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.

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.

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.

Resist Acineto rapid immunological test for the detection of acquired carbapenemase producers among Acinetobacter spp.

The Resist Acineto from Coris Bioconcept is a novel immunochromatographic test for detection of the major acquired carbapenemases (OXA-23, OXA-40, OXA-58, and NDM) identified in Acinetobacter spp. This rapid and easy-to-perform test showed an excellent specificity and sensitivity, with positive and negatives predictive values of 100% in both cases.

Dissemination of ArmA- and OXA-23-co-producing Acinetobacter baumannii Global Clone 2 in Switzerland, 2020-2021.

Following the observation of an increased number of isolation of OXA-23- and ArmA-producing Acinetobacter baumannii at the national level, our aim was to evaluate whether some given clone(s) might actually be spreading and/or emerging in Switzerland. To evaluate this possibility, our study investigated and characterized all A. baumannii isolates harboring both the blaOXA-23 and armA genes that had been collected at the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) from 2020 to 2021. Most isolates were obtained from infections rather than colonization with the majority being obtained from respiratory specimens. Pulsed-field gel electrophoresis (PFGE) analysis of 56 isolates identified nine profiles. Then, whole-genome sequencing that was performed on a subset of 11 isolates including at least one representative isolate of each PFGE profile identified three STs; one each of ST25 and ST1902, and nine ST2 (a member of Global Clone 2 (GC-2). The blaOXA-23 gene was always found embedded within Tn2006 structures, as commonly described with GC-2 (ST2) isolates. Susceptibility testing showed that most of those isolates, despite being highly resistant to all carbapenems and all aminoglycosides, remained susceptible to colistin (94.6%), sulbactam-durlobactam (87.5%), and cefiderocol (83.9% or 91.1% according to EUCAST or CLSI breakpoints, respectively). Overall, this study identified that the A. baumannii co-producing OXA-23 and ArmA are increasing in incidence in Switzerland, largely due to the dissemination of the high-risk GC-2. This highlights the importance of the monitoring of such MDR A. baumannii strains, in order to contribute to reduce their potential further spread.

Multifactorial resistance mechanisms associated with resistance to ceftazidime-avibactam in clinical Pseudomonas aeruginosa isolates from Switzerland.

Background: Increasing reports of multidrug resistance (MDR) in clinical Pseudomonas aeruginosa have led to a necessity for new antimicrobials. Ceftazidime-avibactam (CZA) is indicated for use against MDR P. aeruginosa across a broad range of infection types and particularly those that are carbapenem resistant. This study sought to determine the molecular mechanisms of CZA and imipenem (IPM)-resistance in clinical P. aeruginosa isolates obtained from Swiss hospitals. Methods: Clinical P. aeruginosa isolates were obtained from inpatients in three hospitals in Switzerland. Susceptibility was determined by either antibiotic disc testing or broth microdilution according to EUCAST methodology. AmpC activity was determined using cloxacillin and efflux activity was determined using phenylalanine-arginine ß-naphthylamide, in agar plates. Whole Genome Sequencing was performed on 18 clinical isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. Genes of interest were extracted from sequenced isolates and compared to reference strain P. aeruginosa PAO1. Results: Sixteen different STs were identified amongst the 18 isolates in this study indicating a high degree of genomic diversity. No carbapenemases were detected but one isolate did harbor the ESBL bla PER-1. Eight isolates were CZA-resistant with MICs ranging from 16-64 mg/L, and the remaining ten isolates had either low/wildtype MICs (n=6; 1-2 mg/L) or elevated, but still susceptible, MICs (n=4; 4-8 mg/L). Ten isolates were IPM-resistant, seven of which had mutations resulting in truncations of OprD, and the remaining nine IPM-susceptible isolates had intact oprD genes. Within CZA-R isolates, and those with reduced susceptibility, mutations resulting in ampC derepression, OprD loss, mexAB overexpression and ESBL (bla PER-1) carriage were observed in various combinations and one harbored a truncation of the PBP4 dacB gene. Within the six isolates with wildtype-resistance levels, five had no mutations that would affect any antimicrobial resistance (AMR) genes of interest when compared to PAO1. Conclusion: This preliminary study highlights that CZA-resistance in P. aeruginosa is multifactorial and could be caused by the interplay between different resistance mechanisms including ESBL carriage, increased efflux, loss of permeability and derepression of its intrinsic ampC.

Resistance to ceftazidime-avibactam in a KPC-2-producing Klebsiella pneumoniae caused by the extended-spectrum beta-lactamase VEB-25.

Carbapenem-resistant Enterobacterales, including KPC-producing Klebsiella pneumoniae, represent a major threat to public health due to their rapid spread. The beta-lactam/beta-lactamase inhibitor (BL/BLI) combination ceftazidime-avibactam (CAZ-AVI) has recently been introduced and shown to exhibit excellent activity toward multidrug-resistant KPC-producing Enterobacterales strains. However, CAZ-AVI-resistant K. pneumoniae isolates are being increasingly reported, mostly corresponding to producers of KPC variants that confer resistance to CAZ-AVI but at a cost of carbapenem resistance. We have characterized here, both phenotypically and genotypically, a clinical CAZ-AVI- and carbapenem-resistant KPC-2 K. pneumoniae isolate co-producing the inhibitor-resistant extended-spectrum beta-lactamase VEB-25.

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.

In vitro-obtained meropenem-vaborbactam resistance mechanisms among clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae isolates.

OBJECTIVES: A novel ß-lactam-ß-lactamase inhibitor (BLBI), meropenem (MEM), combined with the boronate-based inhibitor vaborbactam (VAB), has recently been introduced for the treatment of infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. The purpose of this study was to select for MEM-VAB resistance using a collection of eight KPC-producing K. pneumoniae clinical isolates, including three that produce KPC variants conferring ceftazidime-avibactam (CAZ-AVI) resistance, and subsequently decipher the corresponding resistance mechanisms. METHODS: Mutants were selected in a stepwise process on agar plates containing different MEM-VAB concentrations. Susceptibility testing was performed by broth microdilution, and complementation assays were performed with wildtype ompK36. Whole genome sequencing was performed on mutants, and KPC copy number was assessed by quantitative polymerase chain reaction . RESULTS: Mutants were obtained from 6/8 tested isolates and reduced susceptibility to all tested ß-lactams, and BLBIs, including CAZ-AVI, imipenem-relebactam, and aztreonam-AVI, were observed. No mutations were identified in the blaKPC. However, mutations in ompK36 were observed in four mutant lineages, and complementation with a wild-type ompK36 resulted in a reduction of minimal inhibitory concentrations to both MEM-VAB and other ß-lactams/BLBIs. blaKPC gene copy numbers were significantly increased in four mutant lineages. Whole genome sequencing identified genomic rearrangements in two lineages comprising mutations in the plasmid replicon encoding gene and duplication of the Tn4401 transposon bearing the blaKPC gene into a ColE-like, high copy number plasmid. CONCLUSIONS: In contrast to what is observed with KPC-producing mutants exhibiting resistance to CAZ-AVI, mainly corresponding to mutated KPC enzymes, here the MEM-VAB-resistant mutants showed permeability defects combined with increased KPC production, resulting from genomic rearrangement.

In Vitro Mechanisms of Resistance Development to Imipenem-Relebactam in KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales, such as KPC-producing Klebsiella pneumoniae, represent a major threat to public health. Novel drug combinations including imipenem-relebactam (IPM-REL) have recently been introduced and have been shown to exhibit excellent activity toward such strains. However, there has recently been reports of the in vivo emergence of IPM-REL resistance in KPC-producing K. pneumoniae. Here, we evaluated, in vitro, the nature of the mutations that lead to IPM-REL resistance in 5 KPC-producing K. pneumoniae strains, including 2 that produce KPC enzymes conferring ceftazidime-avibactam resistance. An in vitro multi-step selection assay was performed and corresponding mutants obtained. Mutations were identified in OmpK36 as well as 2 different mutant derivatives of KPC. Mutant strains exhibited decreased susceptibility to ß-lactams, including the carbapenems, and meropenem-vaborbactam (MEM-VAB). Expression of blaKPC gene variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to carbapenems and decreased susceptibility to CAZ-AVI, and enzymatic assays showed that the inhibitory activity of both AVI and REL was significantly lowered for both KPC mutants compared to parental enzymes. Complementation assays showed that OmpK36 plays a major role in IPM-REL resistance as well resistance to other ß-lactams and ß-lactam/ß-lactamase inhibitor combinations. Overall, this study showed that (i) IPM-REL resistant strains can be obtained from CAZ-AVI-susceptible or -resistant KPC producers, (ii) selection of IPM-REL resistance has a collateral effect on MEM-VAB susceptibility - indicative of shared resistance mechanisms, (iii) and mutations in the KPC sequence may be obtained using IPM-REL selection leading to the possibility of vertical and horizontal transfer of this resistance trait.

Molecular analysis of OXA-48-producing Escherichia coli in Switzerland from 2019 to 2020.

OXA-48-type ß-lactamases are the most prevalent carbapenemase-type in Enterobacterales in Switzerland, predominantly found in Escherichia coli and Klebsiella pneumoniae. Bacteria-producing OXA-48-type enzymes are endemic in some parts of the world, including Europe and North Africa, and are a frequent cause of nosocomial infections. Despite the emergence of numerous OXA-48-type variants, the original variant, OXA-48, remains the most prevalent in E. coli. This study describes the epidemiology of OXA-48-producing E. coli isolates submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) between January 2019 and December 2020.

In vitro activity of sulbactam-durlobactam against carbapenem-resistant Acinetobacter baumannii and mechanisms of resistance.

OBJECTIVES: Multidrug-resistant Acinetobacter baumannii (MDR-Ab), particularly strains producing oxacillinase (OXA)-type carbapenemases, have rapidly emerged in health care settings as a frequent cause of serious infections with limited treatment options. This study evaluated the in vitro activity of sulbactam (SUL) combined with durlobactam (DUR) against a collection of carbapenemase-producing A. baumannii, and investigated the mechanisms of resistance. METHODS: Susceptibility testing was performed on 100 isolates by either broth microdilution or by the Epsilometer test. Isolates were screened for the insertion sequence ISAba1 upstream of the intrinsic chromosomal blaADC by polymerase chain reaction (PCR). Whole genome sequencing was performed on 25 SUL-DUR resistant isolates, and analyses were performed using the Center for Genomic Epidemiology platform. Target gene sequences were compared to A. baumannii American Type Culture Collection (ATCC) 17978. RESULTS: SUL-DUR exhibited excellent activity against A. baumannii isolates with susceptibility levels as follows: amikacin, 18%; colistin, 91%; cefepime, 5%; imipenem, 0%; minocycline, 46%; SUL, 3%; sulbactam-cefoperazone, 8%; SUL-DUR, 71% (based on a breakpoint at 4 mg/L). Twenty-five non-New Delhi metallo-ß-lactamase (NDM)-producing isolates had SUL-DUR MIC values >4 mg/L, amongst which 14 isolates showed substitutions in penicillin-binding protein (PBP)3, previously shown to be associated with SUL-DUR resistance. Substitutions that have not previously been described were detected in SUL-DUR targets, namely PBP1a, PBP1b, PBP2, and PBP3. By contrast, there was no evidence of the involvement of permeability or efflux. CONCLUSIONS: SUL-DUR exhibited excellent in vitro antibacterial activity against carbapenemase-producing A. baumannii isolates. Amongst the 25 resistant isolates, we identified a number of mechanisms which may be contributing factors, in particular PBP substitutions and the production of specific beta-lactamases.

Evidence that faecal carriage of resistant Escherichia coli by 16-week-old dogs in the United Kingdom is associated with raw feeding.

We report a survey (August 2017 to March 2018) and risk factor analysis of faecal carriage of antibacterial-resistant (ABR) Escherichia coli in 223 16-week-old dogs in the United Kingdom. Raw feeding was associated with the presence of fluoroquinolone-resistant (FQ-R) E. coli and those resistant to tetracycline, amoxicillin, and streptomycin, but not to cefalexin. Whole genome sequencing of 36 FQ-R E. coli isolates showed a wide range of sequence types (STs), with almost exclusively mutational FQ-R dominated by ST744 and ST162. Comparisons between E. coli isolates from puppies known to be located within a 50 × 50 km region with those isolated from human urinary tract infections (isolated in parallel in the same region) identified an ST744 FQ-R lineage that was carried by one puppy and caused one urinary tract infection. Accordingly, we conclude that raw feeding is associated with carriage of ABR E. coli in dogs even at 16 weeks of age and that bacteria carried by puppies are shared with humans. We therefore suggest that those who feed their dogs raw meat seriously consider the potential ABR-transmission threat their pet may become as a result and deploy appropriate hygiene practices in mitigation.

New Delhi Metallo-ß-Lactamase-Producing Enterobacterales Bacteria, Switzerland, 2019-2020.

Carbapenemase-producing Enterobacterales (CPE) bacteria are a critical global health concern; New Delhi metallo-ß-lactamase (NDM) enzymes account for >25% of all CPE found in Switzerland. We characterized NDM-positive CPE submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance during a 2-year period (January 2019-December 2020) phenotypically and by using whole-genome sequencing. Most isolates were either Klebsiella pneumoniae (59/141) or Escherichia coli (52/141), and >50% were obtained from screening swabs. Among the 108 sequenced isolates, NDM-1 was the most prevalent variant, occurring in 56 isolates, mostly K. pneumoniae (34/56); the next most prevalent was NDM-5, which occurred in 49 isolates, mostly E. coli (40/49). Fourteen isolates coproduced a second carbapenemase, predominantly an OXA-48-like enzyme, and almost one third of isolates produced a 16S rRNA methylase conferring panresistance to aminoglycosides. We identified successful plasmids and global lineages as major factors contributing to the increasing prevalence of NDMs in Switzerland.

Limited phylogenetic overlap between fluoroquinolone-resistant Escherichia coli isolated on dairy farms and those causing bacteriuria in humans living in the same geographical region.

BACKGROUND: Our primary aim was to test whether cattle-associated fluoroquinolone-resistant (FQ-R) Escherichia coli found on dairy farms are closely phylogenetically related to those causing bacteriuria in humans living in the same 50 × 50 km geographical region suggestive of farm-human sharing. Another aim was to identify risk factors for the presence of FQ-R E. coli on dairy farms. METHODS: FQ-R E. coli were isolated during 2017-18 from 42 dairy farms and from community urine samples. Forty-two cattle and 489 human urinary isolates were subjected to WGS, allowing phylogenetic comparisons. Risk factors were identified using a Bayesian regularization approach. RESULTS: Of 489 FQ-R human isolates, 255 were also third-generation-cephalosporin-resistant, with strong genetic linkage between aac(6')Ib-cr and blaCTX-M-15. We identified possible farm-human sharing for pairs of ST744 and ST162 isolates, but minimal core genome SNP distances were larger between farm-human pairs of ST744 and ST162 isolates (71 and 63 SNPs, respectively) than between pairs of isolates from different farms (7 and 3 SNPs, respectively). Total farm fluoroquinolone use showed a positive association with the odds of isolating FQ-R E. coli, while total dry cow therapy use showed a negative association. CONCLUSIONS: This work suggests that FQ-R E. coli found on dairy farms have a limited impact on community bacteriuria within the local human population. Reducing fluoroquinolone use may reduce the on-farm prevalence of FQ-R E. coli and this reduction may be greater when dry cow therapy is targeted to the ecology of resistant E. coli on the farm.

KPC-Mediated Resistance to Ceftazidime-Avibactam and Collateral Effects in Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales, such as Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae, represent a major threat to public health due to their rapid spread. Novel drug combinations such as ceftazidime-avibactam (CZA), combining a broad-spectrum cephalosporin along with a broad-spectrum ß-lactamase inhibitor, have recently been introduced and have been shown to exhibit excellent activity toward multidrug-resistant KPC-producing Enterobacterales strains. However, CZA-resistant K. pneumoniae isolates are now being increasingly reported, mostly corresponding to producers of KPC variants. In this study, we evaluated in vitro the nature of the mutations in the KPC-2 and KPC-3 ß-lactamase sequences (the most frequent KPC-type enzymes) that lead to CZA resistance and the subsequent effects of these mutations on susceptibility to other ß-lactam antibiotics. Single-step in vitro selection assays were conducted, resulting in the identification of a series of mutations in the KPC sequence which conferred the ability of those mutated enzymes to confer resistance to CZA. Hence, 16 KPC-2 variants and 10 KPC-3 variants were obtained. Production of the KPC variants in an Escherichia coli recombinant strain resulted in a concomitant increased susceptibility to broad-spectrum cephalosporins and carbapenems, with the exceptions of ceftazidime and piperacillin-tazobactam, compared to wild-type KPC enzymes. Enzymatic assays showed that all of the KPC variants identified exhibited an increased affinity toward ceftazidime and a slightly decreased sensitivity to avibactam, sustaining their impact on CZA resistance. However, their respective carbapenemase activities were concurrently negatively impacted.