In vitro evidence of the synergistic interaction of ceftopibrole and other antibiotics against multidrug-resistant Gram-negative isolates.

The purpose of the present study was to investigate the in vitro activity of ceftobiprole in combination with other antimicrobials against 27 selected Gram-negative isolates, including ESBL-producing E. coli and KPC-OXA-48-producing K. pneumoniae. Ceftobiprole activity in combination with amikacin, colistin, levofloxacin, piperacillin/tazobactam and rifampin was evaluated by time-kill curves and gradient-cross method (except colistin). Among the 27 strains tested with gradient strips most were resistant to ceftobiprole. Synergy was observed in some cases with piperacillin/tazobactam. There was at least one synergistic combination towards 9 isolates belonging to different species. No antagonism was observed with any of the antibiotic tested. In time-kill curves, performed for 12 selected isolates, synergistic interaction was more frequent, occurring with 32/60 combinations. The most interesting results of our study are the bactericidal effects of ceftobiprole in combination with colistin or piperacillin/tazobactam tested against Gram-negative isolates, including KPC and OXA-48-producing K. pneumoniae.

Extensively drug-resistant ArmA-producing Acinetobacter baumannii in an Italian intensive care unit.

We describe the spread of 12 carbapenem-resistant Acinetobacter baumannii isolates in hospitalized patients. All strains showed an extensively drug-resistant phenotype and high-level of aminoglycoside resistance, harboring the ArmA gene and blaoxa-23 downstream of ISAba1 (transposon Tn2008 arrangement) where both were located on the chromosome. These strains carry a class 1 integron containing the gene cassette aacA4-catB8-aadA1. Molecular analysis revealed that all isolates belonged to the same sequence type (ST) 2 clone. The spread of ArmA-producing A. baumannii strains limit the treatment options showing the dramatic situation which requires novel therapies to limit high mortality rates.

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.

Detection of the IncX3 plasmid carrying blaKPC-3 in a Serratia marcescens strain isolated from a kidney-liver transplanted patient.

Dissemination of resistance to carbapenems among Enterobacteriaceae through plasmids is an increasingly important concern in health care worldwide. Here we report the first description of an IncX3 plasmid carrying the blaKPC-3 gene in a strain of Serratia marcescens isolated from a kidney-liver transplanted patient at the transplantation centre ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione, Palermo, Italy). To localize the transposable element containing the resistance-associated gene Next-Generation Sequencing of the bacterial DNA was performed. S. marcescens was positive for blaKPC-3 and blaSHV-11 genes. The molecular analysis demonstrated that the blaKPC-3 gene of this bacterial strain was located in one copy of the Tn-3-like element Tn4401-a carried in a plasmid that is 53 392 bp in size and showed the typical IncX3 scaffold. Our data demonstrated the presence of a new blaKPC-3 harbouring the IncX3 plasmid in S. marcescens. The possible dissemination among Enterobacteriaceae of this type of plasmid should be monitored and evaluated in terms of clinical risk.

Successful ceftazidime-avibactam treatment of MDR-KPC-positive Klebsiella pneumoniae infection in a patient with traumatic brain injury: A case report.

RATIONALE: Carbapenem-resistant Enterobacteriaceae infections are a serious health care problem, because of the high mortality. Carbapenem resistance is mainly caused by carbapenemases production, including Klebsiella pneumoniae carbapenemase (KPC). Ceftazidime-avibactam is a new cephalosporin/ß-lactamase inhibitor combination for the treatment of complicated urinary, intra-abdominal infections, and nosocomial pneumonia caused by gram negative, or other serious gram-negative infections. PATIENT CONCERNS: We showed the case of a 27-year-old patient, hospitalized for traumatic brain injury and chest trauma, with KPC-producing Klebsiella pneumoniae infection. DIAGNOSES: Blood and bronchial aspirate culture analysis detected an infection caused by MDR Klebsiella pneumoniae, resistant to meropenem, ertapenem, piperacillin/tazobactam, amoxicillin/clavulanic acid, aztreonam, ceftazidime, cefotaxime, cefepime, amikacin, ciprofloxacin, trimethoprim/sulfamethoxazole, colistin while it showed an intermediate sensitivity to gentamicin and was sensitive to ceftazidime-avibactam. Molecular analyses revealed that the isolate belonged to the epidemic clone sequence type 258 (ST258) carrying blaKPC-3, blaTEM-1, and blaSHV-11genes. INTERVENTIONS: After various combined antibiotic therapies without improvements, he was treated with ceftazidime-avibactam, on a compassionate-use basis. OUTCOMES: With ceftazidime-avibactam monotherapy clinical and microbiological clearance was obtained. A week after the end of the therapy microbiological analysis was repeated and a positive rectal swab for KPC-Klebsiella pneumoniae was found, becoming negative after 1 month. Moreover, the patient did not show any relapses for up to 18 weeks. LESSONS: This case indicates that ceftazidime-avibactam monotherapy could be efficacious against KPC positive Klebsiella pneumoniae infections.

In vitro activity of fosfomycin trometamol and other oral antibiotics against multidrug-resistant uropathogens.

Clinical midstream and urinary catheter isolates (n = 106) of extended-spectrum ß-lactamase (ESBL)-positive Escherichia coli, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae, Proteus mirabilis and meticillin-resistant Staphylococcus saprophyticus were tested against fosfomycin using the agar dilution method, the broth microdilution method and the gradient test described by the Clinical and Laboratory Standards Institute. Nitrofurantoin, co-trimoxazole, amoxicillin/clavulanic acid, cefuroxime, levofloxacin and ciprofloxacin were tested using the gradient test alone. Breakpoints from the European Committee on Antimicrobial Susceptibility Testing 2015 guidelines were used. Fosfomycin inhibited all of the ESBL-positive E. coli, P. mirabilis and meticillin-resistant S. saprophyticus strains isolated from urine, as well as 82% of KPC-producing K. pneumoniae isolates. Substantial agreement for fosfomycin activity was found for the three test methods, particularly for Enterobacteriaceae. This study confirmed that fosfomycin has good in vitro activity against more common multidrug-resistant uropathogens. Fosfomycin could be a reliable empirical therapeutic option for uncomplicated urinary tract infections caused by these organisms, and a valid option for sparing parenteral antibiotics, such as carbapenems.

Colistin Increases the Cidal Activity of Antibiotic Combinations Against Multidrug-Resistant Klebsiella pneumoniae: An In Vitro Model Comparing Multiple Combination Bactericidal Testing at One Peak Serum Concentration and Time-Kill Method.

The lack of treatment for multidrug-resistant (MDR) Enterobacteriaceae often leads to the use of double or triple antibiotic combinations to increase the option of clinical success. This study analyzes multiple combination bactericidal testing (MCBT) to screen double and triple antibiotic combinations, at standard peak serum concentration, for bactericidal activity against 21 MDR Klebsiella pneumoniae isolates. This method was compared with time-killing curves. The full bactericidal activity against all strains was obtained only by adding colistin. MCBT has a potential to become a rapid method for testing multiple antibiotic combinations for MDR microorganisms when colistin is used, providing clinicians with in vitro cidal data within 48 hr of strain isolation.

Carbapenem and multidrug resistance in Gram-negative bacteria in a single centre in Italy: considerations on in vitro assay of active drugs.

In intensive care units (ICUs), the most important causes of nosocomial bacterial infections are mainly multidrug-resistant (MDR) and extensively drug-resistant (XDR) Acinetobacter baumannii and Klebsiella pneumoniae strains. Mortality related to these infections is very high due to lack of effective therapy and the severity of patient conditions. This study aimed to assess the prevalence of carbapenem resistance genes in 77 carbapenem-resistant Gram-negative bacteria isolated from severe infections (bloodstream, pulmonary and urinary tract) during the period 1 January to 31 July 2013 in a general ICU in Catania, Italy, and to examine their susceptibility to tigecycline and colistin using two different methods. In total, 52 A. baumannii belonging to the same sequence type (ST) 2 clone and carrying the bla(OXA-23) gene as well as 25 K. pneumoniae carrying bla(KPC-3) were isolated. Four distinct pulsotypes were identified in K. pneumoniae, which correlated with four distinct STs: ST258 and ST512, spread worldwide, and ST147 and ST395 detected for the first time in Italy. A. baumannii isolates showed an XDR profile and were fully susceptible only to colistin; all KPC-producing K. pneumoniae isolates were MDR, whilst colistin was active against 19 of 25 strains. These results show that broth microdilution (BMD) is a reliable in vitro susceptibility test for colistin, above all K. pneumoniae, whilst both the gradient test and BMD are suitable for tigecycline susceptibility testing of A. baumannii.