Helena Žemličková

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus isolated from the nasopharynx of healthy children attending day-care centres in the Czech Republic

Nasopharyngeal carriage of potential pathogens was studied in 425 healthy 3- to 6-year-old children attending 16 day-care centres (DCCs) in nine Czech cities during the winter 2004-2005. The overall carriage of pathogens was 62.8% (Streptococcus pneumoniae, 38.1%; Haemophilus influenzae, 24.9%; Moraxella catarrhalis, 22.1%; Staphylococcus aureus, 16%). An age-related downward trend was observed for colonization with respiratory pathogens in contrast to Staph. aureus whose carriage was significantly higher among older children. The following serotypes of colonizing S. pneumoniae were the most predominant: 23F (20.6%), 6A (15.1%), 6B (12.7%), 18C (7.8%), 15B and 19F (6% each). The majority (94.3%) of H. influenzae isolates were non-typable; among capsulated isolates, serotype b was not found. Decreased susceptibility to penicillin was determined in 3% of pneumococci; 4.6% of H. influenzae strains and 85.1% of M. catarrhalis strains produced beta-lactamase. As for non-beta-lactam antibiotics, pneumococci resistant to trimethoprim-sulphamethoxazole were the most common (15.7%) among the attendees.

Comparative activity of carbapenem testing: the COMPACT study

OBJECTIVES Doripenem is a new carbapenem recently introduced into Europe. The COMParative Activity of Carbapenem Testing (COMPACT) study compared the susceptibility of common Gram-negative bacilli causing serious infections in hospitalized patients with doripenem, imipenem and meropenem. METHODS Gram-negative isolates (4498 total: 2171 Pseudomonas species; 1910 Enterobacteriaceae; and 417 other Gram-negative bacilli) were collected from 80 centres in 16 countries in Europe, the Middle East and Africa during 2008-09. The MICs of doripenem, imipenem and meropenem were determined using Etest methodology and broth microdilution. Susceptibility was interpreted according to CLSI, EUCAST and FDA breakpoints. RESULTS The MIC(90)s of doripenem, imipenem and meropenem for all isolates were 8, ≥64 and 32 mg/L, respectively. Doripenem had the lowest MIC(90) for Pseudomonas species at 16 mg/L, with imipenem and meropenem values of ≥64 mg/L. Enterobacteriaceae were highly susceptible to all three carbapenems, with MIC(90)s of doripenem, imipenem and meropenem of 0.06, 0.5 and 0.12 mg/L, respectively. Other Gram-negative isolates, predominantly Acinetobacter baumannii, were resistant to all three carbapenems (MIC(90) ≥64 mg/L). Susceptibility to doripenem was observed in 14.9% of isolates resistant to imipenem and/or meropenem. CONCLUSIONS Doripenem showed excellent activity against Gram-negative isolates; generally it was more active than imipenem and at least as good as meropenem. Against Pseudomonas species, doripenem was more active than both imipenem and meropenem, with doripenem susceptibility observed for some imipenem- and/or meropenem-resistant isolates.

Subpopulations of Staphylococcus aureus Clonal Complex 121 Are Associated with Distinct Clinical Entities

We investigated the population structure of Staphylococcus aureus clonal complex CC121 by mutation discovery at 115 genetic housekeeping loci from each of 154 isolates, sampled on five continents between 1953 and 2009. In addition, we pyro-sequenced the genomes from ten representative isolates. The genome-wide SNPs that were ascertained revealed the evolutionary history of CC121, indicating at least six major clades (A to F) within the clonal complex and dating its most recent common ancestor to the pre-antibiotic era. The toxin gene complement of CC121 isolates was correlated with their SNP-based phylogeny. Moreover, we found a highly significant association of clinical phenotypes with phylogenetic affiliations, which is unusual for S. aureus. All isolates evidently sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, conjunctivitis) clustered in clade F, which included the European epidemic fusidic-acid resistant impetigo clone (EEFIC). In comparison, isolates from deep-seated infections (abscess, furuncle, pyomyositis, necrotizing pneumonia) were disseminated in several clades, but not in clade F. Our results demonstrate that phylogenetic lineages with distinct clinical properties exist within an S. aureus clonal complex, and that SNPs serve as powerful discriminatory markers, able to identify these lineages. All CC121 genomes harboured a 41-kilobase prophage that was dissimilar to S. aureus phages sequenced previously. Community-associated MRSA and MSSA from Cambodia were extremely closely related, suggesting this MRSA arose in the region.

Species identification of Campylobacter jejuni ssp. jejuni and C. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR.

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains — C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination.

Global phylogeography and evolutionary history of Shigella dysenteriae type 1.

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries1. A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission2. This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries1,3,4 and the first isolation of Sd1 in Japan in 18975. Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

Carriage of methicillin-resistant Staphylococcus aureus in veterinary personnel

A survey of 280 attendees at a veterinary meeting in the Czech Republic in 2008 revealed a carriage rate of 0.7% for methicillin-resistant Staphylococcus aureus (MRSA). The two strains isolated were of distinct genetic lineages, carried type IV SCCmec determinants and were negative for Panton-Valentine leukocidin genes. The MRSA positivity rates for veterinarians in the Czech Republic is considerably lower than reported elsewhere.

Staphylococcus aureus spa type t437 : identification of the most dominant community-associated clone from Asia across Europe

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among large epidemiological studies in Europe. Nevertheless, the overall numbers identified in some Northern European reference laboratories have increased during the past decade. To determine whether the S. aureus t437 clone is present in other European countries, and to assess its genetic diversity across Europe, we analysed 147 S. aureus t437 isolates from 11 European countries collected over a period of 11 years using multiple locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and MLST. Additionally 16 S. aureus t437 isolates from healthy carriers and patients from China were included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population represents a genetically tight cluster, irrespective of the year, country and site of isolation. This underpins the view that S. aureus CC59 has been introduced into several European countries, not being restricted to particular geographical regions or specific host environments. The European S. aureus t437 isolates thus bear the general hallmarks of a high-risk clone.

Isolation from a Nonclinical Sample of Leclercia adecarboxylata Producing a VIM-1 Metallo-β-Lactamase

Leclercia adecarboxylata belongs to the family Enterobacteriaceae and is rarely isolated from clinical material, especially from immunocompromised patients. This bacterium is usually susceptible to most commonly used antibiotics, including beta-lactams. However, a few cases of antibiotic-resistant L

C-Geranylated flavonoids from Paulownia tomentosa fruits with antimicrobial potential and synergistic activity with antibiotics

Abstract Context C-6-Geranylated flavonoids possess promising biological activities. These substances could be a source of lead compounds for the development of therapeutics. Objective The study was designed to evaluate their antibacterial and antileishmanial activity. Materials and methods C-6-Geranylated flavanones were tested in micromolar concentrations against promastigote forms of Leishmania brazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC50 values (after 72 h) were calculated and compared with that of miltefosine. Flow cytometry and DNA fragmentation analysis were used the mechanism of the effect. Geranylated flavanones or epigallocatechin gallate were combined with oxacillin, tetracycline, and ciprofloxacin, and the effects of these two-component combinations were evaluated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were established (after 24 h), the synergy was measured by the checkerboard titration technique, and the sums of the fractional inhibitory concentrations (∑FICs) were computed. Results 3′-O-Methyl-5′-O-methyldiplacone and 3′-O-methyldiplacone showed good antileishmanial activities (IC50 8–42 μM). 3′-O-Methyl-5′-hydroxydiplacone activates the apoptotic death at leishmanias, the effect of 3′-O-methyl-5′-O-methyldiplacone has another mechanism. The test of the antibacterial activity showed good effects of 3′-O-methyldiplacol and mimulone against MRSA (MIC 2–16 μg/mL), and in six cases, the results showed synergistic effects when combined with oxacillin. Synergistic effects were also found for the combination of epigallocatechin gallate with tetracycline or oxacillin. Conclusion This work demonstrates anti-MRSA and antileishmanial potential of geranylated flavanones and uncovers their promising synergistic activities with antibiotics. In addition, the mechanism of antileishmanial effect is proposed.

Identification of Plesiomonas spp.: serological and MALDI-TOF MS methods.

Biochemical and serological profiles of isolates of Plesiomonas shigelloides were assayed using standard procedures in isolates from various clinical samples. Seventy-four isolates, including P. shigelloides type strain, were further characterized by MALDI-TOF MS using 3-methoxy-4-hydroxycinnamic acid as matrix. Multiple ions in the 3- to 12-kDa mass range were found in the spectra of each strain, from which the “species-identifying” unique biomarker ions were identified. After creating the species-specific patterns, a spectral database was generated for reliable, rapid, reproducible and accurate identification of Plesiomonas strains. The classical strain description (biochemical and serological) was thus complemented with the metabolic (proteomic) characterization.