An Alicaligenes faecalis (FL-424/98) resistant to expanded-spectrum cephalosporins and aztreonam was isolated from the urine of an inpatient at the Intensive Care Unit of the Varese Hospital (Northern Italy) after antimicrobial chemotherapy with cefazolin, vancomycin, and amikacin. Clavulanic acid restored the activity of expanded-spectrum cephalosporins, suggesting the production of an extended-spectrum β-lactamase (ESβL). A crude extract of FL-424/98 showed the presence of two β-lactamase activities focusing at pH 5.3 and 7.6, respectively. The ESβL activity, purified by means of three Chromatographic steps, was found to correspond to the pI 5.3 enzyme. Determination of kinetic parameters confirmed that the enzyme efficiently hydrolyzed expanded-spectrum cephalosporins and aztreonam. A colony-blot hybridization revealed the presence of blaPER-related sequences in FL-424/98, and sequencing confirmed the identity of this determinant with blaPER-1, previously detected in Pseudomonas aeruginosa, Acinetobacter, and Salmonella clinical isolates from Turkey. Finding of blaPER-1 in a species that can be part of the resident human microbiota raises the possibility that it could be an efficient shuttle for spreading of this resistance gene among other opportunistic pathogens that are normally members of the resident microbiota. Kinetic parameters determined for the PER-1 enzyme with some cephalosporin substrates were somewhat different from those previously reported.
Two epidemiologically unrelated carbapenem-resistant Acinetobacter baumannii isolates were investigated as representatives of the first Italian isolates producing the OXA-24 carbapenemase. Both isolates were of European clonal lineage II and carried an identical OXA-24-encoding plasmid, named pABVA01. Comparative analysis revealed that in pABVA01, bla(OXA-24) was part of a DNA module flanked by conserved inverted repeats homologous to XerC/XerD binding sites, which in other Acinetobacter plasmids flank different DNA modules, suggesting mobilization by a novel site-specific recombination mechanism.
This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of blaKPC-53, located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.
Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-beta-lactamases, which confer high-level resistance to carbapenems and most other beta-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were >/=32 micro g/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-beta-lactamase activity, while molecular analysis detected in each isolate a bla(VIM-1) determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the beta-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the bla(VIM-1) determinant (named In110) showed that the bla(VIM-1)-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a bla(VIM-1)-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.
A mong molecular assays currently developed for detection and identification of pathogens (and their antimicrobial resistance genes) in positive blood cultures (BCs) (1), the BioFire FilmArray blood culture identification (BCID) panel (bioMérieux, Marcy l'Etoile, France), a multiplex PCR assay with ,2 min of hands-on time and an ;1-h turnaround time, allows for syndromic diagnosis of bloodstream infection (BSI) (2, 3).Previously, the panel could identify 24 etiological agents of BSI (11 Gram-negative bacteria, 8 Gram-positive bacteria, and 5 yeast species), as well as three antimicrobial resistance genes (mecA, vanA/B, and bla KPC , which encodes Klebsiella pneumoniae carbapenemase).Now, the BioFire FilmArray BCID2 panel encompasses 43 molecular targets associated with BSI, including 15 Gram-negative bacteria, 11 Gram-positive bacteria, 7 yeast species, and 10 antimicrobial resistance genes (https://www.biomerieux-diagnostics.com/biofire-bcid-panel).The last targets include genes encoding carbapenemases (IMP, KPC, OXA-48-like, NDM, and VIM), colistin resistance (mcr-1), extendedspectrum b-lactamase (ESBL) (CTX-M), methicillin resistance (mecA/C and, specifically for methicillin-resistant Staphylococcus aureus [MRSA], mecA/C and MREJ [mec right-extremity junction]), or vancomycin resistance (vanA/B).Unlike BCID, no published studies to date reported on BCID2 performance.This study evaluated and compared the accuracy of BCID2 with that of BCID to identify bacterial species and relative antimicrobial resistance genes directly from positive BCs.We used archived samples from positive BCs (1.5 ml thereof mixed with 100 ml of dimethyl sulfoxide and stored at 280°C), which had prospectively been processed with the BD Bactec 9240 (BD Diagnostic Systems, Sparks, MD), BacTAlert 3D (bioMérieux), or BacTAlert Virtuo (bioMérieux) system at two hospital microbiology laboratories from January 2018 to August 2020.For BCID2 testing, samples were thawed and allowed to reach room temperature, and a 200-ml aliquot was lysed and processed following the manufacturer's instructions.We compared BCID2 results with those of conventional identification or detection (i.e., MALDI BioTyper analysis and, relative to antimicrobial resistance genes, PCR sequencing, both performed on microbial isolates subcultured from positive BC samples), which was considered the reference method in this evaluation, or with those of the BCID.
The aim of antimicrobial resistance surveillance is to monitor temporal trends and provide clinicians with data to define empirical treatment protocols. The surveillance methods adopted in different settings can be significantly different and, therefore, no reference can be made to a single set of standards. This paper outlines the main features of analysis and reporting of antimicrobial resistance data according to the guidelines issued by the US Clinical and Laboratory Standards Institute, and the surveillance systems adopted in Europe. In this article the strengths and weaknesses of the various types of analyses will be discussed highlighting the critical aspects to be taken into account in surveillance data reporting.
