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.

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.

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.

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.

CTX-M-15-Producing Shewanella Species Clinical Isolate Expressing OXA-535, a Chromosome-Encoded OXA-48 Variant, Putative Progenitor of the Plasmid-Encoded OXA-436.

Shewanella spp. constitute a reservoir of antibiotic resistance determinants. In a bile sample, we identified three extended-spectrum-ß-lactamase (ESBL)-producing bacteria (Escherichia coli, Klebsiella pneumoniae, and Shewanella sp. strain JAB-1) isolated from a child suffering from cholangitis. Our objectives were to characterize the genome and the resistome of the first ESBL-producing isolate of the genus Shewanella and determine whether plasmidic exchange occurred between the three bacterial species. Bacterial isolates were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), standard biochemical tools, and antimicrobial susceptibility testing. Shewanella sp. JAB-1 and ESBL gene-encoding plasmids were characterized using PacBio and Illumina whole-genome sequencing, respectively. The Shewanella sp. JAB-1 chromosome-encoded OXA-48 variant was cloned and functionally characterized. Whole-genome sequencing (WGS) of the Shewanella sp. clinical isolate JAB-1 revealed the presence of a 193-kb plasmid belonging to the IncA/C incompatibility group and harboring two ESBL genes, blaCTX-M-15 and blaSHV-2ablaCTX-M-15 gene-carrying plasmids belonging to the IncY and IncR incompatibility groups were also found in the E. coli and K. pneumoniae isolates from the same patient, respectively. A comparison of the blaCTX-M-15 genetic environment indicated the independent origin of these plasmids and dismissed in vivo transfers. Furthermore, characterization of the resistome of Shewanella sp. JAB-1 revealed the presence of a chromosome-carried blaOXA-535 gene, likely the progenitor of the plasmid-carried blaOXA-436 gene, a novel blaOXA-48-like gene. The expression of blaOXA-535 in E. coli showed the carbapenem-hydrolyzing activity of OXA-535. The production of OXA-535 in Shewanella sp. JAB-1 could be evidenced using molecular and immunoenzymatic tests, but not with biochemical tests that monitor carbapenem hydrolysis. In this study, we have identified a CTX-M-15-producing Shewanella species that was responsible for a hepatobiliary infection and that is likely the progenitor of OXA-436, a novel plasmid-encoded OXA-48-like class D carbapenemase.

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.

Analysis of OXA-204 carbapenemase-producing Enterobacteriaceae reveals possible endoscopy-associated transmission, France, 2012 to 2014.

OXA-48-like beta-lactamase producing bacteria are now endemic in several European and Mediterranean countries. Among this carbapenemase family, the OXA-48 and OXA-181 variants predominate, whereas other variants such as OXA-204 are rarely reported. Here, we report the molecular epidemiology of a collection of OXA-204-positive enterobacterial isolates (n = 29) recovered in France between October 2012 and May 2014. This study describes the first outbreak of OXA-204-producing Enterobacteriaceae in Europe, involving 12 isolates of an ST90 Escherichia coli clone and nine isolates of an ST147 Klebsiella pneumoniae clone. All isolates co-produced the cephalosporinase CMY-4, and 60% of them co-produced the extended-spectrum beta-lactamase CTX-M-15. The blaOXA-204 gene was located on a 150-kb IncA/C plasmid, isolated from various enterobacterial species in the same patient, indicating a high conjugative ability of this genetic vehicle.

Recurrent obstructive acute pyelonephritis: A rare form of Actinotignum (Actinobaculum) schaalii infection in a HIV-1 infected patient.

Actinobaculum schaalii is a rarely reported, anaerobic, Gram-positive bacterium which role as uropathogen is emerging. We report here the case of a 47 year old HIV-1 infected woman presented with five recurrent episodes of obstructive pyelonephritis in the context of multiple renal stones. No bacteria was found until the fifth episode, during which prolonged urinary cultures as well as 16S rDNA sequencing allowed the diagnosis of A. schaalii infection. She had developed a life-threatening condition with severe renal failure. A right nephrectomy was performed and found that the intrarenal stones were attributed to the antiretroviral therapy. The renal parenchyma corresponded to an end-stage renal disease with chronic pyelonephritis without abcesses or granules. The situation improved after six months of amoxicillin therapy.

Derepressed transfer properties leading to the efficient spread of the plasmid encoding carbapenemase OXA-48.

The current emergence of the carbapenemase OXA-48 among Enterobacteriaceae is related to the spread of a single IncL/M-type plasmid, pOXA-48a. This plasmid harbors the blaOXA-48 gene within a composite transposon, Tn1999, which is inserted into the tir gene, encoding a transfer inhibition protein. We showed that the insertion of Tn1999 into the tir gene was involved in a higher transfer frequency of plasmid pOXA-48a. This may likely be the key factor for the successful dissemination of this plasmid.

Should we, and how to, optimize cefiderocol administration during severe nosocomial pneumonia due to carbapenem-resistant Acinetobacter baumanii? A viewpoint.

INTRODUCTION: Acinetobacter baumannii is classified by the Center for Disease Control and Prevention (CDC) as an "urgent threat" due to its ability to acquire and develop resistance to multiple classes of antibiotics. As a result, it is one of the most concerning pathogens in healthcare settings, with increasing incidence of infections due to carbapenem-resistant Acinetobacter baumannii (CRAB) associated with high morbidity and mortality rates. Therefore, there are ongoing efforts to find novel treatment options, one of which is cefiderocol. We aim to review available evidence on cefiderocol use for severe nosocomial pneumonia due to carbapenem-resistant Acinetobacter baumannii METHODS: A comprehensive review was conducted from 2017 to 2023, covering articles from databases such as Pubmed, Scopus, and Embase, along with conference proceedings from ECCMID 2023. The primary focus was on severe nosocomial pneumonia due A. baumannii and cefiderocol. DISCUSSION: Cefiderocol, targeting periplasmic space Penicillin-Binding Proteins (PBPs) via siderophore transport pathways, exhibits promise against multi-drug resistant Gram-negative bacilli. Its effectiveness in treating CRAB pneumonia remains debated. The CREDIBLE trial reported higher mortality with cefiderocol compared to the best available treatment, while other cohort studies showed contrasting outcomes. Patient variations and pharmacokinetic factors may underlie these discrepancies. The recommended cefiderocol dosage regimen may fall short of desired pharmacokinetic targets, especially in critically ill patients and lung infections. Pulmonary factors hindering cefiderocol's entry into bacteria through iron transporters are overlooked in clinical breakpoints. Optimized dosing or combination regimens may enhance infection site exposure and outcomes. CONCLUSION: Further research is needed to determine the optimal cefiderocol dosage and administration (mono vs. dual therapy, continuous vs. intermittent infusion), in severe Acinetobacter baumannii nosocomial pneumonia.

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.

Characterization of OXA-204, a carbapenem-hydrolyzing class D ß-lactamase from Klebsiella pneumoniae.

A Klebsiella pneumoniae clinical isolate recovered in Tunisia showed resistance to all ß-lactams and decreased susceptibility to carbapenems. K. pneumoniae 204 expressed the carbapenem-hydrolyzing ß-lactamase OXA-204, differing from OXA-48 by two amino acid substitutions (Gln98His and Thr99Arg) (class D ß-lactamase [DBL] numbering). OXA-48 and OXA-204 shared similar resistance profiles, hydrolyzing carbapenems but sparing broad-spectrum cephalosporins. The bla(OXA-204) gene was located on a ca. 150-kb IncA/C-type plasmid, which also carried the bla(CMY-4) gene. The bla(OXA-204) gene was associated with an ISEcp1 element, whereas the bla(OXA-48) genes are usually associated with IS1999.

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.

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.

Wild coastline birds as reservoirs of broad-spectrum-ß-lactamase-producing Enterobacteriaceae in Miami Beach, Florida.

A high rate of broad-spectrum-ß-lactamase-producing Escherichia coli isolates was identified from seagull and pelican feces collected in the Miami Beach, Florida, area. The most commonly identified resistance determinants were CMY-2 and CTX-M-15. Those wild birds might be therefore considered vehicles for wide dissemination of multidrug-resistant Enterobacteriaceae in the United States.

OXA-48-like carbapenemases: the phantom menace.

OXA-48-type carbapenem-hydrolysing class D ß-lactamases are increasingly reported in enterobacterial species. To date, six OXA-48-like variants have been identified, with OXA-48 being the most widespread. They differ by a few amino acid substitutions or deletions (one to five amino acids). The enzymes hydrolyse penicillins at a high level and carbapenems at a low level, sparing broad-spectrum cephalosporins, and are not susceptible to ß-lactamase inhibitors. When combining permeability defects, OXA-48-like producers may exhibit a high level of resistance to carbapenems. OXA-163 is an exception, hydrolysing broad-spectrum cephalosporins but carbapenems at a very low level, and being susceptible to ß-lactamase inhibitors. The bla(OXA-48)-type genes are always plasmid-borne and have been identified in association with insertion sequences involved in their acquisition and expression. The current spread of the bla(OXA-48) gene is mostly linked to the dissemination of a single IncL/M-type self-transferable plasmid of 62 kb that does not carry any additional resistance gene. OXA-48-type carbapenemases have been identified mainly from North African countries, the Middle East, Turkey and India, those areas constituting the most important reservoirs; however, occurrence of OXA-48 producers in European countries is now well documented, with some reported hospital outbreaks. Since many OXA-48-like producers do not exhibit resistance to broad-spectrum cephalosporins, or only decreased susceptibility to carbapenems, their recognition and detection can be challenging. Adequate screening and detection methods are therefore required to prevent and control their dissemination.

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.