates against antibiotics used in the clinical practice in Portugal.MethodsMinimum inhibitory concentrations (MICs) were determined for LpnSg1 clinical (n = 100) and related environmental (n = 7) isolates, collected between 2006–2022 in the context of the National Legionnaire´s Disease Surveillance Programme, against azithromycin, clarithromycin, erythromycin, levofloxacin, ciprofloxacin, moxifloxacin, rifampicin, doxycycline, tigecycline, and amoxicillin/clavulanic acid, using three different assays. Isolates were also PCR-screened for the presence of the lpeAB gene.ResultsTwelve isolates had azithromycin MICs above the EUCAST tentative highest WT MIC, 9 of which were lpeAB negative; for erythromycin and clarithromycin, all isolates tested within the susceptible range. The number of isolates with MICs above the tentative highest WT MIC for the remaining antibiotics was: ciprofloxacin: 7; levofloxacin: 17; moxifloxacin: 8; rifampicin: 11; doxycycline: 82; tigecycline: 4. EUCAST breakpoints are not available for amoxicillin/clavulanic acid. We estimated the ECOFFs and one isolate had a MIC eightfold higher than the E-test ECOFF. Additionally, a clinical isolate generated three colonies growing on the E-test inhibition zone that resulted in MICs fourfold higher than for the parental isolate.ConclusionsWe report, for the first time, elevated MICs against first-line and other antibiotics (including azithromycin, fluoroquinolones and amoxicillin/clavulanic acid commonly used to treat pneumonia patients in Portugal) in Portuguese L. pneumophila strains. Results point towards decreased susceptibility in circulating strains, justifying further investigation.

Staphylococcus aureus nasal colonization and susceptibility profile to antimicrobials in hemodialysis patients using a protocol of seven collections

BackgroundPatients colonized with Staphylococcus aureus in their nasal passages have a higher risk of acquiring infection, especially if they are immunocompromised or have comorbidities such as chronic renal failure undergoing hemodialysis (HD). ObjectiveThis study aimed to report the prevalence of nasal carriage of S. aureus among HD patients utilizing a seven-week sampling protocol and to assess the susceptibility of these isolates to various antimicrobial agents. MethodsOver seven consecutive weeks, nasal swab samples were collected from 47 HD patients, resulting in a total of 329 samples. The microorganisms were identified using biochemical methods and subjected to antimicrobial susceptibility testing via disk diffusion and microdilution techniques. ResultsOut of all the patients analyzed, 25 individuals (53.19%) were found to be colonized by S. aureus, with 21 of them displaying intermittent colonization. Additionally, 38% showed positive results for S. aureus in only the 6th or 7th week of sampling. Within the 58 isolates, 17.2% (n=10) exhibited methicillin (oxacillin)-resistance and 25.86% (n=15) displayed elevated vancomycin MIC values (2 µg/ml). Based on the results, daptomycin and gentamicin were found to be effective treatment options. However, 31% of the isolates (n=18) exhibited a MIC of 1 µg/ml for daptomycin. ConclusionOver half of the patients were colonized by S. aureus, but mostly on an intermittent basis. The identification of oxacillin resistance and high vancomycin and daptomycin MICs serve as warnings for possible future complications in managing bacteremia caused by S. aureus in these patients.

Macrolide resistance in Mannheimia haemolytica isolates associated with bovine respiratory disease from the German national resistance monitoring program GERM-Vet 2009 to 2020.

Data collected from the German national resistance monitoring program GERM-Vet showed slowly increasing prevalence of macrolide resistance among bovine respiratory disease (BRD)-associated Pasteurellacae from cattle over the last decade. The focus of this study was to analyze the genetic basis of antimicrobial resistance (AMR) and the prevalence of multidrug-resistance (MDR)-mediating integrative and conjugative elements (ICEs) in 13 German BRD-associated Mannheimia haemolytica isolates collected between 2009 and 2020 via whole-genome sequencing. Antimicrobial susceptibility testing (AST) was performed via broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute for the macrolides erythromycin, tilmicosin, tulathromycin, gamithromycin, tildipirosin, and tylosin as well as 25 other antimicrobial agents. All isolates either had elevated MICs or were resistant to at least one of the macrolides tested. Analysis of whole-genome sequences obtained by hybrid assembly of Illumina MiSeq and Oxford Nanopore MinION reads revealed the presence of seven novel Tn7406-like ICEs, designated Tn7694, and Tn7724- Tn7729. These ICEs harbored the antimicrobial resistance genes erm(T), mef (C), mph(G), floR, catA3, aad(3")(9), aph(3')-Ia, aac(3)-IIa, strA, strB, tet(Y), and sul2 in different combinations. In addition, mutational changes conferring resistance to macrolides, nalidixic acid or streptomycin, respectively, were detected among the M. haemolytica isolates. In addition, four isolates carried a 4,613-bp plasmid with the β-lactamase gene blaROB - 1. The detection of the macrolide resistance genes erm(T), mef (C), and mph(G) together with other resistance genes on MDR-mediating ICEs in bovine M. haemolytica may explain the occurrence of therapeutic failure when treating BRD with regularly used antimicrobial agents, such as phenicols, penicillins, tetracyclines, or macrolides. Finally, pathogen identification and subsequent AST is essential to ensure the efficacy of the antimicrobial agents applied to control BRD in cattle.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

P28 Evaluation of one in-house and three commercial phenotypic tests for detection of KPC, NDM and OXA-48-like carbapenemase in Klebsiella pneumoniae: is routine detection possible?

Abstract Background Phenotypic characterization of the OXA-48-like carbapenemase XDR and pandrug-resistant (PDR) Klebsiella pneumoniae in clinical isolates is essential for making informed empirical decisions and critical for strengthening antimicrobial stewardship programmes. This study focused on assessing one in-house and three commercial phenotypic tests for detection of KPC, NDM and OXA-48-like carbapenemases in an attempt to study the feasibility of making detection of OXA-48-like carbapenemases a routine activity of a diagnostic microbiology laboratory. Methods A total of 40 representative non-duplicate XDR and PDR clinical Klebsiella pneumoniae isolates were screened on the basis of susceptibility profile, resistance to piperacillin/tazobactam; susceptible, intermediate, or resistant to third generation cephalosporins; elevated MIC for meropenem. These isolates were then subjected to one in-house test (OXA-48 disc test) and three commercial tests: Xpert® Carba-R (Cepheid), MASTDISCS® Combi Carba Plus (D73C), Immunochromatographic assay (ICT); KPC/IMP/NDM/OXA-48 Combo test (LFIA), Medomics. Results Xpert ®Carba-R identified OXA-48 in 62.5% (n=25) isolates, NDM in 17.5% (n=7), KPC+NDM co-carriage in 15% (n=6) and KPC in one isolate. Both Xpert® Carba-R and ICT were considered as possible gold standards and the outcomes of the other tests were compared with these two. With Xpert® Carba-R as the gold standard, high sensitivity of 96.15% was detected in OXA-48-like isolates, followed by NDM (92.85%) and NDM+KPC (85.7%). The lowest sensitivity was seen with the OXA-48 disc test (48% in OXA-48, 53.8% in NDM) followed by D73C test (64% in OXA-48, 38.46% in NDM). These two tests could not detect co- carriage of NDM and KPC in six isolates, although in two cases one of the two was detected. Specificity was high among all the tests (96.29%–93.33%). With ICT as the gold standard, 100% sensitivity was reported for NDM and KPC isolates, followed by OXA-48 and KPC+NDM (96% and 83.33%, respectively). The specificity was higher with ICT (99%) than Xpert® Carba-R (96.29%) as in detection of co- carriage of NDM and KPC. In terms of cost ICT ($16 per test) was much cheaper than Xpert® Carba-R($52 per test). Conclusions We recommend immunochromatographic assay (ICT) as an excellent tool for detection of carbapenemases (KPC/IMP/NDM/OXA-48). OXA-48 disc test and MASTDISCS® Combi Carba plus (D73C) cannot be considered as good standard since they could not detect co-carriage of NDM and KPC and had lower sensitivity.

7-Year (2015-21) longitudinal surveillance of lefamulin in vitro activity against bacterial pathogens collected worldwide from patients with respiratory tract infections including pneumonia and characterization of resistance mechanisms.

Lefamulin (Xenleta™), a pleuromutilin antibiotic, was approved for the oral and IV treatment of community-acquired bacterial pneumonia (CABP) in adults in 2019/2020. This study evaluated the in vitro activity of lefamulin and comparators against 19 584 unique bacterial isolates collected from patients with community-acquired respiratory tract infections and hospitalized patients with pneumonia within the global SENTRY Antimicrobial Surveillance Program during 2015-21. Isolates were susceptibility tested by the CLSI broth microdilution method, and resistance mechanisms were investigated in isolates with elevated lefamulin MICs. Lefamulin exhibited potent antibacterial activity against the most common and typical CABP pathogens tested, including Streptococcus pneumoniae [MIC50/90, 0.06/0.25 mg/L; 99.9% susceptible (S)], Staphylococcus aureus (MIC50/90, 0.06/0.12 mg/L; 99.6% S), Haemophilus influenzae (MIC50/90, 0.5/2 mg/L; 99.1% S) and Moraxella catarrhalis (MIC50/90, 0.06/0.12 mg/L; 100.0% S). Potent activity was also observed against the less common pneumonia pathogens: β-haemolytic (MIC50/90 of 0.03/0.06 mg/L) and viridans group Streptococcus spp. (MIC50/90 of 0.06/0.25 mg/L) and Haemophilus parainfluenzae (MIC50/90 of 1/4 mg/L). Lefamulin's activity was not adversely affected by resistance to macrolides, penicillin, tetracyclines, fluoroquinolones and other resistance phenotypes. Non-susceptibility/resistance to lefamulin was rare and primarily determined by ribosomal protection through vga(A) variants in S. aureus, overexpression of AcrAB-TolC efflux pump in H. influenzae or modifications in L3, L4 and 23SrRNA in Streptococcus spp. Based on the coverage of the most important CABP pathogens and lacking cross-resistance, lefamulin may represent a valuable empirical treatment option for ambulatory and hospitalized patients with CABP, particularly in settings with high prevalence of resistance.

Deciphering mechanisms affecting cefepime-taniborbactam in vitro activity in carbapenemase-producing Enterobacterales and carbapenem-resistant Pseudomonas spp. isolates recovered during a surveillance study in Spain.

To characterize the resistance mechanisms affecting the cefepime-taniborbactam combination in a collection of carbapenemase-producing Enterobacterales (CPE) and carbapenem-resistant Pseudomonas spp. (predominantly P. aeruginosa; CRPA) clinical isolates. CPE (n = 247) and CRPA (n = 170) isolates were prospectively collected from patients admitted to 8 Spanish hospitals. Susceptibility to cefepime-taniborbactam and comparators was determined by broth microdilution. Cefepime-taniborbactam was the most active agent, inhibiting 97.6% of CPE and 67.1% of CRPA (MICs ≤ 8/4 mg/L). All isolates with cefepime-taniborbactam MIC > 8/4 mg/L (5 CPE and 52 CRPA) and a subset with MIC ≤ 8/4 mg/L (23 CPE and 24 CRPA) were characterized by whole genome sequencing. A reduced cefepime-taniborbactam activity was found in two KPC-ST307-Klebsiella pneumoniae isolates with altered porins [KPC-62-K. pneumoniae (OmpA, OmpR/EnvZ), KPC-150-K. pneumoniae (OmpK35, OmpK36)] and one each ST133-VIM-1-Enterobacter hormaechei with altered OmpD, OmpR, and OmpC; IMP-8-ST24-Enterobacter asburiae; and NDM-5-Escherichia coli with an YRIN-inserted PBP3 and a mutated PBP2. Among the P. aeruginosa (68/76), elevated cefepime-taniborbactam MICs were mostly associated with GES-5-ST235, OXA-2+VIM-2-ST235, and OXA-2+VIM-20-ST175 isolates also carrying mutations in PBP3, efflux pump (mexR, mexZ) and AmpC (mpl) regulators, and non-carbapenemase-ST175 isolates with AmpD-T139M and PBP3-R504C mutations. Overall, accumulation of these mutations was frequently detected among non-carbapenemase producers. The reduced cefepime-taniborbactam activity among the minority of isolates with elevated cefepime-taniborbactam MICs is not only due to IMP carbapenemases but also to the accumulation of multiple resistance mechanisms, including PBP and porin mutations in CPE and chromosomal mutations leading to efflux pumps up-regulation, AmpC overexpression, and PBP modifications in P. aeruginosa.

Bedaquiline, Delamanid, Linezolid, Clofazimine, and Capreomycin MIC Distributions for Drug Resistance Mycobacterium tuberculosis in Shanghai, China.

New antituberculosis drugs have recently been approved for the treatment of multidrug-resistant tuberculosis TB (MDR-TB). We aimed to describe the distributions of bedaquiline, delamanid, linezolid, clofazimine, and capreomycin MIC values for M. tuberculosis. M. tuberculosis clinical isolates were originally isolated from 2020 to 2021 from 1452 different pulmonary tuberculosis patients of the Shanghai Pulmonary Hospital in China. The drug susceptibility testing was performed using the Sensititre custom plates (SHTBMY) (TREK Diagnostic Systems, Thermo Fisher Scientific In., USA) consisting of a 96-well microtitre plate containing 4 (bedaquiline, delamanid, clofazimine, capreomycin) antimicrobial agents. MICs were determined for linezolid using a microdilution method. Based on the latest definitions, 156 (10.74%) were MDR-TB, 93 (6.40%) were pre-XDR-TB, and 27 (1.86%) were XDR-TB. The rate of BDQ resistance in cases of MDR-TB was 7.69%, while it was observed to be 10.75% in cases of pre-XDR-TB, and significantly higher at 37.04% in cases of XDR-TB. The lowest rate of drug resistance against M. tuberculosis was DLM (0.14%). For LZD, 11 (0.76%) clinical isolates were resistant, based on the CLSI breakpoint of 1μg/mL. The five strains with a MIC value of >32 for LZD resistance were XDR-TB isolates. Among all MDR, pre-XDR, and XDR isolates tested, LZD' MIC50 increased from 0.25 and 0.5 to 1μg/mL. The MIC90 value of LZD against XDR-TB isolates was 32μg/mL. For CFZ, six isolates with elevated MICs of ≥2μg/mL. CFZ's MIC50 and MIC90 values in all isolates were 0.12μg/mL and 0.25μg/mL, respectively. The study findings indicate that BDQ, DLM, CFZ, and LZD may exhibited excellent in vitro activity against MDR-TB isolates. Detection of resistance to BDQ and LZD was alarming for XDR-TB isolates. It is necessary to perform universal drug sensitivity testing for M. tuberculosis, especially MDR-TB and XDR-TB patients.

Evaluation of a Method for Standardized Antimicrobial Susceptibility Testing with Mycoplasma hyorhinis Field Isolates.

Organizations like the Clinical and Laboratory Standards Institute (CLSI) or the European Committee of Antimicrobial Susceptibility Testing (EUCAST) provide standardized methodologies for antimicrobial susceptibility testing of a wide range of nonfastidious and fastidious bacteria, but so far not for Mycoplasma spp. of animal origin. Recently, a proposed method for the standardized broth microdilution testing of Mycoplasma hyorhinis using commercial Sensititre microtiter plates was presented. In this study, we evaluated this broth microdilution method with 37 field isolates and tested their susceptibility toward the following antimicrobial agents: doxycycline, enrofloxacin, erythromycin, florfenicol, gentamicin, marbofloxacin, tetracycline, tiamulin, tilmicosin, tulathromycin, and tylosin. The isolates originated from different countries, isolation sites, and years. The broth microdilution method was carried out using a modified Friis broth as the culture and test medium. For macrolides and lincosamides, a bimodal distribution with elevated MIC values could be observed for almost half of the tested field isolates, deducing reduced susceptibility toward these substances. With a recently published protocol, we were able to test a variety of field isolates, and consistent data could be obtained. Using this method, monitoring studies of Mycoplasma hyorhinis isolates can be carried out in a comparable manner, and the observed susceptibility profiles can be screened for possible changes in MIC values in the future.

Zinc effects on bacteria: insights from Escherichia coli by multi-omics approach.

A long-term exposure of bacteria to zinc oxide and zinc oxide nanoparticles leads to major alterations in bacterial morphology and physiology. These included biochemical and physiological processes promoting the emergence of strains with multi-drug resistance and virulence traits. After the removal of zinc pressure, bacterial phenotype reversed back to the original state; however, certain changes at the genomic, transcriptomic, and proteomic level remained. Why is this important? The extensive and intensive use of supplements in animal feed effects the intestinal microbiota of livestock and this may negatively impact the health of animals and people. Therefore, it is crucial to understand and monitor the impact of feed supplements on intestinal microorganisms in order to adequately assess and prevent potential health risks.

In vitro synergy of the combination of sulbactam-durlobactam and cefepime at clinically relevant concentrations against A. baumannii, P. aeruginosa and Enterobacterales.

Sulbactam-durlobactam is a potent combination active against Acinetobacter baumannii; however, it lacks activity against other nosocomial pathogens. Cefepime is a common first-line therapy for hospital/ventilator-associated pneumonia caused by Gram-negative pathogens including Pseudomonas aeruginosa and Enterobacterales. With increasing resistance to cefepime, and the significant proportion of polymicrobial nosocomial infections, effective therapy for infections caused by Acinetobacter baumannii, P. aeruginosa and Enterobacterales is needed. This study investigated the in vitro synergy of sulbactam-durlobactam plus cefepime against relevant pathogens. Static time-kills assays were performed in duplicate against 14 cefepime-resistant isolates (A. baumannii, n = 4; P. aeruginosa, n = 4; Escherichia coli, n = 3; Klebsiella pneumoniae, n = 3). One WT K. pneumoniae isolate was included. Antibiotic concentrations simulated the free-steady state average concentration of clinically administered doses in patients. Sulbactam-durlobactam alone showed significant activity against A. baumannii consistent with the MIC values. Sulbactam-durlobactam plus cefepime showed synergy against one A. baumannii isolate with an elevated MIC to sulbactam-durlobactam (32 mg/L). Against all P. aeruginosa isolates, synergy was observed with sulbactam-durlobactam plus cefepime. For the Enterobacterales, one E. coli isolate demonstrated synergy while the others were indifferent due to significant kill from sulbactam-durlobactam alone. The combination of sulbactam-durlobactam plus cefepime showed synergy against one of the K. pneumoniae and additive effects against the other two K. pneumoniae tested. No antagonism was observed in any isolates including the WT strain. Synergy and no antagonism was observed with a combination of sulbactam-durlobactam and cefepime; further in vivo pharmacokinetic/pharmacodynamics data and clinical correlation are necessary to support our findings.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

In vitro activity of new combinations of β-lactam and β-lactamase inhibitors against the Mycobacterium tuberculosis complex.

As meropenem-clavulanic acid is recommended for the treatment of drug-resistant tuberculosis, the repurposing of new carbapenem combinations may provide new treatment options, including oral alternatives. Therefore, we studied the in vitro activities of meropenem-vaborbactam, meropenem-clavulanic acid, and tebipenem-clavulanic acid. One hundred nine Mycobacterium tuberculosis complex (MTBC) clinical isolates were tested, of which 69 were pan-susceptible and the remaining pyrazinamide- or multidrug-resistant. Broth microdilution MICs were determined using the EUCAST reference method. Meropenem and tebipenem were tested individually and in combination with vaborbactam 8 mg/L and clavulanic-acid 2 and 4 mg/L, respectively. Whole-genome sequencing was performed to explore resistance mechanisms. Clavulanic acid lowered the modal tebipenem MIC approximately 16-fold (from 16 to 1 mg/L). The modal meropenem MIC was reduced twofold by vaborbactam compared with an approximately eightfold decrease by clavulanic acid. The only previously described high-confidence carbapenem resistance mutation, crfA T62A, was shared by a subgroup of lineage 4.3.4.1 isolates and did not correlate with elevated MICs. The presence of a β-lactamase inhibitor reduced the MTBC MICs of tebipenem and meropenem. The resulting MIC distribution was lowest for the orally available drugs tebipenem-clavulanic acid. Whether this in vitro activity translates to similar or greater clinical efficacy of tebipenem-clavulanic acid compared with the currently WHO-endorsed meropenem-clavulanic acid requires clinical studies. IMPORTANCE Repurposing of already approved antibiotics, such as β-lactams in combination with β-lactamase inhibitors, may provide new treatment alternatives for drug-resistant tuberculosis. Meropenem-clavulanic acid was more active in vitro compared to meropenem-vaborbactam. Notably, tebipenem-clavulanic acid showed even better activity, raising the potential of an all-oral treatment option. Clinical data are needed to investigate whether the better in vitro activity of tebipenem-clavulanic acid correlates with greater clinical efficacy compared with the currently WHO-endorsed meropenem-clavulanic acid.

Toward a Method for Harmonized Susceptibility Testing of Mycoplasma bovis by Broth Microdilution.

Mycoplasma bovis is a fastidious pathogen of cattle causing massive economic losses in the calf and dairy industries worldwide. Since there is no approved standard method for antimicrobial susceptibility testing (AST) of M. bovis, the Clinical and Laboratory Standards Institute has requested the development of a suitable method. Therefore, this study aimed at developing a method for harmonized broth microdilution AST of M. bovis. For this, 131 M. bovis field isolates and M. bovis strain DSM 22781T were collected and macrorestriction analysis was performed to select 15 epidemiologically unrelated M. bovis strains for method validation steps. To select a suitable broth for AST of M. bovis, growth determinations were performed using five media and growth curves were compiled. Then, susceptibility testing was performed considering the exact (precondition of five identical MICs) and essential (MIC mode, accepting a deviation of ±1 dilution step) MIC agreements to evaluate the reproducibility of MIC values using a panel of 16 antimicrobial agents. Subsequently, the remaining field isolates were tested and the suitability of quality control (QC) strains was assessed. Growth experiments showed that SP4 broth was the only one of the five media that yielded sufficient growth of M. bovis. Therefore, it was selected as the test medium for AST and homogeneous MIC values were obtained (exact and essential agreements of 36 to 100% and 92 to 100%, respectively). For all other isolates tested, easy-to-read MIC endpoints were determined with this medium. High overall MIC50 and/or MIC90 values were observed for aminoglycosides and macrolides, and some isolates showed elevated MICs of fluoroquinolones, gentamicin, and/or tiamulin. Since the MICs of four commonly used QC strains were partially not within their ranges, a 20-fold MIC testing of M. bovis DSM 22781T was performed and met the criteria for a new QC strain. For harmonized AST of M. bovis, SP4 broth seems to be suitable with an incubation time of 72 ± 2 h and further validation of M. bovis DSM 22781T as a future QC strain is recommended.

In Vitro Activity of Cefiderocol against a Global Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates.

Background: Cefiderocol is a novel siderophore cephalosporin with potent activity against multi-drug-resistant Gram-negative pathogens including carbapenem-resistant Acinetobacter baumannii (CRAB). Methods: The susceptibility of 313 non-duplicate CRAB isolates with defined carbapenem resistance mechanisms from a global collection to cefiderocol, ceftazidime, ceftazidime/avibactam, ceftolozane/tazobactam, ciprofloxacin, colistin, imipenem/relebactam, meropenem, meropenem/vaborbactam, minocycline, and piperacillin/tazobactam was determined using the broth microdilution method. Isolates were obtained from various body sites from patients in 47 countries in five world regions between 2012 and 2016. The identification of carbapenem resistance mechanisms and assignment to A. baumannii international clonal lineages were based on whole genome sequencing. Results: Cefiderocol showed greater activity than comparator antimicrobials of the β-lactam class, including novel β-lactams combined with β-lactamase inhibitors, ciprofloxacin, and minocycline. Cefiderocol MIC50 and MIC90 values were 0.5 mg/L and 4 mg/L, respectively, while colistin had comparable activity with a higher MIC50 at 1 mg/L and a lower MIC90 value of 2 mg/L. Many isolates with elevated cefiderocol MICs ≥ 4 mg/L represented A. baumannii international clone (IC) 1 and harbored a metallo-β-lactamase. Conclusions: While cefiderocol is a useful addition to the limited armamentarium of drugs targeting this problematic pathogen, a considerable part of CRAB isolates had elevated MIC values in a range of 4 -> 32 mg/L, including all isolates with a metallo-β-lactamase.

Susceptibility patterns of amphotericin B, itraconazole, posaconazole, voriconazole and caspofungin for isolates causing invasive mould infections from the SENTRY Antifungal Surveillance Program (2018-2021) and application of single-site epidemiological cutoff values to evaluate amphotericin B activity.

We evaluated the activity of amphotericin B, itraconazole, posaconazole, voriconazole and caspofungin against 1468 invasive moulds collected worldwide from 2018 to 2021. Most (>92%) of the Aspergillus spp. isolates were wildtype (WT) to amphotericin B, caspofungin and the azoles. Azole-non-wildtype A. fumigatus rates were higher in Europe (9.5%) and North America (9.1%) than Latin America (0.0%; only 12 isolates) and the Asia-Pacific region (5.3%). Amphotericin B and caspofungin were active against azole-non-wildtype A. fumigatus isolates. Posaconazole and amphotericin B were the most active agents against the Mucorales. Among the less common moulds, several expressed a pan-azole-resistant phenotype; many of these species also showed elevated MIC values (MIC, >2 mg/L) for amphotericin B and caspofungin. Although most isolates of Aspergillus spp. remain WT to the azoles, azole resistance is increasing in both North America and Europe. Amphotericin B and caspofungin exhibit potentially useful activity against azole-resistant A. fumigatus.

Antimicrobial susceptibility of western Canadian Brachyspira isolates: Development and standardization of an agar dilution susceptibility test method.

The re-emergence of Brachyspira-associated disease in pigs since the late 2000s has illuminated some of the diagnostic challenges associated with this genus; notably, the lack of standardized antimicrobial susceptibility testing (AST) methods and interpretive criteria. Consequently, laboratories have relied heavily on highly variable in-house developed methods. There are currently no published investigations describing the antimicrobial susceptibility of Brachyspira isolates collected from pigs in Canada. The first objective of this study was therefore to develop a standardized protocol for conducting agar dilution susceptibility testing of Brachyspira spp., including determining the optimal standardized inoculum density, a key test variable that impacts test performance. The second objective was to determine the susceptibility of a collection of western Canadian Brachyspira isolates using the standardized methodology. After assessing multiple media, an agar dilution test was standardized in terms of starting inoculum (1-2 × 108 CFU/ml), incubation temperature and time, and assessed for repeatability. The antimicrobial susceptibility of a collection of clinical porcine Brachyspira isolates (n = 87) collected between 2009-2016 was then determined. This method was highly reproducible; repeat susceptibility testing yielded identical results 92% of the time. Although most of the isolates had very low MICs to the commonly used antimicrobials to treat Brachyspira-associated infections, several isolates with elevated MICs (>32 μg/ml) for tiamulin, valnemulin, tylosin, tylvalosin, and lincomycin were identified. Overall, this study underscores the importance of establishing CLSI approved clinical breakpoints for Brachyspira to facilitate the interpretation of test results and support the evidence-based selection of antimicrobials in swine industry.

Antioxidant-Mediated Modulation of Bacterial Antibiotic Susceptibility

Background: Fluoroquinolones like Ciprofloxacin are broad-spectrum antibiotics. They act on targeted bacteria by interfering with DNA replication, leading to the generation of free radicals which ultimately contribute to bacterial death. Conversely, dietary antioxidant supplements are commonly prescribed during the course of infection treatment. Co-administration of a multivitamin preparation alongside fluoroquinolones might play a role in fluoroquinolone treatment failure. Objectives: Thus, the present study aimed to investigate the effect of antioxidants like vitamin E, vitamin C, and vitamin B12 on the action of ciprofloxacin against various standard bacterial strains and ciprofloxacin-sensitive clinical isolates. Methods: The antibacterial activity of ciprofloxacin in the absence and presence of these antioxidants was assessed using the Kirby-Bauer disc diffusion method and by determining the minimum inhibitory concentration. Results: All strains tested in the study demonstrated sensitivity to ciprofloxacin alone. However, when vitamin C, E, or B12 was incorporated into the test medium, all strains exhibited significantly smaller zones of inhibition and elevated MIC ranges compared to ciprofloxacin alone. Conclusion: The generation of reactive oxygen species during the antibacterial action of ciprofloxacin on bacterial cells was either diminished or attenuated after supplementing the media with vitamin C, E, or B12. These antioxidants acted as free radical scavengers, protecting bacterial cells from the effects of generated free radicals. As the presence of antioxidants in the environment modulates the action of ciprofloxacin, the ingestion of these antioxidants should be closely monitored during the treatment course.

Assessing the in vivo efficacy of rational antibiotics and combinations against difficult-to-treat Pseudomonas aeruginosa producing GES β-lactamases.

We evaluated the in vivo efficacy of human-simulated regimens (HSRs) of cefiderocol, ceftazidime/avibactam, meropenem and ceftazidime/avibactam/meropenem combination against Guiana-extended spectrum (GES)-producing Pseudomonas aeruginosa isolates. Eighteen P. aeruginosa isolates producing GES-1 (n = 5), GES-5 (n = 5) or miscellaneous GESs (combinations of GES-19, GES-20 and/or GES-26; n = 8) were evaluated. In vitro MIC testing was determined using broth microdilution. In a validated murine thigh infection model, HSRs of cefiderocol 2 g q8h as a 3 h IV infusion, ceftazidime/avibactam 2.5 g q8h as a 2 h IV infusion, meropenem 2 g q8h as a 3 h IV infusion or ceftazidime/avibactam/meropenem were administered. Change in bacterial burden relative to baseline and the proportion of isolates in each genotypic group meeting 1-log10 kill endpoint were assessed. Modal MICs (mg/L) ranged from 0.125 to 1 for cefiderocol, 4 to >64 for ceftazidime/avibactam and 2 to >64 for meropenem. Cefiderocol produced >1-log10 of kill against all 18 tested isolates. Meropenem was active against all GES-1 isolates whereas activity against GES-5 and miscellaneous GESs was lacking, consistent with the MICs. Ceftazidime/avibactam was active against all GES-1 and GES-5 isolates and retained activity against 62.5% of miscellaneous GESs including isolates with elevated MICs. For isolates where ceftazidime/avibactam failed, the addition of meropenem restored the in vivo efficacy. As monotherapy, cefiderocol was active in vivo against all tested isolates. The activities of meropenem or ceftazidime/avibactam alone were variable; however, a combination of both was active against all isolates. Cefiderocol and ceftazidime/avibactam/meropenem could be valuable therapeutic options for GES-producing P. aeruginosa infections. Clinical confirmatory data are warranted.