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Hisashi Shoji,1,* Daniel A Vázquez-Sánchez,1,* Aida Gonzalez-Diaz,1,2 Meritxell Cubero,1,2 Fe Tubau,1,2 Salud Santos,2,3 Dolores García-Somoza,1,2 Josefina Liñares,1,2 José Yuste,2,4 Sara Martí,1,2 Carmen Ardanuy1,2 1Microbiology Department, Hospital Universitari de Bellvitge, IDIBELL, University of Barcelona, Barcelona, Spain; 2CIBER de Enfermedades Respiratorias, ISCIII, Madrid, Spain; 3Respiratory Medicine Department, Hospital Universitari de Bellvitge, IDIBELL, University of Barcelona, Barcelona, Spain; 4Pneumococcal Reference Laboratory, Centro Nacional de Microbiología, ISCIII, Madrid, Spain *These authors contributed equally to this work Background: Streptococcus pneumoniae is an important pathogen in chronic obstructive pulmonary disease (COPD). We aimed at showing the recent changes in the epidemiology of pneumococcal diseases in patients with COPD, especially after the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13).Methods: From 2013 to 2016, strains causing invasive pneumococcal disease (IPD), non-bacteremic pneumococcal pneumonia (non-BPP), and acute exacerbation of COPD (AE-COPD) were prospectively included. Antimicrobial susceptibility testing, serotyping, and genotyping were analyzed.Results: We collected 345 pneumococci from 286 COPD patients (57 IPD, 78 non-BPP, and 210 AE-COPD). The most frequent serotypes were serotypes 3 (14.0%), 8 (14.0%), and 12F (8.8%) in IPD; serotypes 3 (16.7%), 11A (9%), 9L/N (7.7%), and 23A (7.7%) in non-BPP; and serotypes 11A (11%), nontypeable (11%), and 6C (10%) in AE-COPD. Resistance rates were highest among AE-COPD strains. Penicillin resistance was associated with serotypes 11A (CC156) and 19A (CC320 and CC230). Compared with previous studies, fluoroquinolone resistance in AE-COPD increased (9.5%), PCV13 serotypes decreased (31.6%, 26.9%, and 16.7% for IPD, non-BPP, and AE-COPD, respectively), and serotype 11A-CC156 in

Objectives: To analyse the epidemiology and genetic evolution of PMEN3 (Spain 9V -156), a penicillin-non-susceptible clone of Streptococcus pneumoniae, causing invasive pneumococcal disease (IPD) in Barcelona during 1987–2016. Methods: WGS was performed on 46 representative isolates and the data were used to design additional molecular typing methods including partial MLST, PCR-RFLP and detection of surface-exposed proteins and prophages, to assign the remaining isolates to lineages. The isolates were also subjected to antimicrobial susceptibility testing. Results: Two hundred and twenty-seven adult cases of IPD caused by PMEN3 were identified. PMEN3 caused mainly pneumonia (84%) and the 30 day mortality rate was 23.1%. Evidence of recombination events was found, mostly in three regions, namely the capsular operon (associated with capsular switching) and adjacent regions containing pbp2x and pbp1a, the murM gene and the pbp2b–ddl region. Some of these genetic changes generated successful new variant serotype lineages, including one of serotype 11A that is not included in the current PCV13 vaccine. Other genetic changes led to increased MICs of b-lactams. Notably, most isolates also harboured prophages coding for PblB-like proteins. Despite these adaptations, the ability of this clone to cause IPD remained unchanged over time, highlighting the importance of its core genetic background. Conclusions: Our study demonstrated successful adaptation of PMEN3 to persist over time despite the introduction of broader antibiotics and conjugate vaccines. In addition to enhancing understanding of the molecular evolution of PMEN3, these findings highlight the need for the development of non-serotype-based vaccines to fight pneumococcal infection.

Background: In this study we describe the clinical and molecular characteristics of an outbreak due to carbapenem-resistant Klebsiella pneumoniae (CR-KP) producing CTX-M-15 and OXA-48 carbapenemase. Isogenic strains, carbapenem-susceptible K. pneumoniae (CS-KP) producing CTX-M-15, were also involved in the outbreak. Results: From October 2010 to December 2012 a total of 62 CR-KP and 23 CS-KP were isolated from clinical samples of 42 patients (22 had resistant isolates, 14 had susceptible isolates, and 6 had both CR and CS isolates). All patients had underlying diseases and 17 of them (14 patients with CR-KP and 3 with CS-KP) had received carbapenems previously. The range of carbapenem MICs for total isolates were: imipenem: 2 to >32 mu g/ml vs. <2 mu g/ml; meropenem: 4 to >32 mu g/ml vs. <2 mu g/ml; and ertapenem: 8 to >32 mu g/ml vs. <2 mu g/ml. All the isolates were also resistant to gentamicin, ciprofloxacin, and cotrimoxazole. Both types of isolates shared a common PFGE pattern associated with the multilocus sequence type 101 (ST101). The bla(CTX-M-15) gene was detected in all the isolates, whereas the bla(OXA-48) gene was only detected in CR-KP isolates on a 70 kb plasmid. Conclusions: The clonal spread of K. pneumoniae ST101 expressing the OXA-48 and CTX-M-15 beta-lactamases was the cause of an outbreak of CR-KP infections. CTX-M-15-producing isolates lacking the blaOXA-48 gene coexisted during the outbreak.

Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the blaOXA-1 gene was located in the variable region of a class I integron that also contains the aac(6′)Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC. [Int Microbiol 2013; 16(4):227-233]Keywords: Klebsiella pneumoniae · sequence type ST14 · gene blaOXA-1 · integrons · nosocomial outbreaks