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Many different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria. A group of international experts came together through a joint initiative by the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC), to create a standardized international terminology with which to describe acquired resistance profiles in Staphylococcus aureus, Enterococcus spp., Enterobacteriaceae (other than Salmonella and Shigella), Pseudomonas aeruginosa and Acinetobacter spp., all bacteria often responsible for healthcare-associated infections and prone to multidrug resistance. Epidemiologically significant antimicrobial categories were constructed for each bacterium. Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA). MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories, XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e. bacterial isolates remain susceptible to only one or two categories) and PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure correct application of these definitions, bacterial isolates should be tested against all or nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided., (© 2011 European Society of Clinical Microbiology and Infectious Diseases. No claim to original US government works.)

Objective: To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates., Methods: E. coli strains recovered at two Belgian hospitals between 2004 and 2006 were selected on the basis of an AmpC-producing phenotype. Plasmid-mediated cephalosporinases coding genes and the sequence of chromosomal ampC genes were identified by PCR/sequencing. The isolates were submitted to phylotyping and genotyping analysis using rep-PCR (Diversilab) and PFGE. A novel chromosomal ampC gene was cloned., Results: Eighty-three out of 6850 E. coli isolates were selected. Seventy-two isolates were found to overexpress their chromosomal cephalosporinases while 11 contained plasmid-mediated cephalosporinases. Among chromosomal AmpC overproducers, 12 were extended-spectrum AmpC (ESAC) expressing isolates which all displayed reduced susceptibility to cefepime. Cloning of a new ESAC allele suggested that L293P mutation was responsible of the extension of the hydrolysis spectrum to cefepime. AmpC overproducers, including ESAC producers, predominantly belonged to phylogenetic group A and B1, while plasmid-mediated AmpC-producing isolates preferentially belong to phylogroup B2 and D. According to rep-PCR, the majority of the E. coli isolates belonging to phylogroup A were clonally related which was further confirmed by PFGE for the 11 ESAC expressing isolates., Conclusions: Chromosomal AmpC overproduction was the most common resistance mechanism, and the occurrence of ESAC was found to be as frequent as plasmid-mediated cephalosporinases. The detection of a new ESAC allele, of an ESAC producing strain belonging to phylogroup D and the existence of a clonal relationship between ESAC producing strains underline the need for study of the clinical relevance of this mechanism of resistance., (Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.)

The public health burden caused by methicillin-resistant Staphylococcus aureus (MRSA) infections is now widely recognized, and is a cause of public alarm. Effective MRSA risk management in the healthcare system as well as in the community should rely on accurate detection of reservoirs and sources of transmission, as well as on close monitoring of the impact of interventions on disease incidence and bacterial dissemination. MRSA carrier screening and disease surveillance, coupled with molecular typing, are key information tools for integrated MRSA control and individual risk assessment. These tools should be tailored to the distinct needs of local interventions and national prevention programmes. Surveillance schemes should primarily inform local staff and serve as quality assurance about MRSA risk management. New technologies, including the use of selective culture media and real-time PCR assays, allow faster detection of MRSA carriers upon admission or during stay in healthcare institutions. More research is needed to ascertain their cost-effectiveness for MRSA control. Likewise, tremendous progress has been made concerning molecular typing methods, with optimization and standardization of sequence-based technologies offering broad applicability and high throughput. However, no single S. aureus typing method is yet providing fully reliable information within the range of discrimination needed for public health action. Further refinement of genotyping methods and international harmonization of surveillance and typing schemes must be achieved to facilitate global MRSA control.

National surveillance of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) isolates by pulsed-field gel electrophoresis (PFGE) typing allowed identification of rarely occurring 'sporadic' isolates with patterns significantly distinct from those of major epidemic clones of methicillin-resistant S. aureus (MRSA) circulating in Belgian hospitals. The aim of the present study was to compare the genetic background, antibiotic susceptibility profile and in vitro growth rates of 36 MRSA isolates with either 'epidemic' or 'sporadic' PFGE profiles to identify factors that could be involved in the epidemic behaviour of S. aureus. Sequence analysis of seven housekeeping genes (multilocus sequence typing) and seven surface-associated genes, combined with staphylococcal cassette chromosome mec (SCCmec) typing and spa typing results, segregated sporadic isolates into four groups: (1) isolates phylogenetically distant from epidemic HA-MRSA clones that possessed several properties of community-acquired MRSA strains; (2) isolates derived from the same methicillin-susceptible S. aureus ancestor as epidemic isolates but possessing a distinct type of SCCmec; and (3) and (4) isolates that were closely related to epidemic strains, either as recent descendants of these or as intermediate evolutionary steps between epidemic HA-MRSA strains and their putative ancestors. Sporadic isolates did not show slower growth in vitro than epidemic isolates. These findings suggest that the SCCmec type and insertion/deletion of other mobile genetic elements may be involved in modulating the epidemic behaviour of MRSA strains of similar genetic background, independently of fitness cost.

Background: Multidrug-resistant tuberculosis (MDR-TB) has become a therapeutic problem in many parts of the world, necessitating the inclusion of second-line anti-tuberculosis drugs in specific treatment regimens., Methods: We studied the susceptibility of 69 MDR Mycobacterium tuberculosis isolates from Rwanda to second-line drugs by the BACTEC 460 method., Results: The results showed that 62 (89.9%) were resistant to rifabutin while a low rate (4.3%) of resistance was registered for ofloxacin; there was one case (1.4%) of resistance each for para-aminosalicylic acid, kanamycin, ethionamide, and clarithromycin., Conclusions: This information is important for devising an appropriate treatment regimen for MDR-TB patients in order to stop the spread of MDR strains and contain the acquisition of additional drug resistance in Rwanda.

Introduction: A cluster of three related cases of tuberculosis (TB) with primary multidrug resistance was investigated at the Centre Hospitalier Universitaire of Kigali (CHUK) in Rwanda. The patients were HIV-1/2 seronegative. Patients 1 and 2 were hospitalized in the same room of CHUK for one month. Patient 3 was a younger sibling of patient 2., Methods: Drug susceptibility of two consecutive Mycobacterium tuberculosis isolates from each patient was tested by the BACTEC 460 radiometric method. DNA fingerprinting was performed using spoligotyping and mycobacterial interspersed repetitive units of variable numbers of tandem repeats (MIRU-VNTR) analysis. All patients initially received the World Health Organization category I regimen., Results: The isolates collected during the first TB episode were resistant to isoniazid, rifampin and ethambutol. After subsequent retreatment regimens with rifampin, isoniazid, streptomycin, pyrazinamide (8 months) and rifampin, isoniazid, streptomycin, pyrazinamide, ciprofloxacin (21 months), patients 1 and 2 developed additional resistance to streptomycin and quinolones. Patient 3 received only the category I regimen and consecutive isolates retained the initial drug susceptibility pattern. All isolates were genetically indistinguishable by spoligotyping and MIRU-VNTR, indicating the same origin., Conclusions: These observations highlight the risk of nosocomial transmission of multidrug-resistant (MDR) TB and the possible selection of secondary resistance to second-line drugs if a single new drug is added at the time of retreatment of MDR TB patients.

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.

The optimal management of bacteraemia related to the presence of totally implantable venous access devices (TIVADs) remains controversial, particularly in terms of whether to remove the infected catheter. The objective of this study was to determine the factors associated with success or failure of treatment of TIVAD-related bacteraemia in patients from whom the infected device was not removed. The outcome of 92 episodes of TIVAD-related bacteraemia and the factors predictive of an unfavourable outcome were evaluated retrospectively. In 32 (35%) episodes, the devices were removed immediately. In 60 episodes, patients were treated with antibiotics infused through the device; treatment was successful in 56% of these cases (66% for infections caused by coagulase-negative staphylococci). Only the presence of sepsis (OR 9.42, 95% CI 1.29-68.92, p 0.0271) and of local signs of infection (OR 9.61, 95% CI 1.98-46.49, p 0.0049) independently predicted the failure of catheter-retaining treatment. Finally, only one-third of the devices were retained. In conclusion, the large number of TIVADs that are removed because of infection justifies reconsidering the criteria for device removal. During catheter-retaining treatment, the presence of local signs of infection or reported sepsis were independent factors for reduced probability of retaining the device.

Major regional variations in the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) are observed across Europe. This study investigated hospital MRSA prevalence in relation to patterns of antimicrobial use and infection control policies in an observational, cross-sectional study that used retrospective data from 2001 and linear regression to model relationships. MRSA prevalence (median 20.8%, n = 173 hospitals) and antimicrobial consumption (median 55.2 defined daily doses/100 bed-days, n = 140 hospitals) both varied significantly according to geographical region (p <0.001). MRSA prevalence and antimicrobial consumption data were provided by 128 hospitals, and showed a strong statistical relationship between macrolide use and MRSA prevalence. Use of (i) third-generation cephalosporins, (ii) all antimicrobial agents, and (iii) all antimicrobial agents except glycopeptides was also associated with MRSA prevalence. Up to 146 hospitals provided data on MRSA prevalence and key infection control parameters. Adjusted linear regression modelling provided strong evidence that infection control policy recommendations associated with lower MRSA prevalence rates were (i) use of alcohol-based solutions for hand hygiene (mean difference 10.3%, 99% CI 1.2-10.3), and (ii) placement of MRSA patients in single rooms (mean difference 11.2%, 99% CI 1.4-20.9). Hospitals with problems in implementing isolation policies had higher resistance levels (mean difference 12%, 99% CI 3.8-20.1). Additional recommendations showed less evidence of association with a low MRSA prevalence. Overall, this study highlighted significant associations between MRSA prevalence, antimicrobial use and various key infection control parameters, all of which showed significant individual variations according to geographical region.

In total, 150 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected during 2002 from a general Belgian hospital were phage-typed at routine test dilution x 100. The majority (45%) belonged to phage group (J)*, while 10% were classified as a new phage type 29/(42E)/54/(D11)*. The isolates belonging to this new type carried the aac(6')-aph(2'') and the aph(3') aminoglycoside resistance genes and showed high-level resistance to oxacillin. Molecular typing revealed that they belonged to the multiresistant clonal pulsed-field gel electrophoresis (PFGE) type D8. PFGE group D, characterised as genotype ST228-MRSA-I, has been present in Belgian hospitals since 1999.

Patient safety in hospital care depends on effective infection control (IC) programmes. The Antimicrobial Resistance Prevention and Control (ARPAC) study assessed the organisation, components and human resources of IC programmes in European hospitals. A questionnaire survey of policies and procedures implemented in 2001 for the surveillance and control of nosocomial infection and antibiotic resistance was completed by 169 acute-care hospitals from 32 European countries, categorised by five geographical regions. A formal IC programme existed in 72% of hospitals, and a multidisciplinary IC committee was operational in 90%. Trained IC nurses (ICNs) were present in 80% of hospitals (ranging from 54% in south-east and central-eastern Europe, to 100% in northern Europe), whereas 74% had one or more trained IC doctors (ICDs) (ranging from 46% in south-east Europe to 84% in western Europe). Median staffing levels were 2.33 ICNs/1,000 beds and 0.94 ICDs/1,000 beds. The intensity of IC programmes scored higher in centres from northern and western Europe than from other European regions. Written guidelines promoted hand hygiene for healthcare workers in 89% of hospitals, education in 85%, and audit in 46%. Guidelines recommended use of alcohol-based solutions (70%) and/or medicated/antiseptic soap (43%) for decontamination of non-soiled hands. Use of alcohol-based solutions varied according to region, from 41% in southern Europe to 100% in northern Europe, compared with use of medicated soap from 77% in southern Europe to 11% in northern Europe (p < 0.01). These findings showed that IC programmes in European hospitals suffer from major deficiencies in human resources and policies. Staffing levels for ICNs were below recommended standards in the majority of hospitals. Education programmes were incomplete and often not supported by audit of performance. Hand hygiene procedures were sub-standard in one-third of centres. Strengthening of IC policies in European hospitals should be a public health priority.

Antimicrobial resistance is a key public health concern in Europe. It is known that there are significant variations in the prevalence of resistance across Europe, and methods to reduce the problem are also assumed to vary significantly. The 'Antibiotic Resistance; Prevention and Control (ARPAC)' Concerted Action project was funded by the European Commission and conducted by four study groups of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). The project established a network of European hospitals and collated data on antimicrobial resistance prevalence, antimicrobial susceptibility testing methods, typing methods employed, antimicrobial use, antibiotic policies and practices, and infection control policies and practices. The ARPAC Consensus Conference, entitled 'Control of antibiotic resistance in European hospitals-informing future evidence-based practice', was held in Amsterdam in November 2004. The conference was co-hosted by the European Commission, ESCMID and the Dutch Working Party on Antibiotic Policy (SWAB). Key ARPAC findings were presented and discussed in the context of the worldwide situation. The conference delivered a set of high-priority recommendations likely to have a significant impact on antimicrobial resistance. This report summarises these recommendations.

In total, 446 healthy individuals (240 in urban and 206 in rural environments, respectively) were selected from 96 households, based on cluster sampling of residential location. Demographic, sociological and environmental data were collected by face-to-face interviews using a standard questionnaire. Infection with Helicobacter pylori was assessed by detection of anti-H. pylori IgG serum antibodies. The prevalence of H. pylori antibodies was 75.4% in the urban population and 72.3% in rural (village) residents (p 0.459). No association was found between infection and age, gender, education level, size of household, economic activity or source of drinking water. The infection rate was higher in children whose parents were both infected, and also in children whose mother was infected (p < 0.001). By logistic regression analysis, the density of occupation of sleeping accommodation (more than three persons sharing a room; 95% odds ratio (OR) = 9.82 (4.13-23.31), p < 0.001), and the mother's status within the household (95% OR = 3.85 (1.53-9.67), p 0.003), were independent predictors for H. pylori infection. The 74% seroprevalence of H. pylori infection found in healthy Beninese individuals is similar to that reported previously from other parts of sub-Saharan Africa. Family contact with infected persons and crowded living conditions were associated with increased risk of infection. These findings are consistent with intra-familial H. pylori transmission and suggest that improvement of living conditions should be protective against infection.

Staphylococcus aureus strains with decreased susceptibility to glycopeptides (GISA) have been associated with increased risk of glycopeptide treatment failure. To assess the prevalence of these strains in hospitalised patients in Belgium, 455 methicillin-resistant S. aureus (MRSA) isolates collected in 2001 were screened by two assays: (i) growth on vancomycin agar screen (VAS; brain heart infusion agar (BHI) + vancomycin 6 mg/L); and (ii) a synergy/antagonism test with aztreonam/cefazolin on Mu3 agar (BHI + vancomycin 3 mg/mL). Isolates growing on VAS or Mu3 agar were characterised further by analysis of population susceptibility profiles. MICs of glycopeptides were determined by agar dilution, broth microdilution and Etest (low and high inocula) methods. The isolates were genotyped by pulsed-field gel electrophoresis (PFGE) and determination of staphylococcal cassette chromosome mec (SCCmec) type. No GISA isolates were found. Three (0.7%) hetero-vancomycin intermediate S. aureus (hVISA) and ten (2.2%) hetero-teicoplanin intermediate S. aureus (hTISA) isolates were identified by population analysis. All but one hetero-GISA isolate belonged to either epidemic PFGE group A/SCCmec type I (69%) or PFGE group D/SCCmec type I (23%), both of which were resistant to gentamicin. The sensitivity and specificity for the detection of hetero-GISA by the two assays were 15.4% and 99.8%, respectively, for VAS, and 84.6% and 95.9%, respectively, for Mu3. The data indicated that hetero-GISA strains were uncommon among Belgian MRSA isolates from hospitalised patients. Use of Mu3 agar was more sensitive, but less specific, than VAS as a screening method.

The Vitek 2 system was assessed against reference methods with 197 methicillin-resistant Staphylococcus aureus from Belgian hospitals and 121 clinically significant blood culture isolates of Staphylococcus spp. Vitek 2 identified 95% of staphylococcal isolates correctly, detected oxacillin resistance with a sensitivity/specificity of 99/96%, and showed acceptable accuracy for susceptibility testing of five of eight other evaluable antibiotics. The median time for reporting results was 2 h 45 min for identification and 7 h for susceptibility tests.

Brevibacterium has been reported as a rare cause of implanted-device infection. In two cases of recurrent Brevibacterium casei bacteraemia associated with infection of surgically implanted intravascular devices, relapse occurred 2 and 5 months, respectively, after completion of therapy with vancomycin via the infected catheter. A second intravenous antibiotic therapy course by the antibiotic-lock technique led to bacteriological cure in one patient. Molecular typing results demonstrated that the recurrent bacteraemia was caused by the same strain. Implanted-device removal may be necessary, in addition to appropriate antibiotics, for successful management of such infections.

Objective: To express the views of a working party held to consider antibiotic resistance surveillance systems, their strengths and weaknesses, and their current and future applications., Methods: The participants, all of whom were experienced in this field, discussed the development of surveillance systems in relation to the increasing prevalence of resistance to antibacterial agents and the current interest in surveillance systems shown by many official bodies, in both the human and veterinary fields. The problems inherent in surveillance systems were considered together with the applications of different systems., Results: The properties of good antibiotic resistance surveillance systems were defined. Surveillance systems vary widely from those with a narrow base, focusing on few organisms in one disease area, to those covering many diseases, many organisms (including normal flora) and many compounds. Whatever their design, they should be able to detect significant differences and shifts in susceptibility to various antibacterial agents, and the information derived from them should reach as many interested parties as possible in a timely manner. In using this information to decide strategies, criteria for action need to be determined by pragmatic consensus. Funding remains a major problem, with few large studies being supported by official bodies in spite of their professed enthusiasm for surveillance. In consequence, many current systems are funded by the pharmaceutical industry and are of necessity restricted in their focus., Conclusions: Antibiotic resistance surveillance studies should and can be well planned and well executed. Many current systems suffer from well-recognized but uncorrected biases. Consortium funding will be necessary for large schemes to be successful. There is no "ideal" surveillance system.

A group of experts met under the auspices of the European Science Foundation, the European Medical Research Council and the European Society of Clinical Microbiology and Infectious Diseases to discuss formulation of a European strategy on the control of antibiotic resistance in Europe. This is a report of the meeting which was used as the basis for a European Commission grant application. The need for a common strategy to make best use of scarce resources was agreed and it was concluded that the first stage (discussed in this article) is to collate existing data on resistance rates, antibiotic consumption, antibiotic stewardship, infection control and molecular typing methods. Consensus reached from analysis of these data can direct us to the most appropriate controlled trials. While it is accepted that there is a widely held perception that the medical profession has been slow to react to the problem of antibiotic resistance, much more work still needs to be carried out before we can recommend, implement and trial definitive control measures. In the meantime, however, all reasonable efforts should be made to reduce antibiotic consumption without compromising patient care, establish cost-effective surveillance systems using existing laboratory generated data, improve hygiene in our hospitals and better define the molecular basis of antibiotic resistance.

Objective: The 'HICPAC guidelines', published in the USA in 1995 stressed the crucial role of restrictive usage of glycopeptides in the strategy to limit the emergence and spread of resistant enterococci. Because controversy still remains in Belgium on the necessity and feasability of restricting glycopeptide usage, the infectious diseases advisory board (IDAB) developed a consensus statement on the judicious use of glycopeptides in Belgium., Methods: The literature on the indications for glycopeptide treatment was reviewed, categorized and discussed by a working party of the IDAB.Consequently, the IDAB reached consensus on the warranted indications for glycopeptide use in Belgium., Results: The opinion of the IDAB-members is reported in a consensus statement specifying the indications for treatment and for prophylaxis with glycopeptide antimicrobials, as well as the situations where glycopeptides should not be used, taking into account the specific epidemiology of bacterial resistance, the availability of antibiotics and the common prescribing practices in Belgium., Conclusions: The IDAB concludes that restrictive usage of glycopeptides must also be a priority in Belgium. Guidelines on the judicious use of these antibiotics adapted to the national situations must contribute to this objective.

Objectives: To determine the diversity of pulsed-field gel electrophoresis (PFGE) types among epidemic strains of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Belgium, France, Germany and The Netherlands over the period 1981-94., Methods: MRSA strains collected in a multicenter survey in Belgium (n = 171) and from reference laboratories in neighboring countries (n = 102) were characterized by PFGE analysis using the SmaI enzyme., Results: In total, 32 PFGE types were found. Epidemic PFGE type 1, first recognized in 1984, accounted for 82% of Belgian strains (87% of hospitals) and 51% of European MRSA strains. Four other internationally epidemic PFGE types (types 8, 10, 11 and 12) were less widely disseminated and more recently detected (1991-94), each recovered from two or three countries. International spread of two PFGE types was linked to transfer of colonized patients to Dutch hospitals from another country where this type was frequently recovered., Conclusions: Genotypic analysis indicated widespread distribution of several outbreak-associated MRSA strains over large European regions, which was in some instances related to interhospital patient transfer. These findings underscore the need for standardized international surveillance and control of MRSA transmission between healthcare institutions across Europe.

Objective: To study the epidemiology of Enterobacter aerogenes infections in Belgian hospitals and determine whether recent trends show an increase in incidence of E. aerogenes infections and antimicrobial resistance., Methods: Data from the bloodstream infection component of the National Surveillance of Hospital Infections (October 1992 to September 1996 data in 45 hospitals) and from a retrospective study on E. aerogenes clinical isolates (1994 and 1995 data in 41 hospitals) were analyzed., Results: E. aerogenes was recovered from clinical specimens with a mean incidence of 4.6 isolates per 10 000 patient-days and caused 0.20 bloodstream infections per 10 000 patient-days during the surveyed periods, respectively. Both rates increased significantly throughout the years. The proportion of E aerogenes within the Enterobacter genus was 35.4% in clinical isolates and 41.2% in bloodstream infections. Both proportions significantly increased over time. Incidence was not statistically different by hospital size but showed major differences between geographic regions. Resistance rates to third-generation cephalosporins and fluoroquinolones increased, and imipenem resistance emerged in several hospitals., Conclusions: This report provides evidence of an increase in E. aerogenes infections in Belgian hospitals and documents an increase in antimicrobial resistance of E. aerogenes strains. These figures provide a baseline for further surveillance data., (1999 European Society of Clinical Microbiology and Infectious Diseases.)

Fifty nosocomial isolates of Serratia marcescens, collected in six Belgian hospitals between 1986 and 1990, were characterized by using pulsed-field gel electrophoresis (PFGE) with XbaI. The results were compared with those previously obtained by three other methods: biotyping, esterase electrophoresis typing and ribotyping with EcoRI and HindIII. Macrorestriction analysis (42 PFGE groups) and esterase typing (42 zymotypes) proved to be the most discriminating, followed by ribotyping (28 ribotypes) and biotyping (10 biochemical profiles). Biotyping would serve as a screen to identify isolates, due to its accessibility. Esterase typing provided a reliable tool to make subdivisions within biotypes because of congruence between biochemical groups and esterase patterns. Additional discrimination was still achieved by ribotyping and PFGE. It is concluded that the combined results of these four markers were useful for distinguishing all epidemic and sporadic isolates.

Oligonucleotide primers were used in the polymerase chain reaction (PCR) to amplify a specific 584-bp DNA fragment, located in the 16S RNA gene of Mycobacterium xenopi. This set of primers, X222 and X224, was able to discriminate between the pathogen and other mycobacterial species as well as non-mycobacterial strains; it detected down to 3 fg of M. xenopi DNA, i.e. about one genome equivalent. These oligonucleotide primers proved suitable for the routine identification of M. xenopi cultures, starting from one single colony on solid medium or from a liquid culture in Middelbrook 12B "Bactec" medium. In addition, a luminescent hybridization assay was designed for use on PCR-amplified DNA. This system, which, for capture, relied on a matrix-bound oligonucleotide (M30) specific for the genus Mycobacterium and, for detection, on a biotinylated xenopi-specific X221 probe, proved fully specific, highly sensitive and rapid for the evaluation of M. xenopi Bactec cultures at low growth index.