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The European NDM-1 Survey Participants: Manuela Caniça (Departamento de Doenças Infecciosas do INSA) Acquired carbapenemases confer extensive antibiotic resistance to Enterobacteriaceae and represent a public health threat. A novel acquired carbapenemase, New Delhi metallo-beta-lactamase 1 (NDM-1), has recently been described in the United Kingdom and Sweden, mostly in patients who had received care on the Indian subcontinent. We conducted a survey among 29 European countries (the European Union Member States, Iceland and Norway) to gather information on the spread of NDM-1-producing Enterobacteriaceae in Europe, on public health responses and on available national guidance on detection, surveillance and control. A total of 77 cases were reported from 13 countries from 2008 to 2010. Klebsiella pneumoniae was the most frequently reported species with 54%. Among 55 cases with recorded travel history, 31 had previously travelled or been admitted to a hospital in India or Pakistan and five had been hospitalised in the Balkan region. Possible nosocomial acquisition accounted for 13 of 77 cases. National guidance on NDM-1 detection was available in 14 countries and on NDM-1 control in 11 countries. In conclusion, NDM-1 is spreading across Europe, where it is frequently linked to a history of healthcare abroad, but also to emerging nosocomial transmission. National guidance in response to the threat of carbapenemase-producing Enterobacteriaceae is available in approximately half of the surveyed European countries. Surveillance of carbapenemase- producing Enterobacteriaceae must be enhanced in Europe and effective control measures identified and implemented.

Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

Introduction of SeqNet.org SeqNet.org is currently an initiative of 44 laboratories from 25 European countries and one laboratory from Lebanon (Table 1), founded in 2004, in collaboration with the Robert Koch Institute at the University of Münster, Germany (http://www.SeqNet.org). Since then, its main objective is to establish a European network of excellence for sequence-based typing of microbial pathogens, having its main focus on Staphylococcus aureus [1]. SeqNet. org comprises a large number of national reference laboratories as well as university laboratories. The principle goal of SeqNet. org is to generate unambiguous, easily comparable typing data in electronic, portable form to be used by infection control at a local level as well as national and European surveillance of sentinel microorganisms, such as methicillin- resistant Staphylococcus aureus (MRSA). spa- typing has been shown to be a useful tool in molecular hospital epidemiology [2, 3]. Veterinary laboratories have recently joined the SeqNet.org initiative as MRSA has become an emerging problem in veterinary medicine [4, 5]. spa-typing data from human and veterinary medicine can be compared using the spa server database [6]. SeqNet.org objectives 1. Organisation and participation in seven international workshops contributed to the harmonisation of sequencing methods for sequence-based typing of MRSA and the capacity for building DNA sequencing in diagnostic microbiology. Further meetings and workshops are planned. 2. SeqNet.org rules require that SeqNet.org laboratories (Table 1) undergo at least one certification trial [7] for sequence- based typing of MRSA. Regular proficiency tests are foreseen. 3. Curatorship of the Ridom spa server and the development and maintenance of a SeqNet.org web-portal allows the transfer of data at an international level. 4. The excellence of data quality needs to be maintained. This is necessary as the access to the spa server will be enlarged in

We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice - MLST/SCCmec typing - and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA. Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Antibiotic resistance, the important public health threat, depends on antibiotic overuse/misuse. Self-medication with antibiotics is of serious medical concern. The aim of the study, as a part of SAR project (Self-medication with antibiotic in Europe) was to survey the incidence of this phenomenon. MATERIAL AND METHODS: In Poland among 938 respondents of the questionnaire 27% adults were treated with antibiotics at least once a year, 13% of the therapies were undertaken without medical consultation. The most frequently prescribed antibiotic was amoxicillin, the most often self-ordered--fusafungine. RESULTS: The main self-medicated symptoms were sore throat and common cold. The duration of self-ordered therapy was less then 5 days. The sources of antibiotics used for self-medication were leftovers and those given by friends or family. CONCLUSION: The results of the study confirmed that antibiotic self-medication is relatively frequent problem in Poland and interventions are required in order to reduce the frequency of antibiotics misuse

Among 1, 011 recently isolated Streptococcus pyogenes isolates from 10 Central and Eastern European centers, the MICs at which 50% of isolates are inhibited (MIC(50)s) and the MIC(90)s were as follows: for telithromycin, 0.03 and 0.06 microg/ml, respectively ; for erythromycin, azithromycin, and clarithromycin, 0.06 to 0.125 and 1 to 8 microg/ml, respectively ; and for clindamycin, 0.125 and 0.125 microg/ml, respectively. Erythromycin resistance occurred in 12.3% of strains. Erm(A) [subclass erm(TR)] was most commonly encountered (60.5%), followed by mef(A) (23.4%) and erm(B) (14.5%). At

Staphylococcus aureus is recognized as one of the major causes of infections in humans occurring in both the community and the hospital. Up to now one of the most serious aspects as far as treatment of S. aureus infections is concerned is resistance to methicillin, which in clinical terms indicates resistance to all beta-lactam antibiotics. The growing incidence of methicillin-resistant S. aureus (MRSA) infections worldwide, their multidrug resistance, several reservoirs of resistant strains, the facility to spread also outside hospitals and to cause outbreaks requires efficacious infection control measures. For this reason microbiological and epidemiological studies are of crucial importance.

Different methods of molecular typing (ribotyping, genomic DNA RFLP and RAPD) were tested on Pseudomonas aeruginosa strains isolated in Polish hospitals in order to elaborate a reliable typing scheme for epidemiological investigations. The combined RAPD analysis with the use of two different primers, RAPD-4 and RAPD-7, was found to have the highest discriminatory power which considering also the easiness and low time-consumption has suggested its high usefulness in studies of outbreaks caused by P. aeruginosa. Ribotyping was shown to be the least discriminatory, however, especially with the use of the PvuII restriction enzyme, this method can be very useful in revealing the genetic structure of P. aeruginosa populations persisting in hospital environments over longer periods. Clonal relations within populations of strains isolated in four different hospitals were revealed. In two of the hospitals P. aeruginosa populations demonstrated a very high diversity which suggested that infections had been caused by strains of different origins, probably introduced from other environments. P. aeruginosa strains from two remaining hospitals were found to form some clonally related clusters what revealed that in these hospitals epidemic strains of this microorganism have been circulating for prolonged periods and infecting predisposed patients.

A total of 674 clinical isolates of Pseudomonas aeruginosa were collected from 30 different hospitals located in 26 cities of Poland. These were 12 big regional hospitals, 7 large teaching hospitals, 4 specialised hospitals curing patients from the whole country, 6 small local hospitals and one regional paediatric hospital. The majority of strains were collected from patients hospitalised at ICUs (25.7%), surgical (21.7%), and internal medicine wards (9.9%). The isolates were recovered from different types of infections, mostly from respiratory tract infections (33.7%), wound infections (22.3%), and urinary tract infections (22.0%). All the isolates were subjected to antimicrobial susceptibility testing by MIC values evaluation. MICs of 13 different antibiotics (beta-lactams, aminoglycosides, chinolones) were determined by the agar dilution method. The general level of resistance of P.aeruginosa observed in the study was high, especially when compared to results of surveys obtained in other countries. Out of the antimicrobials used the highest activity in vitro was observed with meropenem, imipenem, piperacillin--tazobactam and ceftazidime. The high in vitro activity of ceftazidime was striking considering the long time of the use of this antibiotic in Polish hospitals. The highest levels of resistance were observed to some of the aminoglycosides. Populations of strains isolated in different wards or hospitals of different size were characterised by different susceptibility patterns.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure agreement between laboratories on the assignment of strains to ‘susceptible’ and ‘resistant’ categories. An international quality assessment was performed to investigate the performance of susceptibility testing of Klebsiella spp. Ninety-five strains of klebsiellae were selected from clinical isolates at the London Hospital Medical College (LHMC). These included strains with a diversity of susceptibility profiles to amoxycillin/clavulanate, piperacillin, ceftazidime, cefuroxime, ciprofloxacin, gentamicin and trimethoprim. The strains were sent to 13 participating laboratories in Europe and the USA and laboratories were asked to test the susceptibility of these strains to these antibiotics by their usual methods. They were also asked to provide details of the method used to test susceptibility. Several different standard recommended testing methods were used. Reporting of susceptibilities was generally accurate, but a number of anomalies were noted. Discrepancies of reporting between the LHMC and the participating laboratories was more marked for resistant strains, particularly in the detection of resistance to cefuroxime and ciprofloxacin, as well as the assignment of susceptibility and resistance to piperacillin and amoxycillin/clavulanate. Some discrepancies could be attributed to the use of different breakpoints, leading to differing assignment of susceptibility. Methodological variations including disc content, inoculum and failure to measure and interpret zone sizes consistently also led to anomalies. This quality assessment programme has helped to identify problems in susceptibility testing which should be investigated further.

In order to compare the prevalence of antibiotic resistance in different geographical areas, it is necessary to ensure that agreement is achieved between laboratories on the assignment of strains to 'susceptible' and 'resistant' categories. An international quality assessment study, involving 15 laboratories in eight countries, was performed to investigate the standard of performance of the susceptibility testing of Haemophilus influenzae. One hundred and fifty strains of H. influenzae were distributed from the London Hospital Medical College (LHMC) to all laboratories who were asked to test the susceptibility of the strains to ampicillin, chloramphenicol, tetracycline, trimethoprim, cephalosporins and ciprofloxacin. Laboratories were also asked to provide the details of methodology to test the susceptibility. Significant discrepancy between the LHMC and the participating laboratories appeared in the detection of resistance to ampicillin (especially β-lactamase-negative strains resistant to ampicillin) as well as the assignment of susceptibility and resistance to chloramphenicol, tetracycline and trimethoprim. Often these reflected the use of inappropriate breakpoints which led to erroneous assignment of susceptibility. Other variations including disc content, medium and supplement, inoculum as well as failure to measure zone sizes properly also led to some repeating anomalies.

One hundred and fifty hospital Staphylococcus aureus strains isolated on Polish hospitals during an outbreaks were used to evaluate usefulness of S. aureus phenotyping methods. According to the expression of resistance to methicillin (a marker of resistance to all beta-lactam antibiotics) all strains were classified as homogeneously of heterogeneously resistant to methicillin (methicillin resistant S. aureus--MRSA) and as susceptible to this antibiotic (methicillin-susceptible S. aureus--MSSA). All strains were analysed according to the resistance patterns to fifteen antistaphylococcal drugs, results of crystal violet test, phage patterns and results of biochemical fingerprinting by PhenePlate (PhP) System (BioSys inova). All MSSA analysed were resistant to penicillin and 22% to tetracycline, and only occasionally resistant to other antimicrobials. They showed sensitive to phages of all international and additional lytic groups. Heterogeneous MRSA were widely susceptible to almost all antimicrobials except tetracycline and were lysed by phages of Ist, IIIrd international groups and additional phages. All these strains produced penicillinases. Homogeneous MRSA were multi-drug resistant and were not typable by phages of Ist and IInd lytic groups and weakly typable by phages of IIIrd and additional groups. PhP Systems divided all used strains into 13 common (with two dominating) and 50 single strain PhP-types. The combination of various phenotyping methods may be useful in epidemiological investigation of hospitals.

Population analysis of methicillin-resistant Staphylococcus aureus and determination of their sensitivity to antimicrobial agents were performed. It was found, that the methicillin-resistant strains belonged to resistance classes II and III. All methicillin-resistant S. aureus were susceptible to ciprofloxacin, ofloxacin, vancomycin and teicoplanin. Differences between classes were observed in susceptibility to erythromycin and gentamicin.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.