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

BACKGROUND: The emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment. METHODS: Sixty-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. RESULTS: All of the samples were resistant to carbapenems, other ß-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 ß-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. CONCLUSION: Our findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

Can Drainage Using a Negative-Pressure Wound Therapy Device Replace Traditional Sample Collection Methods?

BACKGROUND: In 2015 a new device for the collection of mediastinal fluid from patients with deep sternal wound infection (DSWI) in the presence of negative-pressure wound therapy (NPWT) became available. The present study was designed to evaluate whether changing sample collection devices increased micro-organism detection in patients undergoing NPWT. METHODS: During 2013-2014, 207 samples were collected and cultured from NPWT patients (n = 23) to demonstrate the presence of DSWI using reticulated polyurethane sponge culture, a swab, and blood culture. In 2015, a new collection device was introduced for specimen collection. A total of 357 samples (n = 17) were collected using the ESwab(™) (Copan, Murrieta, CA) for deep and superficial wound sample collection. In addition, blood culture devices were used for collecting mediastinal fluid aspirated directly from the wound and biologic fluid obtained from the NPWT device. Fisher exact test was performed to test the rate of independence rate of micro-organism identification using the NPWT sponge device and taking blood culture results as a reference for micro-organism identification. RESULTS: After the introduction of the new collection device in our hospital, an overall increase in the detection of micro-organisms (46.7%) was reported. During 2013-2014 our traditional microbiologic collection method did not detect a pathogen in 30.4% of patients. During 2015, the new sample collection approach, direct from the NPWT device, improved micro-organism detection by 10.4% and reduced DSWIs with undetected pathogens to 17.6% (p < 0.01). CONCLUSIONS: As a result of proficiency gained in the last year, the most representative specimen in wound infection was represented by mediastinal fluid collected directly from the wound and the NPWT device. Given the correlation between the blood culture of micro-organisms detected using the ESwab device from the wound, mediastinal drainage, and drainage from the NPWT device, we can assume that the NPWT device may replace the other biologic sampling devices.

Use of Dithiothreitol to Dislodge Bacteria From the Biofilm on an Aortic Valve in the Operating Theatre: A Case of Infective Endocarditis Caused by Staphylococcus aureus and Proteus mirabilis.

This is the first reported case of 2 biofilm-producing bacteria, Staphylococcus aureus and Proteus mirabilis, identified from an aortic valve using an innovative device with dithiothreitol solution, able to dislodge bacterial biofilm. The method is usable in the operating theatre and recommended in infective endocarditis nonresponders to empiric therapy.