The molecular detection of carbapenem markers with a two-levels amplification screening protocol: epidemiological and resistome insights.

Objectives: Carbapenem-resistance is a challenging healthcare concern and require specific stewardship programs. Monitoring workflows include the identification from surveillance samples, such as rectal swabs. Although culture assays represent the gold standard, data report a significant effectiveness in detecting carbapenemases genes directly from rectal swabs. The aim of this study was to evaluate the REALQUALITY Carba-Screen kit (AB ANALITICA, Padova, Italy) in detecting carbapenemases genes directly from rectal swabs, also comparing its effectiveness to culture assays results. A next-generation sequencing (NGS) was performed to investigate the positive samples about resistance markers and sequence type (ST). Methods: A number of 136 rectal swabs were collected from the University Hospital Policlinico of Catania critical wards. The samples simultaneously underwent culture and molecular assays (REALQUALITY Carba-Screen kit). The molecular method included two-steps. The first step (1 h and 6 min) rapidly excluded negative samples, while the second one (1 h and 6 min) included only positive samples for a resistance confirmation. All the positive culture samples underwent NGS analysis. Results: Statistical evaluations demonstrated high sensitivity (100%) and detection rates (92.6%) for the REALQUALITY Carba-Screen kit, which mostly correlated to the standard workflow. All the culture positive results matched the positive molecular results, which were mainly confirmed by the NGS resistome analysis. The identified ST appeared to be diversified and different from the clinically significative strains of the same setting, furnishing interesting epidemiological evidence. Conclusion: The molecular detection allowed a coordinate approach in a high-prevalence multi-drug-resistance area. The rapid identification with a multi-step procedure accelerated the infection control procedures, while the preliminary negative results reduced the overtreatment episodes. The molecular method efficacy was confirmed through the NGS. In conclusion, the molecular screening could initially lead to a more conservative approach, which may be reevaluated after a culture result about the microorganisms' identification and susceptibility profile.

Identification and Antibiotic-Susceptibility Profiling of Infectious Bacterial Agents: A Review of Current and Future Trends.

Antimicrobial resistance is one of the most worrying threats to humankind with extremely high healthcare costs associated. The current technologies used in clinical microbiology to identify the bacterial agent and profile antimicrobial susceptibility are time-consuming and frequently expensive. As a result, physicians prescribe empirical antimicrobial therapies. This scenario is often the cause of therapeutic failures, causing higher mortality rates and healthcare costs, as well as the emergence and spread of antibiotic resistant bacteria. As such, new technologies for rapid identification of the pathogen and antimicrobial susceptibility testing are needed. This review summarizes the current technologies, and the promising emerging and future alternatives for the identification and profiling of antimicrobial resistance bacterial agents, which are expected to revolutionize the field of clinical diagnostics.

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.

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.

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.