Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Histological variety of gastric carcinoma and Helicobacter pylori cagA and vacA polymorphism.

BACKGROUND: Helicobacter pylori infection may increase the risk of both histotypes of gastric carcinoma (GC): intestinal (IGC) and diffuse (DGC). Polymorphism of the main H. pylori virulence factors, cagA and vacA, could have a different impact on the histological variety of GC. METHODS: Two hundred and twenty-six H. pylori colonies were examined from 28 GC patients: 15 with IGC and 13 with DGC. DNA was extracted from bacteria and a PCR was performed using primers specific for cagA, the 3' cagA variable region and s and m regions of vacA. RESULTS: There were 214 cagA+ strains and 55.61% of them were isolated from IGC cases; there were 12 cagA- colonies and they were all isolated from DGC (P<0.001). Most patients were infected by strains with more than one cagA structural type. No strains with a particular cagA type were found to be significantly more frequent in either histological variety of GC. A percentage of 43.90 of strains with vacA subtype s1/m1 were isolated from IGC, whereas 80.95% of vacA subtype s1/m2 strains were isolated from DGC cases (P<0.001). CONCLUSION: Polymorphism of the main virulence genes of H. pylori may play different roles in the pathogenesis of IGC and DGC.

Nucleotide variability of Toscana virus M segment in strains isolated from clinical cases.

Toscana virus (TOSV), a member of the Bunyaviridae family, is an important etiologic agent of neurologic infection transmissible to humans by bites of the Phlebotomus spp. In consideration of the variations in the antigenic properties of Bunyaviruses and their potential genetic variability, we analysed a large region (2500nt) of the Toscana virus M segment coding for the non-structural protein (NSm) and the G(N) and G(C) glycoproteins in several strains isolated from patients with meningitis from 1998 to 2004 in the region of Tuscany in Italy. The sequences were compared with the reference strain of Toscana virus isolated from phlebotomus (ISS Phl. 3) and revealed some changes in amino acids, particularly in the G(C) protein, that are probably involved in recognition and binding to the cell receptor. The analyses were aimed at identifying the amino acid changes commonly to all of the clinical isolates potentially related to TOSV virulence.

Development of a mouse model for the study of Toscana virus pathogenesis.

Toscana virus (TOSV) has recently been recognized as an emerging virus transmitted by phlebotomus vectors, responsible for acute neurological diseases in Mediterranean countries. In our study, we demonstrated that adult Balb/c mice were susceptible to TOSV when infected intracerebrally (i.c.) or subcutaneously (s.c.) with a neuroadapted strain of the virus. We have shown that by performing serial passages of a wild type human isolate of TOSV in mouse brains, selection occurs for a highly virulent variant which replicates efficiently in the central nervous system (CNS) of i.c.-injected mice, causing acute encephalitis and death. Immunohistochemical analysis and TUNEL assay of post-mortem organs showed that TOSV replication was highly restricted to neurons in which it induced apoptotic death; however, virus antigen-positivity was also observed in the spleen and lymph nodes. In s.c.-injected mice, virus was detectable in the spleen and lymph nodes, whereas only few meningeal cells and neurons were affected, allowing for the mouse survival the infection. The presence of TOSV in spleen and lymph node cells in both s.c.- and i.c.-treated mice suggests their possible involvement in the diffusion of the infection. This animal model may be helpful for the development of prophylactic measures against TOSV infections.

A Mediterranean arbovirus: the Toscana virus.

Toscana virus (Bunyaviridae family, Phlebovirus genus) is a sandfly fever virus responsible for human neurological infections. Sandfly viruses are transmitted by insect vectors (Phlebotomus species) and the infection is present in climatic areas that allow the life cycle of the vector. The arthropode-borne Toscana virus is the etiologic agent of meningitis, meningoencephalitis, and encephalitis. The frequency of this neuropathic infection increases in the summer months, peaking in August in the endemic Mediterranean areas (Italy, Portugal, Spain, and Cyprus). Infection diagnosis is carried out by molecular assays and immunoenzymatic tests, which are rapid and sensitive. Recent studies have investigated the antigenic properties of the viral proteins (nucleoprotein N and surface glycoproteins G1 and G2), to better understand their immunogentic role.

Epidemiological, clinical and laboratory aspects of sandfly fever.

PURPOSE OF REVIEW: Sandfly fever viruses are still a significant health problem in many regions of the world, such as Africa, the Mediterranean basin, the Middle East, and Central Asia. This review provides an update on the advances in knowledge about epidemiological, clinical, and laboratory aspects of infections caused by Toscana, Sicilian and Naples viruses. RECENT FINDINGS: Diagnosis of Toscana virus infection has been facilitated by new molecular methods and by immunoenzymatic tests based on the recombinant nucleoprotein. Gene analysis has allowed identification of circulating Toscana variants possibly involved in the protean pathomorphism and extreme variability of the clinical picture. New attention has been addressed to the antigenic properties of the viral proteins (the nucleoprotein N and the surface glycoproteins G1 and G2), in order to understand their immunogenetic role. High genetic divergence within the serocomplexes belonging to each of the Sicilian and the Naples viruses has suggested that infection with one genotype may not completely immunize against infection with all other genotypes in a given serocomplex. These findings could serve as a basis for vaccine development and may account for reports of multiple episodes of sandfly fever in the same host. Recently, the performance of analysis models based on weather data and reported vector surveys has allowed the prediction of the risk of acquiring sandfly infection in the endemic geographic areas. SUMMARY: Recent developments include a better knowledge of the epidemiological, clinical, and laboratory aspects of sandfly infection, while the search for effective drugs and vaccines is still in progress.

Serological survey of Toscana virus infections in a high-risk population in Italy.

Toscana virus is the most important agent responsible for meningitis in central Italy. We report a serosurveillance study, using an immunoenzymatic assay, of 360 serum samples harvested from a high-risk population occupationally exposed to Toscana virus in two regions of Italy, Tuscany and Piedmont. The results indicates a seroprevalence of Toscana virus of 77.2% in the forestry workers, particularly in the Tuscany region. This fact is strictly correlated with the ecological niches specific for the survival of Toscana virus arthropod vector.

Increasing diversity within Chlamydiae.

In recent years, 16S ribosomal DNA analyses has allowed the recognition of new chlamydia organisms, requiring the creation of new species, genera, and families within this unique, deep lineage of prokaryotes. The trachoma and psittaci groups chlamydiae are now recognized as separate genera, Chlamydia and Chlamydophila, respectively, and biovars of each group have been elevated to the species rank. Simkania and Parachlamydia have been associated with human respiratory infections, while Waddlia seems to be implicated in abortion in bovins. DNA amplification studies targeting the 16S rDNA have revealed a richer diversity within chlamydiae, identifying new lineages from both environmental and clinical samples. Further studies will be of interest to both examine the ecology and evaluate the clinical importance of these novel chlamydiae. Herein, we provide a summary of literature and our data about the novel chlamydial lineages.

Outbreak and persistence of Chlamydia pneumoniae infection in an Italian family.

We describe an outbreak of familial infection of Chlamydia pneumoniae, an etiological agent for respiratory tract infections. In a family member detection of C. pneumoniae on a pharyngeal swab by polymerase chain reaction was positive until four months after the onset of symptoms, despite a course of antibiotics known to be effective against Chlamydia species

New parachlamydial 16S rDNA phylotypes detected in human clinical samples.

Chlamydiales are important intracellular bacterial pathogens, causing a wide variety of diseases in vertebrates, including humans. Besides the well-known species in the family Chlamydiaceae, new chlamydial organisms have recently been discovered, forming three new families: Parachlamydiaceae, Simkaniaceae and Waddliaceae. Parachlamydia acanthamoebae and Simkania negevensis are currently investigated as emerging human respiratory pathogens. Additional chlamydial lineages have been discovered by 16S rDNA-based molecular studies, and their implication in human infections is poorly known. By using a pan-chlamydia 16S rDNA PCR, we have searched for the presence of chlamydiae in 228 clinical samples that all previously had been shown to be PCR-negative for Chlamydophila pneumoniae: 170 respiratory samples, 45 atheromatic plaques and 13 peripheral blood mononuclear cell samples. Nine respiratory samples tested positive. Sequence analysis has allowed us to assign four sequences to Chlamydophila psittaci, three sequences to Chlamydophila felis, and two sequences to two novel phylotypes belonging to the Parachlamydiaceae. These latter sequences showed similarity values of more than 93% with each other and with the P. acanthamoebae sequence, thus belonging to novel, unrecognized species. In conclusion, this report showed that a variety of non-C. pneumoniae chlamydial respiratory infection is present in humans, and that new parachlamydiae distinct from P. acanthamoebae may be detected in human clinical samples. Future studies will be of interest in order to estimate the diversity of these novel chlamydiae in both clinical and environmental samples, as well as their possible clinical implication in human and animal infections.

Fast duplex one-step RT-PCR for rapid differential diagnosis of entero- or toscana virus meningitis.

Acute meningitis is the most common neurologic disease that involves the central nervous system. The spectrum of infectious agents that cause neurologic infection is remarkably broad and numerous viruses are the most frequent cause of the aseptic meningitis syndrome. We applied a multiplex one-step method for the rapid detection of the genomic RNA of different neurotropic viruses: particles in the genus Enterovirus and Toscana virus, which are the most representative aetiologic agents in our country during the spring-summer period. We have evaluated the sensitivity and the specificity of the multiplex one-step test on positive controls and on RNA extracted from clinical samples harvested from 475 patients with meningitis hospitalized during the 1996-2001 period. The multiplex one-step RT-nPCR protocol allows for the detection of enterovirus and Toscana virus RNA in a single sample, by using, at the same time, a very small clinical sample volume. In our study we were able to diagnose 192 cases of meningitis by Toscana virus and 31 cases by enteroviruses out of 475 cases of meningitis utilizing the described one-step multiplex method.

Search for Chlamydia pneumoniae genes and their expression in atherosclerotic plaques of carotid arteries.

Samples of atherosclerotic tissue from 58 patients undergoing carotid surgery were analysed by tissue culture and PCR for Chlamydia pneumoniae; PCR was performed to detect Omp1, 16S rRNA and HSP-70 genes. To understand the active pathogenic role of C. pneumoniae, a reverse transcriptase-PCR (RT-PCR) assay was applied to detect the specific RNAs expressed either in the replicative form, or in the cryptic form found in chronic infection. The C. pneumoniae omp1 gene, encoding the major outer-membrane protein (MOMP), was detected in 13 of 58 samples. Among these, the result was confirmed in 11 samples after amplification of a further target, the 16S rRNA, and the presence of the HSP-70 gene, encoding heat-shock protein 70, was revealed in only five cases. All the samples were negative for evidence of specific RNAs by RT-PCR. The presence of genomic DNA and absence of specific RNAs in atherosclerotic tissue samples suggests a lack of an active metabolic or persistent infective role for C. pneumoniae. Thus, traces of C. pneumoniae DNA in these samples could be due to a degradative pathway of the host defensive cellular and biochemical mechanisms.