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.

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 bla KPC-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 bla KPC-3 and bla SHV-11 genes. The molecular analysis demonstrated that the bla KPC-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 bla KPC-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.

Klebsiella pneumoniae represent a common invasive infection etiological agent, whose potential carbapenem-resistance and hypermucoviscosity complicate the patient’s management. Infection development often derives from gastrointestinal colonization; thus, it is fundamental to monitor asymptomatic K. pneumoniae colonization through surveillance protocols, especially for intensive care and immunocompromised patients. We described a six-month routine screening protocol from the Policlinico of Catania (Italy), while blood samples were collected from the same patients only in cases of a systemic infection suspicion. All the patients who had dissemination episodes were furtherly investigated through next-generation sequencing, analyzing both colonizing and disseminating strains. This study documents emerging invasive sequence types such as ST101, ST307, and ST395, mainly revealing blaNDM or blaKPC genes, along with siderophores and hyperproduction capsule markers as virulence factors. Most of the detected factors are presumably related to a specific plasmid content, which are extremely varied and rich. In conclusion, active surveillance through sequencing is essential to enhance awareness of local epidemiology within high-risk multi-drug resistance areas. A random sequencing analysis on the most warning microorganisms could enhance sequence typing (ST) awareness within specific settings, allowing for better prevention control strategies on their eventual persistence or diffusion.

Background/Objectives. Carbapenemase production is the most diffused carbapenem-resistance mechanism among Enterobacterales, with Klebsiella pneumoniae carbapenemase (KPC), Verona-imipenemase (VIM), New-Delhi metallo-β-lactamase (NDM), imipenemase (IMP), and oxacillinase (OXA-48) being reported as the main types within Europe. Particularly, Southern Italy holds a concerningly high percentage of carbapenemases-producing Enterobacterales diffused among different hospital settings. These strains may colonize critical patients’ gastrointestinal tracts, often causing disseminations and severe complications. Scientific data recently reported carbapenemase variants’ worldwide diffusion and several double-carbapenemases reports. The diagnostic routine needs devices whose detection rates are extended to similar epidemiological conditions, avoiding a lack of specificity and potential negative results. Methods. We planned a retrospective study including carbapenem- and/or ceftazidime/avibactam-resistant Enterobacterales (62) which were tested with the KPC/IMP/NDM/VIM/OXA-48 Combo Test Kit (KINVO, Medomics Medical Technology, Nanjing, Jiangsu, China) based on the lateral flow assay (LFA) method. Results. We compared its results to the phenotypic antimicrobial susceptibility testing (AST) MIC results, obtaining a 100% agreement rate. The LFA kit reported carbapenemases in all the tested strains, also identifying cases of KPC variants and double-carbapenemases production. Conclusions. Our data demonstrated how LFAs may represent a reliable alternative requiring minimum economic and personnel resources along with simple result interpretations. Future studies will be necessary to further investigate the system effectiveness on a larger isolates’ number and a broad carbapenemase variant spectrum.

Background/Objectives. Multi-drug-resistant (MDR) microorganisms pose a significant challenge in healthcare settings, particularly with beta-lactam-resistant Gram-negative bacteria and glycopeptide-resistant enterococci. Culture represents the most reliable technique in determining their presence within surveillance swabs. However, it requires a long time-to-result (TTR) and shows low sensitivity. Molecular techniques integrate diagnostic procedures, allowing TTR reduction and precise identification of genes. Methods. During our usual surveillance campaign, we had the opportunity to evaluate the Allplex Entero-DR assay (Seegene Inc., Seoul, Republic of Korea) and the Entero-DR Plus assay (Arrow Diagnostics srl, Genova, Italy) molecular kits for the detection of extended-β-lactamases (ESBL), carbapenem- and vancomycin-resistant genes, as well as Acinetobacter spp. and Pseudomonas aeruginosa spp. identification directly from rectal swabs. A comparison between these tests and the culture-based routine completed the study. Results. The analysis included 300 rectal swabs from the University Hospital Policlinico (Catania, Italy). One hundred and eighty-eight samples (62.6%) resulted as positive for at least one Allplex™ target, reaching optimal sensitivity and negative predictive value (100%). Our results underlined the ubiquitous blaCTX-M and van genes presence and demonstrated the diffusion of double-carbapenemases genes and metallo-β-lactamases-producing strains. In our epidemiological setting, few data were collected about carbapenem-resistant P. aeruginosa and Acinetobacter spp., which require further evaluations on simultaneous respiratory colonization and higher sample numbers. Conclusions. Our analysis highlighted the importance of combining conventional and advanced diagnostic methods in investigating MDR pathogens. The right approach should be based on the prevalence and variability of resistance mechanisms within a specific epidemiological area. Remarkably, molecular screenings may exclude negative samples within high-risk areas due to a significant negative predictive value.