Anna Anselmo

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.

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.

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

ABSTRACT 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.

Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012–2014

BackgroundThe emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment.MethodsSixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes.ResultsAll of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis.ConclusionOur findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

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 (β-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.

Pericarditis Caused by Hyperinvasive Strain of Neisseria meningitidis, Sardinia, Italy, 2015.

To the Editor: Invasive meningococcal disease is usually defined by the occurrence of meningitis or septicemia. Pericarditis might occur during the course of invasive infection. This clinical picture, defined as disseminated meningococcal disease with pericarditis (1) or secondary meningococcal pericarditis, was reported in 1918 (2). In 1939, primary or isolated meningococcal pericarditis (1,3) was described. In this form of pericarditis, pericardial or blood cultures are positive for Neisseria meningitidis but there is no meningeal involvement or clinical meningococcemia (4). Since its description, several cases of primary meningococcal pericarditis have been reported (5). Although its pathogenesis remains largely undefined, it has been hypothesized that the onset of primary pericarditis occurs after a transient bacteremia or as a consequence of involvement of the lower respiratory tract (4). Blaser et al. reported that serogroup C meningococci are usually associated with this disease, especially in adults. However, serogroups B, W, and Y have also been identified (4). We report a case-patient with primary meningococcal pericarditis caused by a serogroup C strain of N. meningitidis. The patient was a 32-year-old man who lived in Sardinia, Italy. He had no predisposing factors, such as immunodeficiency or other chronic disorders. Disease onset occurred on August 29, 2015. Clinical manifestations were fever (temperature 38°C), hypotension, epigastralgia, arthralgia, asthenia, chest pain, and reduced vesicular murmur. The left ventricle was widely hypokinetic, and a light ST increase was observed. A blood culture was positive for N. meningitidis. The patient was given piperacillin/tazobactam (4.5 g 3×/d) and metronidazole (500 mg 3×/d) for 4 days. After 4 days, treatment with ceftriaxone (2 g 2×/d) for 4 days was started. Because of persistent fever (38.8°C), levofloxacin (500 mg 2×/d) for 23 days was also started on day 7. On day 10, ceftriaxone was replaced with piperacillin/tazobactam (4.5 g 4×/d) for 21 days. A major bilateral pleural effusion was detected on the left side. On day 11, the fever had resolved. The outcome was favorable for this patient. Drug resistance of the strain was determined by using the MIC Test Strip Method (Liofilchem, Abruzzi, Italy). Breakpoints used were those recommended by the European Committee on Antimicrobial Susceptibility Testing version 5.0 (http://www.eucast.org/). The strain was susceptible to cefotaxime, ceftriaxone, ciprofloxacin, penicillin G, and rifampin. Serogroup was determined by using slide agglutination with commercial antisera (Remel Europe, Ltd., Dartford, UK) and confirmed by PCR (6). Whole-genome sequencing was conducted to obtain molecular data and enable comparison with other meningococci of the same serogroup that were isolated in Italy. Multilocus sequence typing (MLST) and typing of porA and fetA genes and Bexsero (meningcoccal group B vaccine) antigen genes (http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm431374.htm) were conducted as described (http://neisseria.org/). Whole-genome sequence was analyzed by using the BIGSdb Genome Comparator Tool (http://pubmlst.org/neisseria/). Genomes of meningococci belonging to the same finetype were compared by using the core genome MLST (cgMLST) approach. The N. meningitidis strain of serogroup C was susceptible to all antimicrobial drugs tested. Although serogroup C was associated with 53 (41%) of 132 invasive meningococcal disease cases in Italy in 2015 (http://www.iss.it/binary/mabi/cont/Report_MBI_20151223_v4.pdf), this serogroup has not been detected in Sardinia since 2010. Molecular analyses showed that the strain belonged to the hypervirulent clonal complex (cc) 11, sequence type (ST) 11. The complete finetype was C:5–1,10–8:F3–6:ST-11(cc11). This finetype has been reported in the United States and several countries in Europe (7), including Italy, and is responsible for several disease outbreaks. In Italy, this finetype represents 61% (70/115) of all serogroup C strains collected during 2012–2015. Two outbreaks caused by this strain were reported in Italy in 2007 (8) and in 2012 (9). The N. meningitidis factor H binding protein and heparin binding protein alleles were 1.13 and 20, respectively. The N. meningitidis adhesin A variant had an insertion sequence that disrupted this gene, as described for ET-15 meningococci (10). On the basis of results of cgMLST, the strain was determined to be related to strains responsible for an outbreak in Italy in 2015. In summary, we report a case of meningococcal pericarditis caused by a strain of N. meningitidis. This strain belongs to hyperinvasive clonal complex cc11 and was identified as C:P1.5–1,10–8:F3–6:ST-11(cc11), an emerging strain in Italy and worldwide. Timely diagnosis and complete molecular characterization of this strain, which causes a rare form of invasive disease (4), is needed for appropriate management of patients with this disease.

Whole-Genome Sequence of Clostridium botulinum A2B3 87, a Highly Virulent Strain Involved in a Fatal Case of Foodborne Botulism in Italy

ABSTRACT Here, we report the genome sequence of a rare bivalent strain of Clostridium botulinum, A2B3 87. The strain was isolated from a foodborne botulism case that occurred in Italy in 1995. The case was characterized by rapid evolution of the illness and failure of conventional treatments.

Draft Genome Sequence of a Bordetella pertussis Strain with the Virulence-Associated Allelic Variant ptxP3, Isolated in Italy

ABSTRACT Despite a universal immunization program, pertussis has persisted and resurged, and is of particular concern for infants in terms of morbidity and mortality. Here, we report the genome sequence of a Bordetella pertussis strain with the virulence-associated allelic variant ptxP3, isolated from a 45-day-old infant.

Draft Genome Sequence of Neisseria gonorrhoeae Sequence Type 1407, a Multidrug-Resistant Clinical Isolate

ABSTRACT Gonorrhea may become untreatable due to the spread of resistant or multidrug-resistant strains. Cefixime-resistant gonococci belonging to sequence type 1407 have been described worldwide. We report the genome sequence of Neisseria gonorrhoeae strain G2891, a multidrug-resistant isolate of sequence type 1407, collected in Italy in 2013.

Draft Genome Sequence of C:P1.5-1,10-8:F3-6:ST-11 Meningococcal Clinical Isolate Associated with a Cluster on a Cruise Ship

ABSTRACT Meningococcal serogroup C strains, in particular those belonging to the ST-11 clonal complex, are known to cause invasive diseases worldwide. We report the genome sequence of a Neisseria meningitidis strain linked to a cluster of cases of invasive meningococcal disease on a cruise ship that was described in 2012.