Photobacteriosis or fish pasteurellosis is a bacterial disease affecting wild and farm fish. Its etiological agent, the gram negative bacteriumPhotobacterium damselaesubsp.piscicida, is responsible for important economic losses in cultured fish worldwide, in particular in Mediterranean countries and Japan. Efforts have been focused on gaining a better understanding of the biology of the pathogenic microorganism and its natural hosts with the aim of developing effective vaccination strategies and diagnostic tools to control the disease. Conventional vaccinology has thus far yielded unsatisfactory results, and recombinant technology has been applied to identify new antigen candidates for the development of subunit vaccines. Furthermore, molecular methods represent an improvement over classical microbiological techniques for the identification ofP. damselaesubsp.piscicidaand the diagnosis of the disease. The complete sequencing, annotation, and analysis of the pathogen genome will provide insights into the pathogen laying the groundwork for the development of vaccines and diagnostic methods.

Plasmids are the main vectors of resistance and virulence genes in Enterobacteriaceae and plasmid typing is essential for the analysis of evolution, epidemiology and spread of antibacterial resistance. The PCR-Based Replicon Typing (PBRT), developed by Carattoli et al. in 2005, was an efficient method for plasmid identification and typing in Enterobacteriaceae. The 2005 PBRT scheme detected 18 replicons in 8 PCR reactions. Recently, the identification of novel replicons and plasmid types requested an update of the PBRT scheme. A commercial PBRT-KIT was devised for the identification of 28 different replicons in 8 multiplex PCRs. Here we report sensitivity and specificity of the PBRT-KIT carried out in comparison with the 2005 PBRT. The analysis of plasmid content was performed on forty-two enterobacterial strains from different sources, containing different replicon content. The 2005 PBRT identified replicons in 76.2% of the strains. The PBRT-KIT detected replicons in 100% of the analyzed strains, demonstrating increasing sensitivity and specificity of the commercial test with respect to the former 2005 PBRT scheme.

Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as the target sequence. These assays had potential cross-species amplification, due to sequence similarity between Leishmania species. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high-resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographical area. DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were amplified by a previously developed qPCR (qPCR-ML) and subjected to HRM analysis; selected PCR products were sequenced using an ABI PRISM 310 Genetic Analyzer. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. The qPCR-ML followed by HRM analysis did not allow discrimination between L. (L.) amazonensis and L. (L.) infantum in 53.4% of cases. Hence, the novel SYBR green-based qPCR (qPCR-ama) has been tested. This assay achieved a detection limit of 0.1 pg of parasite DNA in samples spiked with host DNA and did not show cross amplification with Trypanosoma cruzi or host DNA. Although the qPCR-ama also amplified L. (L.) infantum strains, the Cq values were dramatically increased compared to qPCR-ML. Therefore, the combined analysis of Cq values from qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples. A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a minicircle sequence rather than targeting a hypothetical species-specific sequence. The fast and accurate discrimination between these species can be useful to provide adequate prognosis and treatment.

The emerging spread of carbapenemase-producing Enterobacterales (CPE) strains, in particular, Klebsiella pneumoniae and Escherichia coli, has become a significant threat to hospitalized patients. Carbapenemase genes are frequently located on plasmids than can be exchanged among clonal strains, increasing the antibiotic resistance rate. The aim of this study was to determine the prevalence of CPE in patients upon their admission and to analyze selected associated factors. An investigation of the antibiotic resistance and genetic features of circulating CPE was carried out. Phenotypic tests and molecular typing were performed on 48 carbapenemase-producing strains of K. pneumoniae and E. coli collected from rectal swabs of adult patients. Carbapenem-resistance was confirmed by PCR detection of resistance genes. All strains were analyzed by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST) was performed on a representative isolate of each PBRT profile. More than 50% of the strains were found to be multidrug-resistant, and the bla_KPC gene was detected in all the isolates with the exception of an E. coli strain. A multireplicon status was observed, and the most prevalent profile was FIIK, FIB KQ (33%). MLST analysis revealed the prevalence of sequence type 512 (ST512). This study highlights the importance of screening patients upon their admission to limit the spread of CRE in hospitals.