Accurate drug susceptibility testing (DST) for Mycobacterium tuberculosis is highly important for both therapy guidance and surveillance of drug resistance. Although liquid medium DST methods are used increasingly and seem most efficient and fast, the high costs hamper widespread implementation. In addition, an inability to check the colony morphology of the growing bacteria is a disadvantage of these methods. Moreover, these methods discriminate only between susceptibility and resistance and do not determine the MIC. In this paper, we describe a low-cost, reproducible, high-throughput, proportional absolute concentration DST method. The method uses a concentration series of antituberculosis drugs, including pyrazinamide in 7H10 medium, distributed semiautomatically in 25-well plates. The performance of this 25-well DST method was evaluated by the World Health Organization and the International Union against Tuberculosis and Lung Disease in 10 rounds of proficiency testing regarding sensitivity, specificity, efficiency, reproducibility, and predictive value for resistance and susceptibility. The performance of the method for these characteristics was 100% for isoniazid and from 96 to 100% for rifampin, 91 to 100% for streptomycin, and 85 to 100% for ethambutol. The method was 100% reproducible for all four drugs. The levels of drug resistance and the MIC distributions for the first-line antituberculosis drugs were determined for all 7,956 M. tuberculosis strains isolated in The Netherlands from 1998 to 2005 and amounted to 7.5% for isoniazid, 1.4% for rifampin, 8.5% for streptomycin, and 1.0% for ethambutol. Pyrazinamide testing was successful for 7,026 (88.3%) of the isolates and showed a resistance level of 0.8%.

A clinical isolate ofHaemophilus influenzae HC234 was found to be resistant to chloramphenicol and tetracycline. It was shown that both resistance markers are tranferable as one unit to other Haemophilus influenzae strains and also to Escherichia coli. Data are presented which indicate that conjugation is the most likely mechanism of resistance transfer. HC234 was shown to carry a single plasmid species with a molecular weight of 38 x 106.Recent investigations have shown the existence of plasmid-mediated drug resistance in Haemophilus influenzae (2,3,5,17). Transfer of resistance, probably by conjugation, was demonstrated for resistance to ampicillin (13,15) and for resistance to kanamycin (3). Recently, we reported on a chloramphenicol (Cm)-resistant strain of H. influenzae isolated from the pharynx of a patient with lymphatic leukemia (10). The resistance to Cm appeared to be due to the production of an enzyme, presumably an acetyltransferase, inactivating this antibiotic. The strain was also resistant to tetracycline (Tc).This report describes experiments on the transfer of the resistance determinants to susceptible strains ofH. influenzae and to a strain of Escherichia coli. Also, the isolation and characterization of plasmid deoxyribonucleic acid (DNA) from the resistant strain are reported. MATERIALS AND METHODSBacterial strains. The strains used in this study are listed in Table 1. The rifampin-and streptomycin-resistant mutant strains HC215-1, HC217-1, and HC218-1 were obtained by selection on plates containing these antibiotics. All strains of H. influenzae investigated in this study were untypable according to Pittman's agglutination method (12). The strain ofE. coli K-12 (SC181) was kindly provided by E. Lederberg (Stanford University).Media. Nutrient broth was prepared in this institute from fresh meat and contained in addition 0.5% NaCl, 1% peptone (Difco) and 0.069% Na2CO3; the pH was 7.5. Nutrient agar contained, in addition, 2% agar (BBL).NXV broth was nutrient broth, supplemented with 4% X factor and 4% V factor. X and V factors were freshly prepared from lysed horse blood and lysed yeast cells, respectively. NXV agar was prepared by adding 2% agar to NSV broth.Stock cultures of H. influenzae were maintained and transferred every 2 weeks in semisolid NXV medium containing 1% agar (BBL).Susceptibility tests were usually carried out on NXV agar. Wellcotest agar, supplemented with X and V factors was used for establishing the susceptibility to cotrimoxazole.Determination of MICs. The minimum inhibitory concentrations (MICs) were determined by agar dilution, using a Steers replicator. As inocula, undiluted overnight cultures (10k to 109 colony-forming units/ml) and also the dilutions 10-', 10-2, and 1-3 were used. The antibiotics used were: Cm (Globenicol, Mycofarm), Tc (Tifaco), ampicillin sodium (Amfipen, Mycofarm), erythromycin (Erythrocin, Ab-

Penicillinase-producing Neisseria gonorrhoeae strains were isolated in the Netherlands with increasing frequency during the period of 1976 to 1979. About 3% of the gonococci isolated in the first half of 1979 produced penicillinase. In contrast to the period of 1976 to 1977, most penicillinase-producing N. gonorrhoeae infections during the period of 1978 to 1979 were contracted in the Netherlands. The results of genetic and molecular studies on 80 penicillinaseproducing N. gonorrhoeae strains were similar to earlier observations of others: resistance plasmids of only two sizes, 4.5 and 3.3 megadaltons (Md), occurred in penicillinase-producing N. gonorrhoeae strains, and these encoded for the TEM-1 enzyme. The 4.5-Md plasmid could be transferred to Escherichia coli when it coexisted with a plasmid of 24 Md. The latter plasmid was present in the vast majority of the strains carrying the 4.5-Md plasmid. One strain carried a cryptic 7.5-Md plasmid in addition to the commonly found 2.5-Md plasmid. Two penicillinase-producing strains of Haemophilus parainfluenzae isolated were found to carry a 3.3-Md plasmid species which was indistinguishable from the 3.3-Md gonococcal resistance plasmids. No plasmid deoxyribonucleic acid was found in two strains of penicillinase-producing Branhamella catarrhalis, and these strains produced a penicillinase different from the TEM-1 enzyme.Penicillinase-producing Neisseria gonorrhoeae (PPNG) strains were isolated for the first time in 1976 in the United Kingdom and the United States (2, 21), and since then they have been found in many other countries (4,5,20,22,35). A significant proportion of PPNG infections encountered in several Western countries seems to have been acquired from foreign sources rather than from local contacts (1). Importation has been mainly from countries in the far east and from the west coast of Africa (1,2,5,18,20,21,27). The origin of these PPNG strains was correlated with two different types of penicillinresistance plasmids: a 4.4-megadalton (Md) plasmid originating in the far east countries and a 3.2-Md plasmid originating in west Africa (9,20,24,25).The emergence of PPNG strains at the end of 1976 (5) and their potential dissemination among the general population is of public health concem in the Netherlands. For this reason, the epidemiology of PPNG strains is studied centrally, and all PPNG strains isolated are asked to be sent to the National Institute of Public Health for confirmation of penicillinase production. Furthermore, the miniimal inhibitory concentrations of various antibiotics are determined for all PPNG strains isolated.To monitor changes at the genetic level, plasmids have been isolated and characterized from about 30% of all PPNG strains isolated in the period 1976 to the beginning of 1979. In this report we describe the results of the epidemiological data, sensitivity testing, and plasmid properties of PPNG strains. Furthermore, several isolates of penicillinase-producing strains of

The Gen-Probe amplified Mycobacterium tuberculosis direct test can give discrepant results directly in respiratory or cultured samples from patients infected with Mycobacterium celatum, leading to inappropriate therapy for, in our case, an immunocompetent patient.Mycobacterium celatum was first described in 1993 (3), and since then, sporadic reports have been published on the isolation of this mycobacterium from immunocompromised patients (1,5,6,8,11,12 1994), and a fatal pulmonary infection in an apparently healthy adult (4). Biochemically, the organism is indistinguishable from the Mycobacterium avium complex, and mycolic acid high-pressure liquid chromatography analysis or genetic analysis is required for proper identification (3). For identification, cross-reactivity with nontuberculous mycobacteria has been described with the AccuProbe culture confirmation test (Gen-Probe, San Diego, Calif.) for the M. tuberculosis complex (2, 10). This note describes the initial misidentification and interpretation of a positive amplified M. tuberculosis direct (AMTD) test (GenProbe) result for a respiratory sample derived from an immunocompetent patient with an M. celatum infection.A 61-year-old man was referred on 5 March 1999 by a general practitioner to the outpatient respiratory disease clinic because of general malaise, a productive cough, and an unexplained 16-kg weight loss in the past 3 years. The chest radiograph showed a pulmonary infiltrate in the right upper lobe, with extensive cavities identified by computed tomography. A bronchial lavage was performed and showed on average more than one acid-fast rod per high-power field (magnification, ϫ1,000. The AMTD test was positive with a value of 1,000,000 relative light units (RLU). Tuberculostatic drug therapy was started with 300 mg of isoniazid, 600 mg of rifampin, and 2,000 mg of pyrazinamide. Liquid culture (MB/BacT; Organon Teknika, Boxtel, The Netherlands) was positive on 25 March 1999 and was subcultured on Löwenstein Jensen agar. The subculture grew very small smooth colonies with a pale yellow pigment and was sent to the Mycobacterium section of the National Institute of Public Health and the Environment (RIVM, Bilthoven, The Netherlands) for drug susceptibility testing by the agar proportion method and species identification. The AccuProbe culture confirmation tests for M. tuberculosis, M. avium complex, Mycobacterium kansasii, and Mycobacterium gordonae were negative. Because of the discrepancy between the AMTD test as performed by the Medical Microbiology department at the University Hospital Maastricht and the AccuProbe results of the RIVM, a culture was sent again to the RIVM. This material again yielded a positive result (509,094 RLU) in the AMTD test in Maastricht and again yielded a negative result in the AccuProbe test at the RIVM. Therefore, it was concluded that the AMTD result was probably a false positive. Drug susceptibility data were only obtained on 26 June 1999 (Table 1) because of the slow growth and the initial suspicion that we were de...

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