In vitro fosfomycin study on concordance of susceptibility testing methods against ESBL and carbapenem-resistant Enterobacteriaceae.

Abstract Objectives The increasing emergence and diffusion of multidrug-resistant (MDR) pathogenic bacteria, both in hospital and community settings, is inducing clinicians to reconsider old antibiotics, such as fosfomycin, to overcome the difficulties posed by these microorganisms. Recent studies have reported good in vitro activity of fosfomycin against extended spectrum s-lactamases (ESBL) and carbapenem-resistant Enterobacteriaceae. The aim of this study was to assess thein vitro activity of fosfomycin by different methods against 120 clinical MDR isolates. Methods Fosfomycin minimum inhibitory concentrations were determined using the agar dilution reference method (AD), gradient test (GT), broth microdilution method (BMD), according to CLSI recommendations, and automated systems (VITEK 2 and BD Phoenix) against 85 carbapenem-resistant Klebsiella pneumoniae and 35 ESBL-producing Escherichia coli. Agreement and discrepancies between the evaluated methods and the reference method were calculated. Results Fosfomycin showed very good activity against ESBL-producing E. coli (88.6%). Excellent agreement (100%) between the three (AD, BMD and GT) susceptibility methods was found for E. coli. No major errors were observed. The fosfomycin resistance rate ranged from 24% (KPC-producing) to 100% (NDM-OXA-48 co-producing) K. pneumoniae. For all carbapenem-resistant K. pneumoniae strains, categorical agreement was >90% for all methods except for VITEK 2, which was 84%. Conclusions When ESBL E. coli isolates are found to be susceptible to fosfomycin with automated systems, it is not necessary to verify these results with the AD reference method; while for resistant strains, the GT can be used. In cases of KPC K. pneumoniae resistant to fosfomycin, the AD method is the only reference method

In vitro activity of tigecycline and comparators against carbapenem-susceptible and resistant Acinetobacter baumannii clinical isolates in Italy

Background: In a recent multi-centre Italian survey (2003-2004), conducted in 45 laboratories throughout Italy with the aim of monitoring microorganisms responsible for severe infections and their antibiotic resistance, Acinetobacter baumannii was isolated from various wards of 9 hospitals as one of the most frequent pathogens. One hundred and seven clinically significant strains of A. baumannii isolates were included in this study to determine the in vitro activity of tigecycline and comparator agents. Methods: Tests for the susceptibility to antibiotics were performed by the broth microdilution method as recommended by CLSI guidelines. The following antibiotics were tested: aztreonam, piperacillin/tazobactam, ampicillin/sulbactam, ceftazidime, cefepime, imipenem, meropenem tetracycline, doxycycline, tigecycline, gentamicin, amikacin, ciprofloxacin, colistin, and trimethoprim/sulphametoxazole. The PCR assay was used to determine the presence of OXA, VIM, or IMP genes in the carbapenem resistant strains. Results: A. baumannii showed widespread resistance to ceftazidime, ciprofloxacin and aztreonam in more than 90% of the strains; resistance to imipenem and meropenem was 50 and 59% respectively, amikacin and gentamicin were both active against about 30% of the strains and colistin about 99%, with only one strain resistant. By comparison with tetracyclines, tigecycline and doxycycline showed a higher activity. In particular, tigecycline showed a MIC90 value of 2 mg/L and our strains displayed a unimodal distribution of susceptibility being indistinctly active against carbapenem-susceptible and resistant strains, these latter possessed OXA-type variant enzymes. Conclusion: In conclusion, tigecycline had a good activity against the MDR A. baumannii strains while maintaining the same MIC90 of 2 mg/L against the carbapenem-resistant strains. © 2008 Mezzatesta et al; licensee BioMed Central Ltd

Gold standard susceptibility testing of fosfomycin in Staphylococcus aureus and Enterobacterales using a new agar dilution panel

Abstract Objectives Many clinical laboratories have difficulty in routinely performing in vitro fosfomycin susceptibility testing using the agar dilution (AD) method, considered to be the gold standard method. The objective of our work was to evaluate a rapid commercial fosfomycin agar dilution panel against clinical Staphylococcus aureus and Enterobacterales strains, in two different centres located in Italy and in the UK. Methods A total of 99 Enterobacterales (mostly Escherichia coli and Klebsiella pneumoniae) and 80 S. aureus clinical isolates was used to evaluate the commercial device, a 12-well panel containing fosfomycin incorporated into CA-MH agar supplemented with 25 mg/L of glucose-6-phosphate (Liofilchem S.r.l., Roseto degli Abruzzi, Italy). Testing was performed in two centres (Italy and UK) and kit results were compared against the gold standard in-house AD MIC method. Results According to the EUCAST breakpoints, fosfomycin inhibited 61% of the S. aureus strains, and 76% of the Enterobacterales isolates tested by the AD reference method. There was a Categorical Agreement (CA) of 100% and an Essential Agreement (EA) of 91.25% for S. aureus; while the Enterobacterales strains showed a CA of 94% and an EA of 97%. No evaluation errors were observed among S. aureus, while 5% Major Error and 1% Very Major Error were observed for the Enterobacterales. Conclusions Our results confirmed the feasibility of determining fosfomycin susceptibility using a commercial AD panel as a routine substitution for the AD test. The few differences observed were only in strains with MICs around the breakpoint used

Colistin Resistant A. baumannii: Genomic and Transcriptomic Traits Acquired Under Colistin Therapy

Even though colistin-based treatment represents the antimicrobial-regimen backbone for the management of multidrug-resistant Gram-negative infections, colistin resistance is still rare, at least as a full resistance, in Acinetobacter baumannii (Ab). We investigated the genomics and transcriptomics of two clinical Extensively Drug Resistance (XDR) colistin-susceptible/resistant (COL-S/R) Ab strain-pairs in which COL-resistance was developed after exposure to colistin therapy. The molecular characterization of the strains showed that all strains belonged to PFGE-A, ST-281, OXA-23 producers, Global Clone-II, and were resistant to imipenem, meropenem, ampicillin/sulbactam, ciprofloxacin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, and susceptible to tigecycline, in agreement with NGS-acquired resistome. COL-R vs. COL-S Ab comparative genomics, mapping on Ab ATCC 17978 and Ab ACICU Reference Genomes, revealed a closely related genomic phylogeny, especially between strain-pair isolates, and distinctive common genomic non-synonymous SNPs (nsSNPs) in COL-R Ab strains. Furthermore, pmrB and pmrC nsSNPs were found. Notably we recovered, for the first time, lpxC and lpxD nsSNPs previously described only in “in-vitro” mutants and associated with colistin resistance in a clinical COL-R Ab. COL-R vs. COL-S Ab comparative transcriptomics evidenced a strain-dependent response to the colistin resistance onset highly variable among the single COL-R strains vs. their COL-S parents and merely seven common over-expressed transcripts, i.e. the PgaB lipoprotein for biofilm-matrix production, the diacylglycerol kinase for the lipid recycling in the membrane-derived oligosaccharide cycle, a membrane non-ribosomal peptide synthetase, the Lipid A phosphoethanol aminotransferase PmrC, and three hypothetical proteins. The transcript analysis of the “COL-R related genes” and the RNA-seq data confirmed pmrCAB over-expression responsible for a greater positive net cell-charge, and lpxACD under-expression in COL-R causing a decreased LPS production, as main mechanisms of colistin resistance. Our study reports the COL-R Ab genomic and transcriptomic signatures reflecting the interplay between several direct and indirect potential adaptations to antimicrobial pressure, including the occurrence of SNP accumulation hotspot loci in genes related to intrinsic or adaptive colistin resistance, surface adhesion proteins and porins, and over-expressed genes involved in different pathways, i.e. biofilm production, oxidative stress response, extensive drug and COL resistance

Management of meningitis caused by multi drug-resistant Acinetobacter baumannii: Clinical, microbiological and pharmacokinetic results in a patient treated with colistin methanesulfonate

This paper reports on a 71- year-old Caucasian male who underwent neurosurgery for an oligodendroglioma, followed by a cranial-sinus fistula and cerebrospinal fluid rhinorrhea. The clinical course was complicated due to an extensively drug-resistant Acinetobacter baumannii meningitis. The patient was treated with colistin methanesulfonate, intrathecal for 24 days and intravenous for 46 days. In addition, the patient received meropenem and teicoplanin to treat a urinary tract infection and a bacterial aspiration pneumonia. Cerebrospinal fluid trough colistin levels resulted above the MIC of A. baumannii. Colistin cerebrospinal fluid concentration did not increase over the treatment period. Meningitis was cured and A. baumannii eradicated. No side effects from the antimicrobial therapy were observed. In conclusion, this case highlights the issues in treating infections caused by resistant Gram negative bacteria and supports previous findings on the efficacy, pharmacokinetic and tolerability of intravenous and intrathecal colistin treatments

Infections with VIM-1 Metallo-β-Lactamase-Producing Enterobacter cloacae and Their Correlation with Clinical Outcome▿

The aim of this study was to ascertain the incidence and clinical significance of metallo-β-lactamases among Enterobacter strains isolated from patients with nosocomial infections. We prospectively collected data on patients with Enterobacter infection during a 13-month period. All of the strains were investigated for antibiotic susceptibility, the presence and expression of metallo-β-lactamases, and clonality. Of 29 infections (11 involving the urinary tract, 7 pneumonias, 3 skin/soft tissue infections, 3 intra-abdominal infections, 3 bacteremias, and 2 other infections), 7 (24%) were caused by Enterobacter cloacae strains harboring a blaVIM-1 gene associated or not with a blaSHV12 gene. Infections caused by VIM-1-producing strains were more frequently associated with a recent prior hospitalization (P = 0.006), cirrhosis (P = 0.03), relapse of infection (P < 0.001), and more prolonged duration of antibiotic therapy (P = 0.01) than were other infections. All of the isolates were susceptible to imipenem and meropenem and had blaVIM-1 preceded by a weak P1 promoter and inactivated P2 promoters. Most VIM-1-producing Enterobacter isolates belonged to a main clone, but four different clones were found. Multiclonal VIM-1-producing E. cloacae infections are difficult to diagnose due to an apparent susceptibility to various beta-lactams, including carbapenems, and are associated with a high relapse rate and a more prolonged duration of antibiotic therapy

Epidemiologic characterization and distribution of carbapenem resistant Acinetobacter baumannii clinical isolates in Italy

This study was aimed at tracing the molecular characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB) clinical isolates in Italy with both pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Two hundred and two CRAB isolates were collected during 2004-2009, in two different surveillance periods, from 22 Italian hospitals that were representative for both distribution and infection. PFGE was performed, and the MLST scheme used was based on the gene sequence as published on the MLST Pasteur website http://www.pasteur.fr/mlst. Representatives of the major European clones I (RUH 875) and II (RUH 134) were used as controls. The two groups of isolates were characterized for their carbapenem resistance genes: 154 of 202 carried bla(OXA-58) alone, 21 of 202 also carried bla(OXA-23) , and 27 of 202 carried bla(OXA-23) alone. No isolates were positive for bla(OXA-24) . Genotype analysis of all isolates identified four distinct patterns by PFGE, which correlated with four distinct sequence types (STs) by MLST. The distribution of these four clusters in Italy confirmed the propensity of A. baumannii for nosocomial cross-transmission in a vast geographical area. We observed that clones A and B had similarities with European clone II and I respectively. By MLST, clone A was ST2, like European clone II, and clone B was ST1, like European clone I. PFGE and MLST showed the same discriminatory power and reproducibility. In addition, the two methods were concordant in defining CRAB Italian clones and in correlating them with the two pan-European clones

Combination of aztreonam, ceftazidime-avibactam and amikacin in the treatment of VIM-1 Pseudomonas aeruginosa ST235 osteomyelitis

We describe a challenging case of a patient with MBL-producing Pseudomonas aeruginosa sternal osteomyelitis following aortic valve replacement with biological prosthesis. The strain exhibited a multidrug-resistance phenotype carrying the blaVIM-1 gene and belonged to the high-risk clone sequence type ST235. The patient was successfully treated with surgical debridement plus antibiotic therapy with ceftazidime/avibactam, aztreonam, and amikacin. Time kill curves showed that this triple antibiotic combination at 1 X MIC was strongly synergic after 8\u2009hours, achieving 99.9% killing, and maintaining this until 48\u2009hours