SummaryBackground Gaps in the diagnostic capacity and heterogeneity of national surveillance and reporting standards in Europe make it diffi cult to contain carbapenemase-producing Enterobacteriaceae. We report the development of a consistent sampling framework and the results of the fi rst structured survey on the occurrence of carbapenemaseproducing Klebsiella pneumoniae and Escherichia coli in European hospitals.

Children with acute otitis media benefit from antimicrobial treatment as compared with placebo, although they have more side effects. Future studies should identify patients who may derive the greatest benefit, in order to minimize unnecessary antimicrobial treatment and the development of bacterial resistance. (Funded by the Foundation for Paediatric Research and others; ClinicalTrials.gov number, NCT00299455.).

Background: The usefulness of induced sputum in searching for causative agents of pneumonia in children has not been studied. Methods: The study involved 101 children, aged 6 months to 15 years, treated for community-acquired pneumonia at Turku University Hospital (Turku, Finland) from January 2006 to April 2007. Nasopharyngeal aspirate samples were first collected through both nostrils. Sputum production was then induced by inhalation of 5.0% hypertonic saline for 5-10 min and a sputum sample was either aspirated or expectorated. The presence and amount of bacteria and viruses in paired nasopharyngeal aspirate and sputum specimens was analysed and compared using semiquantitative bacterial culture and quantitative PCR techniques. Results: A good quality sputum specimen was obtained from 76 children. The possible causative agent was found in 90% of cases. Streptococcus pneumoniae (46%) and rhinovirus (29%) were the most common microbes detected. Newly discovered viruses human bocavirus and human metapneumovirus were detected in 18% and 13% of the children, respectively. One-quarter of all bacterial findings were only detected in sputum, and the amount of bacteria in the remainder of the sputum specimens compared with nasopharyngeal aspirate was higher in 14% and equal in 70%. The amount of rhinovirus in sputum was higher than in nasopharyngeal aspirate in 82%. Conclusions: Sputum induction provides good quality sputum specimens with high microbiological yield in children with community-acquired pneumonia. Induced sputum analysis can be useful in the microbiological diagnosis of childhood community-acquired pneumonia.

Broad-range amplification of bacterial DNA from clinical specimens has proved useful for the diagnosis of various bacterial infections, especially during antimicrobial treatment of the patient. Optimal sample processing protocols for diagnostic broad-range bacterial PCR should release DNA from an array of target organisms with equal efficiencies and wash out inhibitory factors from various sample types without introducing bacterial DNA contamination to the amplification reaction. In the present study, two physical cell wall disintegration methods, bead beating and sonication, for enhanced detection of organisms with difficult-to-lyse cell walls were studied. The analytical sensitivities of several commercially available DNA purification kits, which were used with and without additional cell disintegration steps, were compared by using dilution series of model bacteria. Selected purification methods were used to process routine clinical specimens in parallel with the standard phenol-ether DNA extraction, and the results obtained by bacterial PCR and sequencing with the two template preparations were compared. The method with the DNA isolation kit with the lowest detection limits from the bacterial suspensions (Masterpure) did not prove to be superior to the standard method when the two methods were applied to 69 clinical specimens. For another set of 68 clinical specimens, DNA purified with a glass fiber filter column (High Pure) with an additional sonication step yielded results well in accord with those obtained by the standard method. Furthermore, bacterial DNA was detected in four samples that remained PCR negative by the standard method, and three of these contained DNA from gram-positive pathogens. Three samples were positive by the standard method only, indicating the limitations of applying any single method to all samples.Direct amplification of bacterial DNA from clinical specimens with broad-range primers provides an alternative approach to the recognition of pathogens infecting normally sterile body compartments. In comparing the molecular diagnosis obtained by broad-range bacterial PCR and partial sequencing of the amplicon to the results of routine bacterial cultures, we found 83% overall agreement for a set of 536 clinical specimens from hospitalized patients (7). The molecular approach proved superior to culture during antimicrobial treatment of the patient and in detection of bacteria with unusual growth requirements. A major drawback of the molecular method in comparison to bacterial culture was the difficulty in detecting species with gram-positive cell walls and mycobacteria. This was associated with problems in breaking bacterial cell walls and releasing bacterial DNA for amplification when standard phenol-ether DNA extraction was used. This finding led us to search for better methods to prepare the clinical specimens for broad-range bacterial PCR assay.In general, an optimal sample processing method should concentrate the DNA, especially that derived from the target organism, and wash out in...

For over a century, a fundamental objective in infection biology research has been to understand the molecular processes contributing to the origin and perpetuation of epidemics. Divergent hypotheses have emerged concerning the extent to which environmental events or pathogen evolution dominates in these processes. Remarkably few studies bear on this important issue. Based on population pathogenomic analysis of 1,200 Streptococcus pyogenes type emm89 infection isolates, we report that a series of horizontal gene transfer events produced a new pathogenic genotype with increased ability to cause infection, leading to an epidemic wave of disease on at least two continents. In the aggregate, these and other genetic changes substantially remodeled the transcriptomes of the evolved progeny, causing extensive differential expression of virulence genes and altered pathogen-host interaction, including enhanced immune evasion. Our findings delineate the precise molecular genetic changes that occurred and enhance our understanding of the evolutionary processes that contribute to the emergence and persistence of epidemically successful pathogen clones. The data have significant implications for understanding bacterial epidemics and for translational research efforts to blunt their detrimental effects. IMPORTANCEThe confluence of studies of molecular events underlying pathogen strain emergence, evolutionary genetic processes mediating altered virulence, and epidemics is in its infancy. Although understanding these events is necessary to develop new or improved strategies to protect health, surprisingly few studies have addressed this issue, in particular, at the comprehensive population genomic level. Herein we establish that substantial remodeling of the transcriptome of the human-specific pathogen Streptococcus pyogenes by horizontal gene flow and other evolutionary genetic changes is a central factor in precipitating and perpetuating epidemic disease. The data unambiguously show that the key outcome of these molecular events is evolution of a new, more virulent pathogenic genotype. Our findings provide new understanding of epidemic disease.

The in vitro susceptibilities of 184 erythromycin-resistant streptococci to a novel ketolide, telithromycin (HMR 3647), were tested. These clinical isolates included 111 Streptococcus pyogenes, 18 group C streptococcus, 18 group G streptococcus, and 37 Streptococcus pneumoniae strains. The MICs for all but eight S. pyogenes strains were <0.5 g/ml, indicating that telithromycin is active in vitro against erythromycin-resistant Streptococcus strains. All strains for which MICs were >1 g/ml had an erm(B) resistance gene and six strains for which MICs were >4 g/ml had a constitutive erm(B) gene (MIC range, 4 to 64 g/ml). Interestingly, for S. pneumoniae strains with a constitutive erm(B) gene, MICs were <0.25 g/ml (MIC range, <0.008 to 0.25 g/ ml). Our in vitro data show that for S. pyogenes strains which constitutively express the erm(B) methylase gene, MICs are so high that the strains might be clinically resistant to telithromycin.Ketolides represent a new generation of macrolides, in which a 3-keto group replaces L-cladinose in the lactone ring. Ketolides have shown to be more active in vitro than other macrolides against various gram-positive bacteria such as Enterococcus species (6, 24), Staphylococcus aureus (6, 13), and Streptococcus species, including erythromycin-resistant Streptococcus pneumoniae and Streptococcus pyogenes strains (6,16,18). On the other hand, methicillin-resistant and some erythromycin-resistant S. aureus strains, as well as some Staphylococcus epidermidis strains (1, 18), seem to be resistant to ketolides (1, 6, 13).In streptococci, there are two well-characterized macrolide resistance mechanisms: target site modification and active drug efflux. Target site modification is mediated by methylases encoded by the erm (erythromycin ribosome methylation) genes (21, 26). Methylation of A2058 of the peptidyl transferase loop of 23S rRNA causes resistance to macrolides as well as to lincosamides and streptogramin B antibiotics (the MLS B resistance phenotype) (26). The expression of the erm genes can be either constitutive or inducible (27). In streptococci, erm genes are carried on both the chromosome and plasmids (2, 21) and are associated with conjugative transposons (25). The activeefflux mechanism, encoded by the mef (macrolide efflux) genes, is more specific and causes resistance only to 14-and 15-member-ring macrolides (the M resistance phenotype) (3,22). Expression of mef genes is constitutive (C. Arpin, M. H. Canron, P. Noury, and C. Quentin, Letter, J. Antimicrob. Chemother. 44:133-134, 1999). The mef genes are chromosomal (11,17) and, at least in the case of S. pyogenes, can be transferred by conjugation (11).The present work was carried out to study the activity of a novel ketolide, telithromycin (HMR 3647), against Streptococcus species with known macrolide resistance determinants. In addition, its activity against nine S. pneumoniae strains with an unknown macrolide resistance mechanism was tested. MATERIALS AND METHODSBacterial strains. Altogether 184 erythromycin-resistant strep...

IntroductionCarbapenemase-producing Enterobacteriaceae (CPE) have rarely been reported in dogs, and never in animals in Finland. However, in April 2015, two meropenem-resistant Escherichia coli were identified from two dogs in one family. Both dogs suffered from chronic otitis externa. Methods: Epidemiological and molecular investigations (pulsed-field gel electrophoresis (PFGE), multilocus sequence typing) were conducted to investigate the source of infection and transmission routes. Results: In both dogs and one family member New Delhi metallo-beta-lactamase (NDM-5)-producing multidrug-resistant ST167 E. coli was found. Whole genome sequencing confirmed that the isolates were identical or only had one or two allelic differences. Additionally, the dogs and humans of the family carried an identical extended-spectrum beta-lactamase (ESBL) CTX-M-group 9 E. coli ST69 strain, indicating interspecies transmission. While the original source remains unclear, human-to-canine transmission is possible. No carbapenems had been administered to the dogs, but exposure to numerous other antimicrobials likely sustained the bacteria and supported its propagation in the canine host. Conclusion: To our knowledge, canine clinical NDM-5 E. coli in Europe, and confirmed CPE transmission between dogs and humans have not been previously reported. The screening of veterinary Enterobacteriaceae isolates for carbapenem resistance is highly recommended.

The Carba NP test was evaluated against a panel of 61 carbapenemase-producing bacterial species (15 producing class A carbapenemases, 15 producing class D carbapenemases, and 31 producing metallo-␤-lactamases) and against 111 isolates with nonwild-type carbapenem susceptibility but not producing carbapenemase. Carbapenemase production was verified by PCR and UV-spectrophotometric measurement of imipenem hydrolysis. No false positives were seen, but there were consistent problems with the detection of OXA-48-like enzymes and also some rarer class A enzymes.