Cristina Pagani

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.

Remitting infections due to community-acquired Panton–Valentine leukocidin-producing Staphylococcus aureus in the Milan area

One of the most important Staphylococcus aureus virulence factors is Panton-Valentine leukocidin (PVL). We describe an outbreak of recurrent cutaneous PVL infections in different members of three family clusters. Molecular investigations were performed to confirm the presence of the mecA and PVL genes and to assign the SCCmec type, sequence type (ST) and clonal relatedness. A strain of PVL-producing methicillin-resistant S. aureus (MRSA) was responsible for infection in two related families (A and B), and a third family (C) was infected with PVL-producing methicillin-sensitive S. aureus (MSSA). Molecular investigations revealed the same clone of community-acquired (CA)-MRSA, PVL positive ST8, and SCCmec IV in families A and B and CA-MSSA PVL positive ST15 in family C. S. aureus PVL may give rise to recurrent uncontrolled infections that are difficult to eradicate, and close family contacts are at high risk for transmission.

Mingling of human and veterinary strains of Staphylococcus aureus: An emerging issue in health-care systems

Aim: Methicillin-resistant Staphylococcus aureus remains a leading cause of hospital and community infections. We report a retrospective molecular characterization of S. aureus strains from different settings: hospital workers and patients, and veterinarian surgeons and pets. Materials and Methods: Eighty-nine S. aureus isolates obtained from nasal swabs of 10 patients, 17 health-care workers (HCWs), 9 pets, and 53 veterinarians were genotypically characterized by means of repetitive extragenic palindromic polymerase chain reaction (Rep PCR) and whole-genome sequencing. Results: Thirteen different sequence types (STs) were detected: ST398, ST22, ST8, ST30, ST15, ST5, ST121, ST45, ST10, ST6, ST34, ST97, and ST1. Two new STs differing from ST22 and ST5 for a single multilocus sequence typing gene were also identified. Rep PCR documented a genetic relationship among isolates obtained from 5 veterinarians and 10 HCWs. Conclusion: The large diversity of S. aureus strains detected may reflect a larger epidemiology within the hospital and community, in which companion animals likely act as a reservoir. We identified the circulation of ST5, ST8, ST15, ST22, ST30, ST45, and ST121 both in the hospital and veterinarian environment. Starting from the idea of a unique setting where our population lives, we consider the relationship between community- and hospital-acquired S. aureus.

Pasteurella spp in Knee Mega Prosthesis Infection: A Case Report

Pasteurella spp is the most frequently pathogen isolated from infected wound inflicted by domestic animal bites. Here we present a case of isolation of Pasteurella pneumotropica like organism from knee mega prosthesis infection occurred in 63 years old female.