Durability of neutralizing antibodies against yellow fever virus after vaccination in healthy adults.

In accordance with the World Health Organization, one dose of yellow fever vaccine may guarantee protection lifelong in healthy adults. However, relatively little information is still available from ad hoc studies. We evaluated the persistence of neutralizing antibodies, which are considered to be an immune correlate of protection, in a large number of military personnel vaccinated up to 47 years before. Overall, 322 individuals were studied. The median time from vaccination to blood collection for neutralizing antibody evaluation was 9 years, ranging from <1 to 47 years. Of the 322 participants, 319 had neutralizing antibodies (99.1 %). The highest median PRNT50 value was observed in those vaccinated ≤1 year before (median PRNT50 = 320). In conclusion, our study confirms on a larger scale that, in healthy adults, neutralizing antibodies may persist as long as 47 years after a single yellow fever vaccines dose.

Seven Years of Culture Collection of Neisseria gonorrhoeae: Antimicrobial Resistance and Molecular Epidemiology.

The emergence of Neisseria gonorrhoeae isolates displaying resistance to antimicrobials, in particular to ceftriaxone monotherapy or ceftriaxone plus azithromycin, represents a global public health concern. This study aimed to analyze the trend of antimicrobial resistance in a 7-year isolate collection retrospective analysis in Italy. Molecular typing on a subsample of gonococci was also included. A total of 1,810 culture-positive gonorrhea cases, collected from 2013 to 2019, were investigated by antimicrobial susceptibility, using gradient diffusion method, and by the N. gonorrhoeae multiantigen sequence typing (NG-MAST). The majority of infections occurred among men with urogenital infections and 57.9% of male patients were men who have sex with men. Overall, the cefixime resistance remained stable during the time. An increase of azithromycin resistance was observed until 2018 (26.5%) with a slight decrease in the last year. In 2019, gonococci showing azithromycin minimum inhibitory concentration above the EUCAST epidemiological cutoff value (ECOFF) accounted for 9.9%. Ciprofloxacin resistance and penicillinase-producing N. gonorrhoeae (PPNG) percentages increased reaching 79.1% and 18.7% in 2019, respectively. The most common sequence types identified were 5,441, 1,407, 6,360, and 5,624. The predominant genogroup (G) was the 1,407; moreover, a new genogroup G13070 was also detected. A variation in the antimicrobial resistance rates and high genetic variability were observed in this study. The main phenotypic and genotypic characteristics of N. gonorrhoeae isolates were described to monitor the spread of drug-resistant gonorrhea.

Antimicrobial susceptibility profiles and genotyping of Neisseria meningitidis of serogroup C, Italy, 2000-2020.

Background: In Italy the introduction of meningococcal C conjugate vaccine in 2005 has led to a significant reduction of invasive meningococcal disease (IMD) caused by Neisseria meningitidis of serogroup C (MenC). However, this serogroup is still responsible of sporadic cases, clusters and local outbreaks. The study aims to investigate the genotype and antimicrobial susceptibility profile of MenC isolates collected in Italy from 2000 to 2020. Methods: Bacterial isolates and biological samples (blood or cerebrospinal fluid) from invasive meningococcal cases are collected and characterized at the National Reference Laboratory for IMD of Istituto Superiore di Sanità. Antimicrobial susceptibility was determined by MIC Test Strip Method and interpreted according to the EUCAST breakpoints guideline. Genotypic characteristics, including multi locus sequence typing (MLST), finetype, and antimicrobial resistance target genes were performed and analyzed using the PubMLST database. Genomic comparison of core genome MLST (cgMLST) of MenC genomes was also carried out. Results: From 2000 to 2020, a total of 665 MenC isolates were investigated for antimicrobial susceptibility and 301 for genotyping. Over two decades, almost all MenC isolates resulted susceptible to antimicrobials with few isolates resulting resistant to ciprofloxacin (N = 2), penicillin G (N = 13), and rifampicin (N = 9), respectively. Molecular typing of MenC obtained from isolates or clinical specimens identified mostly the genotype C:P1.5-1,10-8:F3-6:ST-11(cc11). However, phylogenetic analysis, performed on genomes from MenC isolates, identified two sub lineages, 11.1 and 11.2, among cc11, of which the sub lineage 11.2 was the predominant. Conclusion: Wider application of the genomic analysis and monitoring of antimicrobial susceptibility represent key aspects of IMD surveillance and to monitor the continued evolution of these hyperinvasive strains.

Reactive vaccination as control strategy for an outbreak of invasive meningococcal disease caused by Neisseria meningitidis C:P1.5-1,10-8:F3-6:ST-11(cc11), Bergamo province, Italy, December 2019 to January 2020.

In Italy, serogroup C meningococci of the clonal complex cc11 (MenC/cc11) have caused several outbreaks of invasive meningococcal disease (IMD) during the past 20 years. Between December 2019 and January 2020, an outbreak of six cases of IMD infected with MenC/cc11 was identified in a limited area in the northern part of Italy. All cases presented a severe clinical picture, and two of them were fatal. This report is focused on the microbiological and molecular analysis of meningococcal isolates with the aim to reconstruct the chain of transmission. It further presents the vaccination strategy adopted to control the outbreak. The phylogenetic evaluation demonstrated the close genetic proximity between the strain involved in this outbreak and a strain responsible for a larger epidemic that had occurred in 2015 and 2016 in the Tuscany Region. The rapid identification and characterisation of IMD cases and an extensive vaccination campaign contributed to the successful control of this outbreak caused by a hyperinvasive meningococcal strain.

Genome analysis of Legionella pneumophila ST23 from various countries reveals highly similar strains.

Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

Molecular characterisation and antibiotic susceptibility of meningococcal isolates from healthy men who have sex with men.

OBJECTIVES: To evaluate and characterise meningococcal carriage among healthy men who have sex with men (MSM) within a screening programme for Neisseria gonorrhoeae infection at the San Gallicano Dermatological Institute, Italy. METHODS: A total of 441 MSM attending the STI/HIV Centre of the San Gallicano Institute, Rome, Italy, in 2016 were routinely screened for N. gonorrhoeae infection by pharyngeal and rectal swabs. N. meningitidis isolates were evaluated for antibiotic susceptibility and characterised by whole genome sequencing. Genetic relationships among the meningococcal carriage isolates were determined using core genome multilocus sequence typing analysis. The soluble domain of AniA (sAniA) protein expression by western blotting was also evaluated. RESULTS: A total of 62 (14.1%, 95% CI 11.1 to 17.6) carriage meningococci were found among 441 MSM. Forty-three viable N. meningitidis isolates were cultivated (42 from pharyngeal and 1 from rectal swabs). All the viable isolates were susceptible to cefotaxime, ceftriaxone, ciprofloxacin and rifampicin. Four isolates were penicillin G-resistant and 73% of those penicillin G-susceptible showed a minimum inhibitory concentration from 0.064 µg/mL to 0.25 µg/mL. Serogroup B was the most frequent (44.2%), followed by Z (16.3%), E (9.3%), and Y and W (2.3%), respectively. Multilocus sequence typing analysis identified 29 sequence types belonging to 12 clonal complexes. The sAniA protein was expressed in 8 out of 28 (29%) screened meningococcal carriage isolates. CONCLUSIONS: Serogroup B meningococcal carriage identified from oral and anal specimens among healthy MSM was the most frequent serogroup identified in this study. Molecular evaluation revealed a degree of similarity among strains belonging to the same clonal complex.

Draft Genome Sequence and Biofilm Production of a Carbapenemase-Producing Klebsiella pneumoniae (KpR405) Sequence Type 405 Strain Isolated in Italy.

Rapid identification and characterization of multidrug-resistant Klebsiella pneumoniae strains is essential to diagnose severe infections in patients. In clinical routine practice, K. pneumoniae is frequently identified and characterized for outbreak investigation. Pulsed-field gel electrophoresis or multilocus sequence typing could be used, but, unfortunately, these methods are time-consuming, laborious, expensive, and do not provide any information about the presence of resistance and virulence genes. In recent years, the decreasing cost of next-generation sequencing and its easy use have led to it being considered a useful method, not only for outbreak surveillance but also for rapid identification and evaluation, in a single step, of virulence factors and resistance genes. Carbapenem-resistant strains of K. pneumoniae have become endemic in Italy, and in these strains the ability to form biofilms, communities of bacteria fixed in an extracellular matrix, can defend the pathogen from the host immune response as well as from antibiotics, improving its persistence in epithelial tissues and on medical device surfaces.

Meningococcal B vaccine antigen FHbp variants among disease-causing Neisseria meningitidis B isolates, Italy, 2014-2017.

BACKGROUND: Typing of Neisseria meningitidis isolates is crucial for the surveillance of invasive meningococcal disease (IMD). We performed a molecular epidemiology study of N. meningitidis serogroup B (MenB) causing IMD in Italy between 2014 and 2017 to describe circulating strains belonging to this serogroup, with particular regards to the two factor H-binding protein (FHbp) subfamilies present in the bivalent MenB vaccine. MATERIALS AND METHODS: A total of 109 culture positive and 46 culture negative MenB samples were collected within the National Surveillance System (NSS) of IMD in Italy and molecularly analyzed by conventional methods. RESULTS: Overall, 71 MenB samples showed the FHbp subfamily A and 83 the subfamily B. The subfamily variants were differently distributed by age. The most frequent variants, A05 and B231, were associated with cc213 and cc162, respectively. All MenB with the FHbp A05 variant displayed the PorA P1.22,14 and 85.7% of them the FetA F5-5. The majority of MenB with the FHbp B231 variant showed the PorA P1.22,14 (65.4%) and 84.6%, the FetA F3-6. CONCLUSION: MenB circulating in Italy were characterized by a remarkable association between clonal complex and FHbp variants, although a high degree of genetic diversity observed over time. A dynamic trend in clonal complexes distribution within MenB was detected. Our results stress the importance of continued meningococcal molecular surveillance to evaluate the potential vaccine coverage of the available MenB vaccines.

Whole genome and phylogenetic analysis of two SARS-CoV-2 strains isolated in Italy in January and February 2020: additional clues on multiple introductions and further circulation in Europe.

Whole genome sequences of SARS-CoV-2 obtained from two patients, a Chinese tourist visiting Rome and an Italian, were compared with sequences from Europe and elsewhere. In a phylogenetic tree, the Italian patient's sequence clustered with sequences from Germany while the tourist's sequence clustered with other European sequences. Some additional European sequences in the tree segregated outside the two clusters containing the patients' sequences. This suggests multiple SARS-CoV-2 introductions in Europe or virus evolution during circulation.

Co-existence of virulence factors and antibiotic resistance in new Klebsiella pneumoniae clones emerging in south of Italy.

BACKGROUND: Endemic presence of Klebsiella pneumoniae resistant to carbapenem in Italy has been due principally to the clonal expansion of CC258 isolates; however, recent studies suggest an ongoing epidemiological change in this geographical area. METHODS: 50 K. pneumoniae strains, 25 carbapenem-resistant (CR-Kp) and 25 susceptible (CS-Kp), collected from march 2014 to march 2016 at the Laboratory of Bacteriology of the Paolo Giaccone Polyclinic University hospital of Palermo, Italy, were characterized for antibiotic susceptibility and fully sequenced by next generation sequencing (NGS) for the in silico analysis of resistome, virulome, multi-locus sequence typing (MLST) and core single nucleotide polymorphism (SNP) genotypes RESULTS: MLST in silico analysis of CR-Kp showed that 52% of isolates belonged to CC258, followed by ST395 (12%), ST307 (12%), ST392 (8%), ST348 (8%), ST405 (4%) and ST101 (4%). In the CS-Kp group, the most represented isolate was ST405 (20%), followed by ST392 and ST15 (12%), ST395, ST307 and ST1727 (8%). The in silico ß-lactamase analysis of the CR-Kp group showed that the most detected gene was blaSHV (100%), followed by blaTEM (92%), blaKPC (88%), blaOXA (88%) and blaCTX-M (32%). The virulome analysis detected mrk operon in all studied isolates, and wzi-2 was found in three CR-Kp isolates (12%). Furthermore, the distribution of virulence genes encoding for the yersiniabactin system, its receptor fyuA and the aerobactin system did not show significant distribution differences between CR-Kp and CS-Kp, whereas the Klebsiella ferrous iron uptake system (kfuA, kfuB and kfuC genes), the two-component system kvgAS and the microcin E495 were significantly (p < 0.05) prevalent in the CS-Kp group compared to the CR-Kp group. Core SNP genotyping, correlating with the MLST data, allowed greater strain tracking and discrimination than MLST analysis. CONCLUSIONS: Our data support the idea that an epidemiological change is ongoing in the Palermo area (Sicily, Italy). In addition, our analysis revealed the co-existence of antibiotic resistance and virulence factors in CR-Kp isolates; this characteristic should be considered for future genomic surveillance studies.

Genomic Characterization of Gonococci from Different Anatomic Sites, Italy, 2007-2014.

In recent decades, Neisseria gonorrhoeae has developed resistance to several antimicrobial classes. Molecular epidemiology approaches are useful for detecting emerging, often resistant, gonococcal clones. In this study, 67 N. gonorrhoeae isolates from different anatomic sites, collected over 8 years in Italy, were analyzed by whole genome sequencing (WGS). WGS was performed using the Illumina NextSeq 500 platform. Phylogenetic analysis was based on core single nucleotide polymorphism (SNP) and core genome multilocus sequence typing (cgMLST). N. gonorrhoeae multi-antigen sequence typing (NG-MAST), MLST, and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were carried out in silico using WGS data. Antimicrobial susceptibility against a four-drug panel was evaluated using a gradient diffusion method. Overall, gonococci clustered in accordance with NG-MAST, MLST, NG-STAR, and antimicrobials susceptibility profiles, but not with the site of isolation, HIV status, and patient sexual orientation. Phylogenetic analysis identified nine clades: two of them were the predominant and including gonococci of G1407 and G2400 genogroups.

Carriage meningococcal isolates with capsule null locus dominate among high school students in a non-endemic period, Italy, 2012-2013.

Meningococcal disease incidence in Italy remains quite low in the overall population except for infants. Within a study on carriage isolates among high school students we aimed to define: i) the prevalence of carriage isolates, ii) the phenotypic and iii) the molecular features of meningococci by Whole Genome Sequencing (WGS). A total of 1697 pharyngeal samples from undergraduate students (age range 14-19 years) were collected from 2012 to 2013 from six larger cities in Italy. One hundred and twenty culture positive meningococci (7%) were analyzed. Carriage isolates were sent to the National Reference Laboratory for invasive meningococcal disease (IMD) for PCR-based serogroup identification, Multilocus Sequence Typing, PorA and FetA typing. Moreover, factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisserial adhesin A (NadA) were typed. Core genome MLST (cgMLST) was performed on a subsample of 75 carriage isolates. Capsule null locus (cnl) predominated (47%), followed by serogroup B (27%). The antimicrobial susceptibility profile revealed an high prevalence of reduced susceptibility to penicillin G (54%) and a full susceptibility to ceftriaxone, ciprofloxacin and rifampicin. Carriage isolates presented a high genetic diversity: the clonal complexes (ccs) cc1136, cc198 and cc41/44, were the predominant. An high heterogeneity was also observed for PorA and FetA types. The fhbp and nhba genes were identified in all the carriage isolates; only 5% of the carriage isolates presented the nadA gene. The core genome MLST analysis revealed that the majority of the cnl isolates clustered in a distinct group. The evidence gathered during this study provides the estimate of carriage isolates in high school students in a non-epidemic period in Italy that was lower than expected. Moreover, the highest proportion of carriage isolates were cnl and, overall, they were molecular heterogeneous.

Cocirculation of Hajj and non-Hajj strains among serogroup W meningococci in Italy, 2000 to 2016.

In Italy, B and C are the predominant serogroups among meningococci causing invasive diseases. Nevertheless, in the period from 2013 to 2016, an increase in serogroup W Neisseria meningitidis (MenW) was observed. This study intends to define the main characteristics of 63 MenW isolates responsible of invasive meningococcal disease (IMD) in Italy from 2000 to 2016. We performed whole genome sequencing on bacterial isolates or single gene sequencing on culture-negative samples to evaluate molecular heterogeneity. Our main finding was the cocirculation of the Hajj and the South American sublineages belonging to MenW/clonal complex (cc)11, which gradually surpassed the MenW/cc22 in Italy. All MenW/cc11 isolates were fully susceptible to cefotaxime, ceftriaxone, ciprofloxacin, penicillin G and rifampicin. We identified the full-length NadA protein variant 2/3, present in all the MenW/cc11. We also identified the fHbp variant 1, which we found exclusively in the MenW/cc11/Hajj sublineage. Concern about the epidemic potential of MenW/cc11 has increased worldwide since the year 2000. Continued surveillance, supported by genomic characterisation, allows high-resolution tracking of pathogen dissemination and the detection of epidemic-associated strains.

Meningococci of Serogroup X Clonal Complex 181 in Refugee Camps, Italy.

Four cases of infection with serogroup X meningococci (MenX) (1 in 2015 and 3 in 2016) occurred in migrants living in refugee camps or reception centers in Italy. All MenX isolates were identified as clonal complex 181. Our report suggests that serogroup X represents an emerging health threat for persons arriving from African countries.

Molecular characterization of a collection of Neisseria meningitidis isolates from Croatia, June 2009 to January 2014.

In the last decade, the incidence of invasive meningococcal disease (IMD) in Croatia remained stable at approximately 1 case per 100 000 inhabitants, affecting mainly children aged ≤5 years. We report the molecular characterization of meningococci causing IMD occurring from June 2009 to January 2014 in Croatia. Genomic DNA from 50 clinical isolates was analysed for serogroup, multilocus sequence typing and allele type of the two outer membrane protein genes, porA and the iron-regulated fetA. Furthermore, 22 of them were characterized by using whole-genome sequencing to define the meningococcal vaccine four-component meningococcal serogroup B (4CMenB) antigen genes factor H-binding protein (fHbp), Neisseria heparin-binding antigen (nhba) and Neisseria adhesin A (nadA) and the antimicrobial target resistance genes for penicillin (penicillin binding protein 2, penA), ciprofloxacin (DNA gyrase subunit A, gyrA) and rifampicin (ß-subunit of RNA polymerase, rpoB). The Etest was used to phenotypically determine the antimicrobial susceptibility of isolated meningococci. The main serogroup/clonal complex combinations were MenB cc41/44, MenC/cc11, MenW/cc174 and MenY/cc23. PorA P1.7-2, FetA F5-5 and F1-5 were the most represented through the serogroups. Meningococci with decreased susceptibility to penicillin (38.9 %) and one strain resistant to ciprofloxacin were identified. Forty-two percent of MenB showed the presence of at least one of the 4CMenB vaccine antigens (fHbp, NHBA, NadA and PorA). Our findings highlight the genetic variability of meningococci causing IMD in Croatia, especially for the serogroup B. Molecular-based characterization of meningococci is crucial to enhance IMD surveillance and to better plan national immunization programmes.

A new genotyping scheme based on MLVA for inter-laboratory surveillance of Streptococcus pyogenes.

A newly developed MLVA seven-loci scheme for Streptococcus pyogenes is described. The method can be successfully applied by using both agarose gel with visual inspections of bands and Lab on Chip technology. The potential of the present MLVA has been tested on a collection of 100 clinical GAS strains representing the most common emm types found in high-income countries plus 18 published gap-free genomes, in comparison to PFGE and MLST. The MLVA analysis defined 30 MLVA types with ten out of the considered 15 emm types exhibiting multiple and specific MLVA types. In only one occasion the same MLVA profile was shared between isolates belonging to two different emm types. A robust congruency between the methods was observed, with MLVA discriminating within clonal complexes as defined by PFGE or MLST. This new MLVA scheme can be adopted as a quick, low-cost and reliable typing method to track the short-term diffusion of GAS clones in inter-laboratory-based surveillance.

Genomic characterization of Italian Clostridium botulinum group I strains.

Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

Draft Genome Sequence of Clostridium botulinum B2 450 Strain from Wound Botulism in a Drug User in Italy.

Here, we report the draft genome sequence of Clostridium botulinum B2 450, responsible for the first reported case of wound botulism in a drug user in Italy.

Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.