Adaptive resistance to cefiderocol in carbapenem-resistant Acinetobacter baumannii (CRAB): Microbiological and clinical issues.

Objectives: Determining the best available therapy for carbapenem-resistant Acinetobacter baumannii (CRAB) infections is a challenge. Cefiderocol is an attractive alternative drug effective against many resistance mechanisms in Gram-negative bacteria. However, its place in the treatment of Acinetobacter baumannii infections remains unclear and much debated, with contradictory results. Methods: We describe here the case of a 37-year-old man with ventilator-associated bacteraemic CRAB pneumonia in an intensive care unit. He was initially treated with a combination of colistin and tigecycline, and was then switched onto colistin and cefiderocol. We then used a new accessible protocol to test 30 CRAB isolates (OXA-23/OXA-24/OXA-58/NDM-1) for adaptive resistance to cefiderocol (ARC) after exposure to this drug. Results: After clinical failure with the initial combination, we noted a significant clinical improvement in the patient on the second combination, leading to clinical cure. No ARC was detected in the two OXA-23 case-CRAB isolates. All NDM-1 CRAB isolates were resistant to cefiderocol in standard tests; the OXA-23, OXA-24 and OXA-58 CRAB isolates presented 84.2 %, 50 % and 0 % ARC, respectively. Conclusions: ARC is not routinely assessed for CRAB isolates despite frequently being reported in susceptible isolates (69.2 %). Subpopulations displaying ARC may account for treatment failure, but this hypothesis should be treated with caution in the absence of robust clinical data. The two main findings of this work are that (i) cefiderocol monotherapy should probably not be recommended for OXA-23/24 CRAB infections and (ii) the characterisation of carbapenemases in CRAB strains may be informative for clinical decision-making.

Multiple host colonization and differential expansion of multidrug-resistant ST25-Acinetobacter baumannii clades.

The Acinetobacter baumannii clonal lineage ST25 has been identified in humans and animals and found associated with outbreaks globally. To highlight possible similarities among ST25 A. baumannii of animal and human origins and to gather clues on the dissemination and evolution of the ST25 lineage, we conducted a phylogenetic analysis on n = 106 human and n = 35 animal A. baumannii ST25 genomes, including 44 sequenced for this study. Resistance genes and their genetic background were analyzed, as well. ST25 genomes are clustered into four clades: two are widespread in South America, while the other two are largely distributed in Europe, Asia and America. One particular clade was found to include the most recent strains and the highest number of acquired antibiotic resistance genes. OXA-23-type carbapenemase was the most common. Other resistance genes such as blaNDM-1, blaPER-7, and armA were found embedded in complex chromosomal regions present in human isolates. Genomic similarity among multidrug resistant ST25 isolates of either animal or human origin was revealed, suggesting cross-contaminations between the two sectors. Tracking the clonal complex ST25 between humans and animals should provide new insights into the mode of dissemination of these bacteria, and should help defining strategies for preserving global health.

Genomic characterization of an NDM-9-producing Acinetobacter baumannii clinical isolate and role of Glu152Lys substitution in the enhanced cefiderocol hydrolysis of NDM-9.

Here, we characterized the first French NDM-9-producing Acinetobacter baumannii isolate. A. baumannii 13A297, which belonged to the STPas25 (international clone IC7), was highly resistant to ß-lactams including cefiderocol (MIC >32 mg/L). Whole genome sequencing (WGS) using both Illumina and Oxford Nanopore technologies revealed a 166-kb non-conjugative plasmid harboring a blaNDM-9 gene embedded in a Tn125 composite transposon. Complementation of E. coli DH5α and A. baumannii CIP70.10 strains with the pABEC plasmid carrying the blaNDM-1 or blaNDM-9 gene, respectively, resulted in a significant increase in cefiderocol MIC values (16 to >256-fold), particularly in the NDM-9 transformants. Interestingly, steady-state kinetic parameters, measured using purified NDM-1 and NDM-9 (Glu152Lys) enzymes, revealed that the affinity for cefiderocol was 3-fold higher for NDM-9 (Km = 53 µM) than for NDM-1 (Km = 161 µM), leading to a 2-fold increase in catalytic efficiency for NDM-9 (0.13 and 0.069 µM-1.s-1, for NDM-9 and NDM-1, respectively). Finally, we showed by molecular docking experiments that the residue 152 of NDM-like enzymes plays a key role in cefiderocol binding and resistance, by allowing a strong ionic interaction between the Lys152 residue of NDM-9 with both the Asp223 residue of NDM-9 and the carboxylate group of the R1 substituent of cefiderocol.

Performance of the Disc Diffusion Method, MTS Gradient Tests and Two Commercially Available Microdilution Tests for the Determination of Cefiderocol Susceptibility in Acinetobacter spp.

Cefiderocol is a siderophore-conjugated cephalosporin with potent activity against multidrug-resistant Gram-negative pathogens including Acinetobacter baumannii. The aim of this study was to evaluate cefiderocol testing methods on a relevant collection of 97 Acinetobacter spp. isolates. Commercialized broth microdilution methods (ComASP®, Liofilchem and UMIC®, Bruker), MIC test strips (Liofilchem) and disc diffusion using discs of three different brands (Mast Diagnostic, Liofilchem and Oxoid-Thermo Fisher Scientific) were compared with the broth microdilution reference method. None of the methods tested fulfilled acceptable criteria (essential agreement [EA] ≥ 90%; bias = ±30%) but both BMD methods achieved acceptable categorical agreement rates (CA = 95.9% [93/97, 95% CI 89.9-98.4] and CA = 93.8% [91/97, 95% CI 87.2-97.1] for ComASP® and UMIC®, respectively) and bias < 30% (-7.2% and -25.2% for ComASP® and UMIC®, respectively). The use of MIC gradient testing is strongly discouraged due to misclassification of 55% (n = 23/42) of resistant strains. Finally, the disc diffusion method could be used to rapidly screen for susceptible strains by setting a critical diameter of 22 mm.

Genomic analysis of CTX-M-115 and OXA-23/-72 co-producing Acinetobacter baumannii, and their potential to spread resistance genes by natural transformation.

OBJECTIVES: To characterize Acinetobacter baumannii strains co-producing the ESBL CTX-M-115 and carbapenem-hydrolysing class D ß-lactamases (CHDLs), and to assess the potential diffusion of their resistance genes by horizontal transfer. METHODS: Nineteen CTX-M-115/CHDL-positive A. baumannii were collected between 2015 and 2019 from patients hospitalized in France. Their whole-genome sequences were determined on Illumina and Oxford Nanopore platforms and were compared through core-genome MLST (cgMLST) and SNP analyses. Transferability of resistance genes was investigated by natural transformation assays. RESULTS: Eighteen strains were found to harbour CHDL OXA-72, and another one CHDL OXA-23, in addition to CTX-M-115, narrow-spectrum ß-lactamases and aminoglycoside resistance determinants including ArmA. cgMLST typing, as well as Oxford Scheme ST and K locus typing, confirmed that 17 out of the 18 CTX-M-115/OXA-72 isolates belonged to new subclades within clonal complex 78 (CC78). The chromosomal region carrying the blaCTX-M-115 gene appeared to vary greatly both in gene content and in length (from 20 to 79 kb) among the strains, likely because of IS26-mediated DNA rearrangements. The blaOXA-72 gene was localized on closely related plasmids showing structural variations that occurred between pdif sites. Transfer of all the ß-lactamase genes, as well as aminoglycoside resistance determinants to a drug-susceptible A. baumannii recipient, was easily obtained in vitro by natural transformation. CONCLUSIONS: This work highlights the propensity of CC78 isolates to collect multiple antibiotic resistance genes, to rearrange and to pass them to other A. baumannii strains via natural transformation. This process, along with mobile genetic elements, likely contributes to the considerable genomic plasticity of clinical strains, and to the diversity of molecular mechanisms sustaining their multidrug resistance.

Efficacy and Safety of Oral Fosfomycin-Trometamol in Male Urinary Tract Infections with Multidrug-Resistant Enterobacterales.

BACKGROUND: Antimicrobial drugs to treat male urinary tract infection (UTI) with multidrug-resistant Enterobacterales are limited. We studied oral fosfomycin-trometamol (FT) in this situation. The objective was to assess the clinical cure rate in patients presenting UTIs treated with oral FT. METHODS: We conducted a single-center observational retrospective study from January 2017 to August 2018. The primary endpoint was clinical cure; and the secondary endpoints were incidence of recurrences, oral FT safety, and microbiological cure. RESULTS: Sixteen male patients were included, presenting 21 UTI episodes. Fourteen patients (88%) have at least one underlying urologic disorder. We described 4 episodes of acute UTI and 17 episodes of chronic bacterial prostatitis (CBP). Sixteen out of twenty-one Enterobacterales were extended spectrum beta-lactamase (ESBL)-producers and all the patients presented a resistance to fluoroquinolones and trimethoprim/sulfamethoxazole. In acute UTI, the regimen was a daily dose of oral FT for a mean duration of 2.5 weeks (+/-7.0 days). Clinical and microbiological recovery was achieved in all patients, with no recurrence after 5.3 months follow-up on average (+/-10.4 days). In CBP, the regimen was one oral dose of fosfomycin every 24-48 h, for a mean duration of 5.5 weeks/UTI episodes (+/-15.3 days). Clinical and microbiological recovery was found in 16/17 cases. Seven of the twelve patients with CBP had relapsed and 3/12 had had a new episode of infection after an average follow-up of 5.8 months. Only 6/21 of patients presented minor or moderate adverse effects, such as digestive disorders. CONCLUSIONS: FT could be an alternative option to carbapenems in the treatment of multidrug-resistant Enterobacterales infections for male UTIs.

Interbacterial Transfer of Carbapenem Resistance and Large Antibiotic Resistance Islands by Natural Transformation in Pathogenic Acinetobacter.

Acinetobacter baumannii infection poses a major health threat, with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intrastrain but also interstrain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4 and AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genomes of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii. IMPORTANCE Acinetobacter baumannii is a multidrug-resistant pathogen responsible for difficult-to-treat hospital-acquired infections. Understanding the mechanisms leading to the emergence of the multidrug resistance in this pathogen today is crucial. Horizontal gene transfer is assumed to largely contribute to this multidrug resistance. However, in A. baumannii, the mechanisms leading to genome recombination and the horizontal transfer of resistance genes are poorly understood. We describe experimental evidence that natural transformation, a horizontal gene transfer mechanism recently highlighted in A. baumannii, allows the highly efficient interbacterial transfer of genetic elements carrying resistance to last-line antibiotic carbapenems. Importantly, we demonstrated that natural transformation, occurring in mixed populations of Acinetobacter, enables the transfer of large resistance island-mobilizing multiple-resistance genes.

High Prevalence of OXA-23 Carbapenemase-Producing Proteus mirabilis among Amoxicillin-Clavulanate-Resistant Isolates in France.

In this multicentric study performed in 12 French hospitals, we reported that 26.9% (14/52) of the amoxicillin-clavulanate-resistant Proteus mirabilis isolates produced the OXA-23 carbapenemase. We found that an inhibition zone diameter of <11 mm around the amoxicillin-clavulanate disc was an accurate screening cutoff to detect these OXA-23 producers. We confirmed by whole-genome sequencing that these OXA-23-producers all belonged to the same lineage that has been demonstrated to disseminate OXA-23 or OXA-58 in P. mirabilis.

Molecular and epidemiological investigation of a colistin-resistant OXA-23-/NDM-1-producing Acinetobacter baumannii outbreak in the Southwest Indian Ocean Area.

Dual resistance to colistin and carbapenems is a milestone reached by certain extensively-drug resistant (XDR) Gram-negative bacteria. This study describes the first outbreak of XDR colistin- and carbapenem-resistant OXA-23-/NDM-1-producing Acinetobacter baumannii (CCRAB) in the European overseas territory of Reunion Island (France, Indian Ocean). Between April 2019 and June 2020, 13 patients admitted to the University Hospital of Reunion Island were involved in the outbreak, of whom eight were infected and six died. The first case was traced to a medical evacuation from Mayotte Island (Comoros archipelago). An epidemiological link could be established for 11 patients. All of the collected CCRAB isolates showed the same resistance profile and co-produced intrinsic ß-lactamases OXA-69 and ADC-191, together with acquired carbapenem-hydrolysing ß-lactamases OXA-23 and NDM-1. A mutation likely involved in colistin resistance was detected in the two-component system PmrAB (D82N in PmrA). All of the isolates were found to belong to STPas1/STOx231 clonal complex and were phylogenetically indistinguishable. Their further characterization by whole-genome sequence analyses (whole-genome multi-locus sequence typing, single nucleotide polymorphisms) provided hints about the transmission pathways. This study pleads for strict application of control and prevention measures in institutions where the risk of imported XDR bacteria is high.

Clinical relevance and antimicrobial susceptibility profile of the unknown human pathogen Corynebacterium aurimucosum.

Introduction. Even though Corynebacterium aurimucosum has been described in 2002, this species has long been underestimated due to the unreliability of conventional identification methods and only a few cases of infections have been reported.Hypothesis/Gap Statement. Little is known about clinical significance and antimicrobial susceptibility profile of this uncommon species.Aim. To evaluate the clinical relevance of C. aurimucosum and its antimicrobial susceptibility profile.Methodology. All C. aurimucosum isolates, collected from 2010 to 2019 in 10 French university hospitals, were retrospectively included. Demographic, clinical and microbiological data were collected for all cases. Antimicrobial susceptibility testing was performed according to the 2019 EUCAST guidelines.Results. Fifty-seven clinical isolates of C. aurimucosum were collected in 57 patients (median age, 65.8 years; male/female sex ratio, 1.1), mostly from urine (28 %), blood culture (28 %) and bone/synovial fluid (19 %) samples. Of them, 14 cases of infection were confirmed, mainly bone and joint infections (50 %) followed by urinary tract infections (UTIs) (21 %), bacteremia (14 %), skin and soft-tissue infections (14 %). C. aurimucosum was recovered in pure culture in 36 % of cases (UTIs and bacteremia) while mixed cultures were observed for other infections. By testing 52 clinical isolates in vitro, this species appeared to be fully susceptible to linezolid and vancomycin while most isolates (>80 %) were susceptible to amoxicillin (MIC90, 2 µg ml-1), gentamicin, tetracycline and rifampicin. Both cefotaxime and ciprofloxacin seemed to have a limited activity (ca. 50 % of susceptible strains). The MIC distribution for ciprofloxacin showed a bimodal profile with a population of highly-resistant strains with MICs >2 µg ml-1. Most isolates (>90 %) were categorized as resistant to penicillin G and clindamycin.Conclusion. C. aurimucosum should be considered as an actual opportunistic pathogen, and treatment with amoxicillin, vancomycin or linezolid should be preferred.

MacAB-TolC Contributes to the Development of Acinetobacter baumannii Biofilm at the Solid-Liquid Interface.

Acinetobacter baumannii has emerged as one of the most problematic bacterial pathogens responsible for hospital-acquired and community infections worldwide. Besides its high capacity to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development. This lifestyle confers additional protection against various treatments and allows it to persist for long periods in various hospital niches. Due to their remarkable antimicrobial tolerance, A. baumannii biofilms are difficult to control and ultimately eradicate. Further insights into the mechanism of biofilm development will help to overcome this challenge and to develop novel antibiofilm strategies. To unravel critical determinants of this sessile lifestyle, the proteomic profiles of two A. baumannii strains (ATTC17978 and SDF) grown in planktonic stationary phase or in mature solid-liquid (S-L) biofilm were compared using a semiquantitative proteomic study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump played a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm. It contributes to maintain wild type (WT) membrane rigidity and provides tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen face and adapt to deleterious conditions occurring in mature biofilms. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

Evaluation of the Immunochromatographic NG-Test Carba 5 for Rapid Identification of Carbapenemase in Nonfermenters.

The immunochromatographic assay NG-Test Carba 5 (NG-Biotech) was evaluated with a collection of 107 carbapenemase-producing nonfermenters (CP-NF) (55 Pseudomonas spp., 51 Acinetobacter spp., and 1 Achromobacter xylosoxidans isolate) and 61 carbapenemase-negative isolates. All KPC, VIM, and NDM carbapenemase producers tested were accurately detected. Of the 16 IMP variants tested, 6 (37.5%) variants were not detected. Considering the epidemiology of CP-NFs in France, the NG-Test Carba 5 would detect 89.4% of CP Pseudomonas spp. but only 12.9% of CP Acinetobacter spp.

ISAba1-dependent overexpression of eptA in clinical strains of Acinetobacter baumannii resistant to colistin.

BACKGROUND: Colistin resistance in Acinetobacter baumannii often results from mutational activation of the two-component system PmrAB and subsequent addition of phospho-ethanolamine (pEtN) to lipooligosaccharide by up-regulated pEtN transferase PmrC. OBJECTIVES: To characterize mechanisms of colistin resistance independent of PmrCAB in A. baumannii. METHODS: Twenty-seven colistin-resistant A. baumannii were collected from 2012 to 2018. Analysis of operon pmrCAB was performed by PCR and sequencing. Seven strains were investigated further by WGS and whole-genome MLST (wgMLST). RESULTS: Seven out of the 27 selected isolates were found to overexpress eptA, a gene homologous to pmrC, likely as a consequence of upstream insertion of an ISAba1 element. Insertion sites of ISAba1 were mapped 13, 18 and 156 bp ahead of the start codon of eptA in five strains, one strain and one strain, respectively. The finding that the isolates did not cluster together when compared by wgMLST analysis supports the notion that distinct insertion events occurred in close, but different, genetic backgrounds. CONCLUSIONS: Activation of eptA and subsequent addition of pEtN to the cell surface represents a novel mechanism of resistance to colistin in A. baumannii.

Carbapenem-Susceptible OXA-23-Producing Proteus mirabilis in the French Community.

Nineteen Proteus mirabilis isolates producing the carbapenemase OXA-23 were recovered over a 2-year period in 19 French hospitalized patients, of whom 12 had community onset infections. The isolates exhibited a slightly reduced susceptibility to carbapenems. Whole-genome analysis revealed that all 19 isolates formed a cluster compared to 149 other P. mirabilis isolates. Because of its susceptibility to carbapenems, this clone may be misidentified as a penicillinase producer while it constitutes a reservoir of the OXA-23-encoding gene in the community.

Sequential emergence of colistin and rifampicin resistance in an OXA-72- producing outbreak strain of Acinetobacter baumannii.

OBJECTIVES: This study reported a hospital outbreak due to an extensively drug-resistant (XDR) OXA-72-producing strain of Acinetobacter baumannii (A. baumannii). METHODS AND RESULTS: The isolates were found to be genotypically indistinguishable by whole-genome multiple locus sequence typing, and to belong to the international clonal complex CC2. One of these isolates sequentially developed a high resistance to colistin and rifampicin under treatment, as a result of mutations in genes pmrB and rpoB, respectively. The blaOXA-72 gene was localised on a 10-kb transferable plasmid, named pAB-STR-1, whose sequence is nearly identical to that of another plasmid previously found in Lithuanian strains, pAB120. CONCLUSION: This report highlighted the need to carefully monitor the emergence of colistin and rifampicin resistance in patients treated for infections with multidrug-resistant A. baumannii.

Rapid detection of colistin resistance in Acinetobacter baumannii using MALDI-TOF-based lipidomics on intact bacteria.

With the dissemination of extremely drug resistant bacteria, colistin is now considered as the last-resort therapy for the treatment of infection caused by Gram-negative bacilli (including carbapenemase producers). Unfortunately, the increase use of colistin has resulted in the emergence of resistance as well. In A. baumannii, colistin resistance is mostly caused by the addition of phosphoethanolamine to the lipid A through the action of a phosphoethanolamine transferase chromosomally-encoded by the pmrC gene, which is regulated by the two-component system PmrA/PmrB. In A. baumannii clinical isolate the main resistance mechanism to colistin involves mutations in pmrA, pmrB or pmrC genes leading to the overexpression of pmrC. Although, rapid detection of resistance is one of the key issues to improve the treatment of infected patient, detection of colistin resistance in A. baumannii still relies on MIC determination through microdilution, which is time-consuming (16-24 h). Here, we evaluated the performance of a recently described MALDI-TOF-based assay, the MALDIxin test, which allows the rapid detection of colistin resistance-related modifications to lipid A (i.e phosphoethanolamine addition). This test accurately detected all colistin-resistant A. baumannii isolates in less than 15 minutes, directly on intact bacteria with a very limited sample preparation prior MALDI-TOF analysis.

Rapid detection and discrimination of chromosome- and MCR-plasmid-mediated resistance to polymyxins by MALDI-TOF MS in Escherichia coli: the MALDIxin test.

Background: Polymyxins are currently considered a last-resort treatment for infections caused by MDR Gram-negative bacteria. Recently, the emergence of carbapenemase-producing Enterobacteriaceae has accelerated the use of polymyxins in the clinic, resulting in an increase in polymyxin-resistant bacteria. Polymyxin resistance arises through modification of lipid A, such as the addition of phosphoethanolamine (pETN). The underlying mechanisms involve numerous chromosome-encoded genes or, more worryingly, a plasmid-encoded pETN transferase named MCR. Currently, detection of polymyxin resistance is difficult and time consuming. Objectives: To develop a rapid diagnostic test that can identify polymyxin resistance and at the same time differentiate between chromosome- and plasmid-encoded resistances. Methods: We developed a MALDI-TOF MS-based method, named the MALDIxin test, which allows the detection of polymyxin resistance-related modifications to lipid A (i.e. pETN addition), on intact bacteria, in <15 min. Results: Using a characterized collection of polymyxin-susceptible and -resistant Escherichia coli, we demonstrated that our method is able to identify polymyxin-resistant isolates in 15 min whilst simultaneously discriminating between chromosome- and plasmid-encoded resistance. We validated the MALDIxin test on different media, using fresh and aged colonies and show that it successfully detects all MCR-1 producers in a blindly analysed set of carbapenemase-producing E. coli strains. Conclusions: The MALDIxin test is an accurate, rapid, cost-effective and scalable method that represents a major advance in the diagnosis of polymyxin resistance by directly assessing lipid A modifications in intact bacteria.