SUMMARY The spread of Enterobacteriaceae , primarily Klebsiella pneumoniae , producing KPC, VIM, IMP, and NDM carbapenemases, is causing an unprecedented public health crisis. Carbapenemase-producing enterobacteria (CPE) infect mainly hospitalized patients but also have been spreading in long-term care facilities. Given their multidrug resistance, therapeutic options are limited and, as discussed here, should be reevaluated and optimized. Based on susceptibility data, colistin and tigecycline are commonly used to treat CPE infections. Nevertheless, a review of the literature revealed high failure rates in cases of monotherapy with these drugs, whilst monotherapy with either a carbapenem or an aminoglycoside appeared to be more effective. Combination therapies not including carbapenems were comparable to aminoglycoside and carbapenem monotherapies. Higher success rates have been achieved with carbapenem-containing combinations. Pharmacodynamic simulations and experimental infections indicate that modification of the current patterns of carbapenem use against CPE warrants further attention. Epidemiological data, though fragmentary in many countries, indicate CPE foci and transmission routes, to some extent, whilst also underlining the lack of international collaborative systems that could react promptly and effectively. Fortunately, there are sound studies showing successful containment of CPE by bundles of measures, among which the most important are active surveillance cultures, separation of carriers, and assignment of dedicated nursing staff.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

Pulsed-field gel electrophoresis (PFGE) is the most common genotypic method used in reference and clinical laboratories for typing methicillin-resistant Staphylococcus aureus (MRSA). Many different protocols have been developed in laboratories that have extensive experience with the technique and have established national databases. However, the comparabilities of the different European PFGE protocols for MRSA and of the various national MRSA clones themselves had not been addressed until now. This multinational European Union (EU) project has established for the first time a European database of representative epidemic MRSA (EMRSA) strains and has compared them by using a new "harmonized" PFGE protocol developed by a consensus approach that has demonstrated sufficient reproducibility to allow the successful comparison of pulsed-field gels between laboratories and the tracking of strains around the EU. In-house protocols from 10 laboratories in eight European countries were compared by each center with a "gold standard" or initial harmonized protocol in which many of the parameters had been standardized. The group found that it was not important to standardize some elements of the protocol, such as the type of agarose, DNA block preparation, and plug digestion. Other elements were shown to be critical, namely, a standard gel volume and concentration of agarose, the DNA concentration in the plug, the ionic strength and volume of running buffer used, the running temperature, the voltage, and the switching times of electrophoresis. A new harmonized protocol was agreed on, further modified in a pilot study in two laboratories, and finally tested by all others. Seven laboratories' gels were found to be of sufficiently good quality to allow comparison of the strains by using a computer software program, while two gels could not be analyzed because of inadequate destaining and DNA overloading. Good-quality gels and inclusion of an internal quality control strain are essential before attempting intercenter PFGE comparisons. A number of clonally related strains have been shown to be present in multiple countries throughout Europe. The well-known Iberian clone has been demonstrated in Belgium,

Streptococcus pyogenes (group A streptococcus [GAS]), a major human pathogen (9), has been studied for decades and may give rise to common throat and skin infections as well as to invasive diseases, such as arthritis, septicemia, cellulitis, puerperal fever, necrotizing fasciitis (NF), and streptococcal toxic shock syndrome (STSS) (14). Since the mid-1980s there have been increasing numbers of reports describing severe manifestations of GAS infections; however, the factors underlying the worldwide resurgence of this pathogen remain unknown (20).The M protein, which is encoded by the emm gene, is an important virulence factor and is also an epidemiological marker that is used throughout the world to characterize GAS isolates (5,(21)(22)(23). The type specificity of the M protein, of which more than 100 different types are known, is largely determined by the epitope located in 40 to 50 amino acid residues at the amino terminus (4,16,27). These regions of M proteins have been shown to evoke antibodies that have strong bactericidal activity and that are not likely cross-reactive with

VIM-1-producing Klebsiella pneumoniae (VPKP) is an emerging pathogen. A prospective observational study was conducted to evaluate the importance of VIM production on outcome of patients with K. pneumoniae bloodstream infections (BSIs). Consecutive patients with K. pneumoniae BSIs were identified and followed up until patient discharge or death. A total of 162 patients were included in the analysis; 67 (41.4%) were infected with VPKP, and 95 were infected with non-VPKP. Fourteen of the patients infected with VPKP were carbapenem resistant (Carb r ) (MIC > 4 g/ml), whereas none of the non-VPKP exhibited carbapenem resistance. The patients infected with a Carb r organism were more likely (odds ratio, 4.08; 95% confidence interval [CI], 1.29 to 12.85; P ‫؍‬ 0.02) to receive inappropriate empirical therapy. The all-cause 14-day mortality rates were 15.8% (15 of 95) for patients infected with VIM-negative organisms, 18.9% (10 of 53) for those infected with VIM-positive carbapenem-susceptible organisms, and 42.9% (6 of 14) for those infected with VIM-positive Carb r organisms (P ‫؍‬ 0.044). In Cox regression analysis, age (hazard ratio [HR], 1.03; 95% CI, 1.01 to 1.06; P ‫؍‬ 0.021), rapidly fatal underlying disease (HR, 2.84; 95% CI, 1.26 to 6.39; P ‫؍‬ 0.012), and carbapenem resistance (HR, 2.83; 95% CI, 1.08 to 7.41; P ‫؍‬ 0.035) were independent predictors of death. After adjustment for inappropriate empirical or definitive therapy, the effect of carbapenem resistance on outcome was reduced to a level of nonsignificance. In patients with K. pneumoniae BSIs, carbapenem resistance, advanced, age, and severity of underlying disease were independent predictors of outcome, whereas VIM production had no effect on mortality. The higher mortality associated with carbapenem resistance was probably mediated by the failure to provide effective therapy.Over the last few years, the reliance on carbapenems has been challenged owing to the wide spread of acquired metallo-␤-lactamases [MBLs] (2,11,20,25,31). Two dominant groups of acquired MBLs have been recognized: the IMP and VIM types. This class of enzymes is characterized by the ability to hydrolyze carbapenems and all available ␤-lactams with the exception of aztreonam. Moreover, since the MBL genes are linked to other resistance determinants within the same integron or plasmid, MBL-producing organisms are commonly multidrug resistant, further compromising our therapeutic options (8,26,28).MBLs have spread throughout the world with an overall trend moving from Pseudomonas aeruginosa into Enterobacteriaceae (9,10,14,16,18,22,23,28). Recently, there have been several reports on the emergence of VIM-producing Klebsiella pneumoniae (VPKP), mainly in Southern Europe (16,20). In Greece, VPKP isolates are endemic in various hospitals and cause life-threatening infections (5,8,29). Based on previous reports (30) and on the National Surveillance System for Antibiotic Resistance Database (www.mednet.gr/whonet/top .htm), the dissemination of such isolates appears to have occurred in a r...

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Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

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