1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2007, p Vol. 45, No /07/$ doi: /jcm Copyright 2007, American Society for Microbiology. All Rights Reserved. Molecular Epidemiological and Antibiotic Susceptibility Characterization of Brucella Isolates from Humans in Sicily, Italy Cinzia Marianelli, 1 * Caterina Graziani, 1 Carmela Santangelo, 2 Maria Teresa Xibilia, 2 Alida Imbriani, 2 Rosa Amato, 3 Domenico Neri, 3 Mario Cuccia, 4 Sebastiano Rinnone, 4 Vincenzo Di Marco, 5 and Franco Ciuchini 1 Dipartimento di Sanità Alimentare ed Animale, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy 1 ; Azienda Ospedaliera Vittorio Emanuele, Dipartimento di Microbiologia, Via Plebiscito 628, Catania, Italy 2 ; Azienda Ospedaliera Gravina, Dipartimento di Microbiologia, Via Portosalvo 1, Caltagirone, Italy 3 ; Azienda Unità Sanitaria Locale No. 3, Dipartimento di Prevenzione, Via Santa Maria La Grande 5, Catania, Italy 4 ; and Istituto Zooprofilattico Sperimentale della Sicilia, Via S. Andrea 96, Barcellona Pozzo di Gotto, Italy 5 Received 18 April 2007/Returned for modification 18 June 2007/Accepted 6 July 2007 Brucellosis is a serious problem in Sicily. Brucella melitensis was identified as the species most frequently isolated in humans in Italy. No data, however, are available about the molecular epidemiological characterization of Brucella isolates from humans. We have conducted this study to molecularly characterize clinical isolates of Brucella spp. and to evaluate their antimicrobial susceptibilities. Twenty Brucella isolates were studied. Differential growth characteristics and DNA polymorphisms such as the restriction patterns of the PCR-amplified omp2a and omp2b genes, rpob nucleotide sequencing, and multiple-locus variable-number tandem repeat analysis of 16 loci (MLVA-16) were used to characterize the strains. In vitro antibiotic susceptibility was determined by the E-test method on two different agar media, and the results were compared. All isolates were identified as B. melitensis biovar 3. rpob nucleotide sequence analysis allowed the identification of two different genotypes of B. melitensis biovar 3. On the other hand, the MLVA-16 typing assay recognized 17 distinct genotypes. All isolates were sensitive to all tested antibiotics (rifampin, doxycycline, ciprofloxacin, ceftriaxone, and trimethoprim-sulfamethoxazole), and the Mueller-Hinton agar plate is recommended for antibiotic susceptibility testing by the E-test method. Our findings identify B. melitensis biovar 3 as the etiological agent isolated in Sicily and encourage the use of both molecular methods, and in particular of the MLVA-16 assay, in epidemiological trace-back analysis. This study represents the first epidemiological data from molecular typing of Brucella strains circulating in Italy and, in particular, in eastern Sicily. Brucellosis is an important health problem affecting animals and humans in many countries in the world and especially in the Mediterranean areas. Brucella infections may cause tremendous economic losses through reproductive failure in animals. The disease is transmitted by humans through the consumption of contaminated foods or by direct/indirect contact with infected animals. Brucella melitensis is the most important zoonotic agent, followed by Brucella abortus and Brucella suis. While most industrialized countries have been remarkably successful in eradicating or controlling bovine brucellosis, some of these countries as well as all developing countries still suffer from small-ruminant brucellosis (24). In developing countries, in fact, brucellosis is almost always present where small ruminants are kept. Most human cases are caused by B. melitensis and particularly by biovars 1 and 3 (1, 4, 6, 27, 29). Brucellosis is endemic in the south of Italy, and in particular in Sicily. Annual reported human cases in Italy have steadily declined in the past 5 years (from 923 cases in 2001 to 316 cases * Corresponding author. Mailing address: Dipartimento di Sanità Alimentare ed Animale, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy. Phone: Fax: Published ahead of print on 18 July in 2005), according to data obtained from the Italian Ministry of Health ( /bollettino.jsp). However, the annual incidence of the disease has not dropped uniformly across the country: Sicily alone reported 92.4% of the 316 cases in 2005, notwithstanding evident underreporting in that area. In Sicily, brucellosis remains a serious problem. The persistence of the disease in the region, despite full awareness of the causes of infection and all possible measures to control it, can be traced back to the presence of infected animals. Sicily is 80% hilly terrain, and livestock are one of the most important resources for the regional economy. Ovine-caprine breeding is the main zootechnical activity with high density per km 2. Most livestock are owned by smallholders and farmers in Sicily. Every breeder produces a local and typical product, preventing standardization of the product, and hygienic conditions are often poor. While there are a few intensive or semi-intensive breeding farms, family breeding farms are the most common (8). Practices common in Sicily include free breeding and semifree breeding; promiscuity between sheep and goats and among sheep, goats, and cattle; seasonal moves; and the exchange of ovine-caprine male and female breeders. These breeding practices increase the possibility of contamination, transmission, and spread of infections. In Italy, eradication plans for bovine brucellosis and sheep and 2923
2 2924 MARIANELLI ET AL. J. CLIN. MICROBIOL. goat brucellosis are implemented according to law. Health and prophylactic measures established by national legislation have led to a substantial decline of brucellosis in Italy and to the eradication of disease from the northern Italian regions. In the south of Italy and especially Sicily, on the other hand, eradication programs have been hampered by the lack of complete control over movement and identification of animals, by the delayed elimination of infected animals, and by the traditional breeding practices described above. Previous studies have reported B. melitensis as the most frequently isolated species in human cases of brucellosis in Italy between 1970 and 1990, accounting for 99% of total cases (7). A significant correlation between human and animal infections, in particular of sheep and goats, indicating brucellosis as a food-borne zoonosis rather than an occupational disease, has been reported in Italy (11, 15). No data are available about the characterization at biovar level of Brucella isolates from humans in Italy. The aim of this paper is (i) to molecularly characterize clinical isolates of Brucella spp. from Sicily, since strategies for disease control and eradication derive primarily from the epidemiological characteristics of the disease, and (ii) to evaluate the antimicrobial susceptibilities in vitro of the Brucella isolates. MATERIALS AND METHODS Human cases. Twenty Brucella strains were isolated from patients with sporadic cases of acute brucellosis hospitalized in Catania, Sicily, from April 2005 to May None of the patients belonged to the occupational-risk category. The diagnosis of brucellosis was made based on compatible clinical signs, agglutination titers of 1:160, and isolation of Brucella spp. from blood. All isolates were obtained from blood culture and stored in skim milk at 20 C. A regimen of combined rifampin (RIF) and doxycycline (DOX) for 6 weeks was used for therapy as recommended by the World Health Organization (30). Blood samples were cultured in vials of the BACTEC 9120 system (Becton Dickinson, Rutherford, NJ) at 37 C for at least 7 days. Subcultures were made on Columbia blood agar plates supplemented with 5% sheep blood (biomerieux, Marcy l Etoile, France). Brucella isolates were then submitted for classical identification procedures: CO 2 requirement, H 2 S production, dye sensitivity, and agglutination with specific antisera (Oxoid Ltd., Hampshire, England) (3). Reference Brucella strains. Four reference Brucella strains were used in this study: B. melitensis biovar 1 strain 16 M, biovar 2 strain 63/9, and biovar 3 strain Ether (Veterinary Laboratories Agency, Surrey, United Kingdom) and the vaccine strain B. abortus RB51 (CZ Veterinaria, S.A., Porrino, Spain). Cultures were grown at 37 C on brucella agar plates supplemented with 5% horse serum. Bacterial DNA extraction. Brucella DNA was extracted using the proteinase K and sodium dodecyl sulfate method. DNA was purified twice with phenol-chloroform using Phase Lock Gel Heavy tubes (Eppendorf AG, Hamburg, Germany). DNA was precipitated and washed, and the pellet was resuspended in 50 l of nuclease-free water. Two hundred nanograms of DNA template was used for PCR amplifications. omp2a and omp2b polymorphism. Two PCR amplifications were performed to amplify genes omp2a and omp2b using, respectively, the primer pairs 2aA/2aB and 2bA/2bB (9). Amplification reaction mixtures were prepared in 50- l volumes using GoTaq DNA polymerase (Promega Corporation, Madison, WI) following the manufacturer s protocol. Amplifications were initiated by denaturing the sample for 3 min at 95 C, followed by 30 cycles at 95 C for 30 s, 58 C for 30 s, and 72 C for 3 min. After the last cycle, samples were incubated for an additional 5 min at 72 C before storage at 4 C. Five microliters of PCR-amplified omp2a and omp2b genes was digested by the restriction enzymes PstI and HinfI, respectively (Promega Corporation, Madison, WI), at 37 C for 3hina20- l reaction volume and using the manufacturer s recommended buffer. The mixtures were then electrophoresed on a 1.5% agarose gel with ethidium bromide. The reference Brucella strains 16 M, 63/9, and Ether were also analyzed. rpob sequencing. The rpob gene was amplified using the primers 1rB and 4134rB previously described (22). PCR amplifications were carried out in 50- l volumes using GoTaq DNA polymerase (Promega Corporation, Madison, WI) following the enclosed protocol. Amplifications were initiated by denaturing the sample for 3 min at 94 C, followed by 30 cycles at 94 C for 30 s, 60 C for 30 s, and 72 C for 4 min. After the last cycle samples were incubated for an additional 7 min at 72 C before they were stored at 4 C. PCR amplifications were analyzed by electrophoresis through a 1% agarose gel with ethidium bromide. All PCR products were purified by Montage PCR centrifugal filter devices (Millipore, Billerica, MA) and directly sequenced with the ABI PRISM 310 Genetic Analyzer equipment using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). PCR primers and sequencing primers previously described (23) were used to sequence the whole rpob gene. The electropherograms were assembled based on the published rpob sequence of B. melitensis 16 M by the ABI Prism SeqScape Software version 2.0 (Applied Biosystems, Foster City, CA). All consensus sequences generated were then compared to the published B. melitensis 16 M rpob gene (accession number AE009516) to detect nucleotide diversity. MLVA-16 (multiple-locus variable-number tandem repeat analysis of 16 loci) typing. PCR assays were performed in a total volume of 25 l using GoTaq Green Master Mix (Promega Corporation, Madison, WI). Sixteen sets of primers previously proposed and divided into two groups (2, 18) were used: panel 1 (loci Bruce06, Bruce08, Bruce11, Bruce12, Bruce42, Bruce43, Bruce45, and Bruce55) and panel 2 (loci Bruce04, Bruce07, Bruce09, Bruce16, Bruce18, Bruce19, Bruce21, and Bruce30). Amplifications were initiated by denaturing the sample for 3 min at 94 C, followed by 30 cycles at 94 C for 30 s, 60 C for 30 s, and 72 C for 50 s. After the last cycle samples were incubated for an additional 7 min at 72 C before they were stored at 4 C. Five microliters of the amplification products was loaded on 2% and 3% standard agarose gels with ethidium bromide for analyzing tandem repeats with a unit length longer (panel 1) and shorter (panel 2) than 10 bp, respectively. Gels were stained with ethidium bromide (0.5 g/ml), visualized under UV light, and photographed. The reference strain B. melitensis 16 M, for which the expected size is known for each variable-number tandem repeat locus, was used. To estimate exactly the band sizes, PCR products were purified and directly sequenced as described above. The electropherograms were assembled, and consensus sequences were generated using the Navigator Sequence Software (Applied Biosystems, Foster City, CA). Testing antimicrobial susceptibility. In vitro activities of RIF, DOX, ciprofloxacin (CIP), ceftriaxone (CRO), and trimethoprim-sulfamethoxazole (SXT) were determined by the E-test method (Biolife Italiana s.r.l., Milan, Italy) in accordance with the procedures recommended by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) for slow-growing bacteria (25). Antimicrobial susceptibility testing was performed by inoculating the isolates in broth cultures adjusted to an 0.5 McFarland turbidity both on Mueller-Hinton agar plates supplemented with 5% sheep blood (Oxoid Ltd., Hampshire, England) and on brucella agar plates supplemented with 5% horse serum. E-test strips of RIF, DOX, CIP, CEF, and SXT were stored at 20 C until use. The E-test strips were applied to the inoculated culture plates separately as recommended by the manufacturer, and the plates were incubated at 37 C for 48 h in a5%co 2 atmosphere. Determination of the MICs by the E-test was performed in duplicate, according to the manufacturer s recommendations, and the MICs were interpreted at the point of intersection between the inhibition zone and the E-test strips. The results were evaluated according to the E-test manufacturer s instructions. The vaccine strain B. abortus RB51 was used as a RIF control. RESULTS During the study period, from April 2005 to May 2006, 20 Brucella strains isolated from blood cultures of patients with acute brucellosis were studied. Seventeen (70.83%) of the strains were recovered from May through August 2005 with a peak in June. All isolates were identified on the basis of colony morphology, staining, growth characteristics, and slide agglutination with monospecific anti-brucella serum. Three isolates showed rough forms and, therefore, could not be typed. All smooth isolates reacted in agglutination tests with both anti-a and anti-m sera, suggesting B. melitensis biovar 3 identification. To better characterize human isolates at biovar level, we turned to a molecular approach. First, we studied the intraspecies polymorphism of omp2a and omp2b genes as suggested by other studies (9). By analysis of the PCR products of the omp2a and omp2b genes digested with the restriction enzymes
3 VOL. 45, 2007 MOLECULAR TYPING OF HUMAN BRUCELLA STRAINS 2925 FIG. 1. Amplification patterns of loci Bruce09 (top) and Bruce16 (bottom) of MLVA panel 2. Lanes 1, 8, and 15, 100-bp DNA ladder; lanes 2 and 9, reference strain 16 M; lanes 3 to 7, strains DG1, AM12, MM14, CM10, and BC6, respectively; lanes 10 to 14, strains MA5, PD11, CV5, BL9, and CS13, respectively. PstI and HinfI (PCR-restriction fragment length polymorphism [RFLP]), all human isolates presented the same restriction patterns and were identified as either biovar 1 or biovar 3, the two biovars being indistinguishable based on PCR-RFLP results. We then analyzed the nucleotide sequence of the rpob gene to differentiate biovars. Comparing those sequences to the published B. melitensis 16 M rpob gene (accession number AE009516), all isolates carried nucleotide alterations of ATC to ATA at codon position 1249 (M1249I), previously identified as a molecular marker of B. melitensis biovar 3 strain Ether and classified as B. melitensis genotype 3 (22). Three out of 20 isolates carried an additional CTT-to-TTT mutation at codon position 670, which led to substitution of amino acid L670F, never before observed in the rpob gene of Brucella strains (22). This mutation was not found to be associated with the RIF resistance phenotype, as demonstrated by the in vitro antimicrobial susceptibility test. In fact, this mutation does not map in the two identified hot regions in which mutations have been associated with the development of the RIF resistance phenotype in Brucella spp., located at the 5 end and close to the center of the rpob gene (23). We identified, therefore, two different B. melitensis rpob genotypes 3. DNA from all 20 human isolates and from B. melitensis strain 16 M was also amplified at 16 loci (MLVA-16 typing assay) (2, 18) to generate multiple band profiles. The reference strain B. melitensis 16 M was used because the expected size for each variable-number tandem repeat locus is known and published (18). Polymorphism among isolates was observed at seven loci: Bruce08 and Bruce12 of panel 1 and Bruce04, Bruce07, Bruce09, Bruce16, and Bruce18 of panel 2. Some amplification patterns are shown in Fig. 1. The MLVA-16 typing assay allowed the 20 Brucella isolates to be grouped into 17 distinct genotypes, as shown in Table 1. The three isolates carrying both L670F and M1249I mutations showed the same multiple band profiles and grouped in genotype 2. Genotype 3 was comprised of two isolates, and genotypes 4 to 18 were comprised of only one isolate each. Susceptibility to the antimicrobials commonly used against Brucella spp. was tested by the E-test on two different agar media, Mueller-Hinton agar plates with 5% sheep blood and brucella agar plates with 5% horse serum; both are commonly used for the culture of Brucella spp. in our laboratory. After 48 h the plates were examined and compared. No significant differences were found between the two culture media used. The Mueller-Hinton agar plate is, however, preferred because it allows clearer detection of zones of inhibition, making it easier to read the intersection of the inhibitory zone edges. MICs obtained are shown in Table 2. The range of MICs of our isolates was compared to that of the reference strain B. melitensis Ether. No significant differences were observed. We also used the reference strain B. abortus RB51 as a RIF antibiotic control because the vaccine strain RB51 is the only known RIF-resistant Brucella reference strain. All isolates were susceptible to all antibiotics; RIF had the highest MICs (between 0.75 and 2 g/ml). DISCUSSION Brucellosis in Sicily, where the prevalence of infection in sheep and goats is the highest in Italy, is an important disease with a considerable number of human cases reported every year. In this study we have characterized 20 Brucella strains isolated from patients hospitalized with symptoms of acute brucellosis in the city of Catania in a 1-year period. The consumption of ricotta (an Italian cheese made with sheep s milk) or desserts containing ricotta was identified as the most likely route of infection. The investigation of phenotypic characteristics, through conventional biotyping tests, allowed the identification of all smooth Brucella isolates as B. melitensis biovar 3. The roughness and the atypical susceptibility to dyes of some our isolates prevented characterization at biovar level. Complications in conventional biotyping characterization of B. melitensis strains have already been reported (4, 10, 20). In order to better understand the epidemiological scenario, we turned to molecular approaches. PCR-RFLP of omp2a and omp2b was not able to accurately identify our isolates, which were identified as B. melitensis biovar 1 or 3. In contrast, rpob sequencing, recently proposed as a molecular typing method for the identification and differentiation of Brucella strains at species and biovar levels (22), allowed us to presumptively identify all isolates as B. melitensis biovar 3. All isolates, in fact, carried the molecular marker M1249I. This method was also able to characterize two distinct rpob genotypes: the first carrying the M1249I mutation only (17 isolates) and the second carrying both L670F and M1249I mutations (three isolates). The MLVA-16 assay, consisting of 16 selected markers that are a combination of moderately variable (eight minisatellite markers, panel 1) and highly discriminant (eight microsatellite markers, panel 2) loci (2, 18), was used to investigate the polymorphism of tandem repeat
4 2926 MARIANELLI ET AL. J. CLIN. MICROBIOL. TABLE 1. MLVA-16 genotypes for 24 B. melitensis human isolates isolates b Repeat copy no. at locus: Bruce06 Bruce08 Bruce11 Bruce12 Bruce42 Bruce43 Bruce45 Bruce55 Bruce04 Bruce07 Bruce09 Bruce16 Bruce18 Bruce19 Bruce21 Bruce30 Genotype Strain a No. of 1 16 M DG GV CM CS BL MM AM BC PD CV MA SP DZ GA GR DN ST a All strains belong to B. melitensis, and all are biovar 3, except for 16 M, which is biovar 1. b Number of isolates studied with identical genotype. Drug TABLE 2. MICs of Brucella species under study MIC ( g/ml) for B. melitensis All isolates Strain Ether (range) RIF DOX CIP CRO SXT loci of Brucella isolates. Although the 20 isolates have identical geographic origins, differences occurred at seven markers and allowed us to identify 17 distinct genotypes (Table 1). The MLVA-16 typing method was previously used to analyze human and animal isolates (2, 18), and a large number of different genotypes have been identified. The strains were collected from different countries, and no data were available about the hypothetical distribution of Brucella genotypes within a restricted area. This information could represent an important insight in order to characterize the significance of such polymorphism in the pathogenesis of Brucella. Our results clearly show that Brucella, despite the high genetic homogeneity within the genus, is highly polymorphic at minisatellite and microsatellite levels. In fact, 17 different genotypes have been identified in analyzing 20 human isolates belonging to the same biovar and coming from a restricted area of endemicity. In all such instances where isolates share the same genotype (genotypes 2 and 3), the epidemiological data are compatible with a common source of infection. Interestingly, the three isolates carrying the additional mutation L670F showed the same MLVA-16 genotype. According to our findings, since 100% of human isolates were identified as B. melitensis biovar 3, we hypothesized that (i) B. abortus strains could not be present in the environment, (ii) B. melitensis strains are more pathogenic than B. abortus strains, or (iii) there could be some particular ecological risk factors in our epidemiological settings which influence the disease in humans and favor infection by B. melitensis biovar 3 strains. We therefore used the recent survey of animal brucellosis carried out in Sicily (12). Most Brucella strains were identified by conventional biotyping tests as B. melitensis biovar 3 (98% of isolates from sheep and goats and 25% of isolates from cattle). B. abortus biovars 1, 3, and 4 were identified in 75% of isolates from cattle. B. abortus is present among animals in Sicily, so we can reject the first hypothesis. B. melitensis is known to be the most pathogenic species, producing the most intense symptoms, the greatest tissue damage, and the highest incidence of localization in body organs, systems, or tissues (21). On the other hand, a recent study analyzing laboratory findings for patients with brucellosis reported that there was no evidence suggesting that B. melitensis is more virulent than B. abortus or that infections due to B. abortus are less severe than the infections with B. melitensis, despite the higher prevalence of B. melitensis in humans (13). Regarding the second hypothesis, we have no evidence that B. melitensis is more pathogenic than B. abortus. Since the existing literature is conflicting, additional studies are needed for greater understanding of this question. Based on animal data summarized
5 VOL. 45, 2007 MOLECULAR TYPING OF HUMAN BRUCELLA STRAINS 2927 above, our findings, the socioeconomic and cultural environment of small rural Sicilian areas with risky breeding practices, the lack of both standard procedures and good hygiene conditions during cheese production steps, and the pouring of raw milk onto finished cheese to improve its taste, we strongly support the third hypothesis. Overall, we infer that, in this ecosystem, small ruminants and their products are the major source of infection and play a key role in the spread of brucellosis both in cattle and in humans. Cattle, in fact, are not the usual reservoir of this species, and yet there are a consistent number of B. melitensis biovar 3 isolates in cattle and a high prevalence of bovine brucellosis in the same areas where ovine-caprine brucellosis is widespread. The link between human and small-ruminant infections is indeed confirmed by the seasonal trend noted in the distribution of our human cases (from May through August with a peak in June), which is easily correlated with the peak of animal cases of brucellosis and also with the peak of consumption of at-risk products. Our data suggest the use of one or both molecular approaches, rpob sequencing and MLVA-16 assay, according to the aims of the investigation, because different information is obtained from each approach. rpob sequencing allowed us to presumptively characterize all isolates as B. melitensis biovar 3, a result that would have otherwise been complicated by the roughness and the atypical dye susceptibility of some Brucella strains. The characterization at biovar level cannot be obtained by the MLVA-16 assay because, as reported by others (2, 18), the B. melitensis group is highly heterogeneous. Therefore, neither panel 1 nor panel 2 is able to cluster the isolates as expected from the biovars within the species B. melitensis. On the other hand, the MLVA-16 assay appears more suitable to assist with investigation of outbreaks: strains clustering together in the same MLVA genotype indicate a common source of infection. Therefore, typing clinical, environmental, and animal isolates through the MLVA-16 assay allows testing of hypotheses on outbreak confirmation, extent of transmission, source, and reservoir. In this study we also determined the antibiotic susceptibilities of our isolates by the E-test method. The E-test was used because it has been reported to be less labor-intensive, less time-consuming, and more practical than the broth microdilution method for testing antibiotic susceptibility of Brucella spp. (14). Susceptibility testing of brucellae is not routinely used in clinical practice, few studies of in vitro antimicrobial susceptibilities of Brucella organisms are available (5, 26, 28), and no standard protocol has been determined yet. We studied susceptibilities of our Brucella isolates on two different culture media: the Mueller-Hinton agar plates widely used for antibiotic susceptibility testing and the brucella agar plates commonly used in the laboratory as Brucella growth medium. RIF, DOX, CIP, CRO, and SXT proved to be very effective antibiotic drugs in vitro, although CRO is not effective in treating brucellosis patients. The highest MICs were found for RIF: two of our isolates showed a MIC of 2 g/ml. According to evaluation based on CLSI standards for slow-growing bacteria, these two isolates should be considered intermediately susceptible. On the other hand, in this study we have characterized the whole rpob gene, recognized to be the RIF antibiotic target in prokaryotes (16, 17, 19), of all our isolates, and these two isolates carried only the B. melitensis biovar 3 molecular marker (M1249I). We can, therefore, assert that all isolates investigated were found to be sensitive to RIF and, in general, to all tested antimicrobial agents. Although no significant differences were observed between two tested culture media, the Mueller-Hinton agar plate is recommended because clearer inhibition zones are visible and the calibrated carrier strip indicating the MIC can be more easily read. Since increasing resistance to RIF and SXT has been reported in many parts of the world, we suggest periodic assessment of susceptibility of strains to those antibiotics used most frequently in treatment, for an early detection of any drug resistance, especially in areas of endemicity. In conclusion, our findings confirm B. melitensis, and in particular biovar 3, as the etiological agent most frequently isolated in humans in Italy and suggest, therefore, that sheep and goat populations are the principal cause of human brucellosis. Moreover, our results provide proof of the high discriminatory power of MLVA-16 typing even if Brucella isolates belong to the same biovar. This method led us to ascertain that in a restricted area in which brucellosis is still endemic, different genotypes of B. melitensis biovar 3 are present. It paves the way for further studies aiming to evaluate if it is possible to correlate different genotypes with different pathogenic patterns. ACKNOWLEDGMENTS We thank Paolo Pasquali for suggestions and revision of the text and Massimiliano Francia for his skilled technical assistance in this project. REFERENCES 1. Aggad, H., and L. Boukraa Prevalence of bovine and human brucellosis in western Algeria: comparison of screening tests. East. Mediterr. Health J. 12: Al Dahouk, S., P. Le Fleche, K. Nockler, I. Jacques, M. Grayon, H. C. Scholz, H. Tomaso, G. Vergnaud, and H. Neubauer Evaluation of Brucella MLVA typing for human brucellosis. J. Microbiol. Methods 69: Alton, G. G., L. M. Jones, R. D. Angus, and J. M. Verger Techniques for the brucellosis laboratory. Institut National de la Recherche Agronomique Publications, Paris, France. 4. Banai, M., I. Mayer, and A. Cohen Isolation, identification, and characterization in Israel of Brucella melitensis biovar 1 atypical strains susceptible to dyes and penicillin, indicating the evolution of a new variant. J. Clin. Microbiol. 28: Baykam, N., H. Esener, O. Ergonul, S. Eren, A. K. Celikbas, and B. Dokuzoguz In vitro antimicrobial susceptibility of Brucella species. Int. J. Antimicrob. Agents 23: Bodur, H., N. Balaban, S. Aksaray, V. Yetener, E. Akinci, A. Colpan, and A. Erbay Biotypes and antimicrobial susceptibilities of Brucella isolates. Scand. J. Infect. Dis. 35: Caporale, V., D. Nannini, A. Giovannini, D. Morelli, and M. Ramasco Prophylaxis and control of brucellosis due to Brucella melitensis in Italy: acquired and expected results, p In Prevention of brucellosis in the Mediterranean countries. Proceedings of the International Seminar of the International Center for Advanced Mediterranean Agronomic Studies, the Commission of the European Communities, and the Ministry of Agriculture and Fisheries. International Center for Advanced Mediterranean Agronomic Studies, Valletta, Malta. 8. Caracappa, S Livestock production and animal health in Sicily, Italy. Parassitologia 41: Cloeckaert, A., J. M. Verger, M. Grayon, and O. Grepinet Restriction site polymorphism of the genes encoding the major 25 kda and 36 kda outer membrane proteins of Brucella. Microbiology 141: Corbel, M. J Identification of dye-sensitive strains of Brucella melitensis. J. Clin. Microbiol. 29: De Massis, F., A. Di Girolamo, A. Petrini, E. Pizzigallo, and A. Giovannini Correlation between animal and human brucellosis in Italy during the period Clin. Microbiol. Infect. 11: Di Marco, V., C. Piratino, I. Bazzana, M. Russo, V. Aronica, A. M. F. Marini., and S. Caracappa A study on the circulation of biovars of
6 2928 MARIANELLI ET AL. J. CLIN. MICROBIOL. brucellae in sheep and goat in Sicily from 1993 to 2002, p Abstr. 14th Int. Congr. Mediterr. Fed. Health Prod. Ruminants, Santiago De Compostela, Spain. 13. Dokuzoguz, B., O. Ergonul, N. Baykam, H. Esener, S. Kilic, A. Celikbas, S. Eren, and B. Esen Characteristics of B. melitensis versus B. abortus bacteraemias. J. Infect. 50: Gur, D., S. Kocagoz, M. Akova, and S. Unal Comparison of E test to microdilution for determining in vitro activities of antibiotics against Brucella melitensis. Antimicrob. Agents Chemother. 43: Iaria, C., F. Ricciardi, F. Marano, G. Puglisi, G. Pappas, and A. Cascio Live nativity and brucellosis, Sicily. Emerg. Infect. Dis. 12: Jin, D. J., and C. A. Gross Mapping and sequencing of mutations in the Escherichia coli rpob gene that lead to rifampin resistance. J. Mol. Biol. 202: Jin, D. J., and C. A. Gross Characterization of the pleiotrophic phenotypes of rifampin-resistant rpob mutants of Escherichia coli. J. Bacteriol. 171: Le Fleche, P., I. Jacques, M. Grayon, S. Al Dahouk, P. Bouchon, F. Denoeud, K. Nockler, H. Neubauer, L. A. Guilloteau, and G. Vergnaud Evaluation and selection of tandem repeat loci for a Brucella MLVA typing assay. BMC Microbiol. 6: Levin, M. E., and G. F. Hatfull Mycobacterium smegmatis RNA polymerase: DNA supercoiling, action of rifampin and mechanism of rifampin resistance. Mol. Microbiol. 8: Lucero, N. E., S. M. Ayala, G. I. Escobar, M. Grayon, and I. Jacques A new variant of Brucella melitensis. Clin. Microbiol. Infect. 12: Madkour, M. M Madkour s brucellosis, 2nd ed. Springer Verlag, Berlin, Germany. 22. Marianelli, C., F. Ciuchini, M. Tarantino, P. Pasquali, and R. Adone Molecular characterization of the rpob gene in Brucella species: new potential molecular markers for genotyping. Microbes Infect. 8: Marianelli, C., F. Ciuchini, M. Tarantino, P. Pasquali, and R. Adone Genetic bases of the rifampin resistance phenotype in Brucella spp. J. Clin. Microbiol. 42: McDermott, J. J., and S. M. Arimi Brucellosis in sub-saharan Africa: epidemiology, control and impact. Vet. Microbiol. 90: National Committee for Clinical Laboratory Standards Performance standards for antimicrobial susceptibility testing. Eighth informational supplement. NCCLS document M 100-S8, vol. 18. National Committee for Clinical Laboratory Standards, Wayne, PA. 26. Orhan, G., A. Bayram, Y. Zer, and I. Balci Synergy tests by E test and checkerboard methods of antimicrobial combinations against Brucella melitensis. J. Clin. Microbiol. 43: Refai, M Incidence and control of brucellosis in the Near East region. Vet. Microbiol. 90: Turkmani, A., A. Ioannidis, A. Christidou, A. Psaroulaki, F. Loukaides, and Y. Tselentis In vitro susceptibilities of Brucella melitensis isolates to eleven antibiotics. Ann. Clin. Microbiol. Antimicrob. 5: Wallach, J. C., S. E. Miguel, P. C. Baldi, E. Guarnera, F. A. Goldbaum, and C. A. Fossati Urban outbreak of a Brucella melitensis infection in an Argentine family: clinical and diagnostic aspects. FEMS Immunol. Med. Microbiol. 8: World Health Organization Joint FAO-WHO Expert Committee on Brucellosis, sixth report. WHO Tech. Rep. Ser. 740:1 132.
JCM Accepts, published online ahead of print on June 009 J. Clin. Microbiol. doi:0./jcm.00-09 Copyright 009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
J Vet Diagn Invest :164 168 (1998) Evaluation of a computerized antimicrobial susceptibility system with bacteria isolated from animals Susannah K. Hubert, Phouc Dinh Nguyen, Robert D. Walker Abstract.
International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 7 (2017) pp. 4008-4014 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.607.415
JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2011, p. 3276 3283 Vol. 49, No. 9 0095-1137/11/$12.00 doi:10.1128/jcm.02538-10 Copyright 2011, American Society for Microbiology. All Rights Reserved. Multiple-Locus
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2002, p. 1475 1480 Vol. 40, No. 4 0095-1137/02/$04.00 0 DOI: 10.1128/JCM.40.4.1475 1480.2002 Copyright 2002, American Society for Microbiology. All Rights Reserved.
Veterinaria Italiana, 46 (1), 13 19 Efficacy of Brucella abortus vaccine strain RB51 compared to the reference vaccine Brucella abortus strain 19 in water buffalo Vincenzo Caporale, Barbara Bonfini, Elisabetta
VICH GL27 (ANTIMICROBIAL RESISTANCE: PRE-APPROVAL) December 2003 For implementation at Step 7 - Final GUIDANCE ON PRE-APPROVAL INFORMATION FOR REGISTRATION OF NEW VETERINARY MEDICINAL PRODUCTS FOR FOOD
CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, July 2003, p. 647 651 Vol. 10, No. 4 1071-412X/03/$08.00 0 DOI: 10.1128/CDLI.10.4.647 651.2003 Copyright 2003, American Society for Microbiology. All Rights
J. Vet. Med. B 52, 75 81 (25) Ó 25 Blackwell Verlag, Berlin ISSN 931 1793 Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise ÔG. CaporaleÕ, Campo Boario, Teramo, Italy The Use of Homologous
International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 4 Number 9 (2015) pp. 957-961 http://www.ijcmas.com Original Research Article Antibiotic Susceptibility Pattern
Antimicrobial susceptibility of Shigella, 2015 and 2016 Helen Heffernan and Rosemary Woodhouse Antibiotic Reference Laboratory, Institute of Environmental Science and Research Limited (ESR); August 2017
Principles and Practice of Antimicrobial Susceptibility Testing Microbiology Technical Workshop 25 th September 2013 Scope History Why Perform Antimicrobial Susceptibility Testing? How to Perform an Antimicrobial
SEROPREVALENCE OF BRUCELLA SPP, LEPSTOSPIRA SPP AND TOXOPLASMA GONDII IN WILD BOARD (SUS SCROFA) FROM SOUTHERN BRAZIL Iara Maria Trevisol 1, Beatris Kramer 1, Arlei Coldebella¹, Virginia Santiago Silva
Research in rabbit science. University of Bari Antonio Camarda Università of Bari Aldo Moro Faculty of Veterinary Medicine Dept of Veterinary Public Health and Animal Sciences email@example.com
AAC Accepted Manuscript Posted Online 30 March 2015 Antimicrob. Agents Chemother. doi:10.1128/aac.00513-15 Copyright 2015, American Society for Microbiology. All Rights Reserved. 1 2 Decrease of vancomycin
European Committee on Antimicrobial Susceptibility Testing Routine and extended internal quality control as recommended by EUCAST Version 5.0, valid from 015-01-09 This document should be cited as "The
Istituto Zooprofilattico Sperimentale dell Abruzzo e del Molise Teramo ITALY www.izs.it SILAB For Africa a LIMS for African Country and Animal Identification Registration Traceability system Ercole Del
Milano, August 31 2015 International Conference Prevention and control of Campylobacter in the poultry production system Dr. Silvio Borrello Direzione generale della sanità animale e dei farmaci veterinari
How to load and run an Agarose gel PSR Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from100 bp to 25 kb. This protocol divided into three stages:
Archives of Razi Institute, Vol. 70, No. 1 (2015) 51-55 Copyright 2014 by Razi Vaccine & Serum Research Institute Short Communication Detection of and abortus strains using a single-stage PCR method Alamian
Quality Assurance of antimicrobial susceptibility testing Derek Brown EUCAST Scientific Secretary ESCMID Postgraduate Education Course, Linz, 17 September 2014 Quality Assurance The total process by which
ORIGINAL ARTICLE Public Health Res Perspect 2017;8(1):65 70 eissn 2233-6052 A Novel PCR Assay for Detecting Brucella abortus and Brucella melitensis Saeed Alamian a, Majid Esmaelizad b, Taghi Zahraei c,
The Basics: Using CLSI Antimicrobial Susceptibility Testing Standards Janet A. Hindler, MCLS, MT(ASCP) UCLA Health System Los Angeles, California, USA firstname.lastname@example.org 1 Learning Objectives Describe information
INFECTION AND IMMUNITY, July 2005, p. 4198 4204 Vol. 73, No. 7 0019-9567/05/$08.00 0 doi:10.1128/iai.73.7.4198 4204.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved. Protective
International Journal of Microbiology and Allied Sciences (IJOMAS) ISSN: 2382-5537 May 2016, 2(4):22-26 IJOMAS, 2016 Research Article Page: 22-26 Isolation of antibiotic producing Actinomycetes from soil
BRUCELLOSIS CPMP/4048/01, rev. 3 1/7 General points on treatment Four species are pathogenic to man: B. melitenis (acquired from goats), B. suis (pigs), B. abortus (cattle) and B. canis (dogs). The bacteria
Bulgarian Journal of Veterinary Medicine, 2015, 18, No 1, 40 48 ISSN 1311-1477; DOI: 10.15547/bjvm.842 Original article ISOLATION OF BRUCELLA MELITENSIS STRAINS FROM SYRIAN BOVINE MILK SAMPLES A. AL-MARIRI
2. Disease Outbreak Investigation Protocol: Brucellosis Case Study MONOGRAPH Protocol for conducting an outbreak investigation A. Goals for outbreak investigation 1. Stop the occurrence of disease with
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 2005, p. 3533 3537 Vol. 49, No. 8 0066-4804/05/$08.00 0 doi:10.1128/aac.49.8.3533 3537.2005 Copyright 2005, American Society for Microbiology. All Rights Reserved.
Bovine Mastitis Products for Microbiological Analysis 121917ss Hardy Diagnostics has everything for your laboratory! SAVE MONEY Now you have a choice for obtaining your supplies for mastitis testing. Hardy
The Veterinary Medicine International Conference 2017 Volume 2017 Conference Paper Control And Preventive Study Of Brucellosis By Using Lipopolysacharide Sub Unit Vaccine Brucella abortus Strain S-19 J.
Abstract Antibiotic and Disinfectant Resistant Bacteria in Rivers of the United States Ronald J. Ash and Jamey L. Iverson Department of Biology, Washburn University,Topeka, KS We examined natural water
Food and Agriculture (FA) Domain Committee MONITORING PROGRESS REPORT 2006 COST - Chair: Konrad Sachse 3rd DC meeting, Antalya (TR), 31 Jan 2 Feb 2007 COST Action Domain Food and Agriculture (FA) Animal
Veterinary Science Preparatory Training for the Veterinary Assistant Floron C. Faries, Jr., DVM, MS Brucellosis & Tuberculosis Eradication Programs Floron C. Faries, Jr., DVM, MS Objectives Discuss the
Informing Public Policy on Agricultural Use of Antimicrobials in the United States: Strategies Developed by an NGO Stephen J. DeVincent, DVM, MA Director, Ecology Program Alliance for the Prudent Use of
DIFFERENTIATION OF BRUCELLAE BY THE AID OF PHAGES J6ZEF PARNAS Department of Microbiology, Academy of Medicine, Lublin, Poland ABSTRACT PARNAS, J6ZEF (Academy of Medicine, Lublin, Poland). Differentiation
Role and responsibility of Animal Health Research Institute in the national veterinary infrastructure Dr. Abdel-khalik M. montasser Chief researcher Brucella Department, AHRI e-mail: email@example.com
Original Article Vol. 21 No.1 The optimum agent for ESBL screening and confirmatory tests:- Srisangkaew S & Vorachit M. 1 The Optimum Agent for Screening and Confirmatory Tests for Extended-Spectrum Beta-Lactamases
Q1. (a) Clostridium difficile is a bacterium that is present in the gut of up to 3% of healthy adults and 66% of healthy infants. C. difficile rarely causes problems, either in healthy adults or in infants.
Towards Rational International Antibiotic Breakpoints: Actions from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) A report to ISC presented by Paul M. Tulkens representative of
Questions and answers about methicillin-resistant Staphylococcus aureus (MRSA) Updated FAQ, 18 November 2014 Methicillin-resistant Staphylococcus aureus (MRSA) are bacteria which are resistant to certain
CHAPTER 20 ANTIBIOTIC RESISTANCE WHY IS THIS IMPORTANT? The most important problem associated with infectious disease today is the rapid development of resistance to antibiotics It will force us to change
L 225/76 19.8.2016 COMMISSION REGULATION (EU) 2016/1396 of 18 August 2016 amending certain Annexes to Regulation (No 999/2001 of the European Parliament and of the Council laying down rules for the prevention,
In the first half of the 20th century, Dr. Guido Fanconi published detailed clinical descriptions of several heritable human diseases. Two disease syndromes were named after him: Fanconi Anemia and Fanconi
Antimicrobial Resistance Consequences of Antimicrobial Resistant Bacteria Change in the approach to the administration of empiric antimicrobial therapy Increased number of hospitalizations Increased length
1 In 1958, scientists made a breakthrough in artificial reproductive cloning by successfully cloning a vertebrate species. The species cloned was the African clawed frog, Xenopus laevis. Fig. 1.1, on page
JCM Accepts, published online ahead of print on 5 December 2012 J. Clin. Microbiol. doi:10.1128/jcm.02835-12 Copyright 2012, American Society for Microbiology. All Rights Reserved. 1 20th November 2012
Mal et al. BMC Microbiology (2016) 16:236 DOI 10.1186/s12866-016-0707-6 RESEARCH ARTICLE Open Access Antimicrobial susceptibility testing of Neisseria gonorrhoeae isolates in Pakistan by Etest compared
Fish Farms Myron Kebus, MS, DVM State Aquaculture Veterinary Epidemiologist DATCP Fish Health National model for fish health programs Requirements: Import permits Health certificates Record-keeping Reportable
Evolution in Action: Antibiotic Resistance HASPI Medical Biology Lab 21 Background/Introduction Evolution and Natural Selection Evolution is one of the driving factors in biology. It is simply the concept
Epidemiology of Opisthorchis felineus in the European Union Edoardo Pozio European Union Reference Laboratory for Parasites Istituto Superiore di Sanità Rome, Italy World distribution and human prevalence
Lab Exercise: Antibiotics- Evaluation using Kirby Bauer method. OBJECTIVES 1. Compare the antimicrobial capabilities of different antibiotics. 2. Compare effectiveness of with different types of bacteria.
Acinetobacter Outbreaks: Experience from a Neurosurgery Critical Care Unit Jumoke Sule Consultant Microbiologist 19 May 2010 Epidemiology of Acinetobacter spp At least 32 different species Recovered from
MEETING OF THE ICAR WORKING GROUP ON MILK RECORDING OF SHEEP 17 th June 2008, Niagara Falls, USA Draft minutes Presents : Jean-Michel Astruc (France), Francis Barillet (France), Antonello Carta (Italy),
Iranian Journal of Pharmaceutical Research (22), (2): 559-563 Received: January 2 Accepted: June 2 Copyright 22 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services
This article reprinted from: Lessem, P. B. 2008. The antibiotic resistance phenomenon: Use of minimal inhibitory concentration (MIC) determination for inquiry based experimentation. Pages 357-362, in Tested
Guideline for Prevention of Brucellosis in Meat Packing Plant Workers Introduction Brucellosis is a disease which may spread from animals to man. There is no evidence for person to person transmission.
Appraisal of the Breeding Plan for Scrapie resistance in the Sarda dairy sheep breed. S. Salaris 1, F. Ingravalle 2, A. Pernisa 1, L. Crasta 1, A. Fraghì 1, C. Ligios 3, S. Murru 4, G. Ru 2, and A. Carta
J. Hyg., Camb. (1982), 88, 21 21 Printed in Great Britain A comparison of the results of the brucellosis radioimmunoassay and other serological tests in experimentally infected cattle BY J. HAYES AND R.
Monitoring gonococcal antimicrobial susceptibility The rapidly changing antimicrobial susceptibility of Neisseria gonorrhoeae has created an important public health problem. Because of widespread resistance
Diseases of Small Ruminants and OIE Standards, Emphasis on PPR Dr Ahmed M. Hassan Veterinary Expert 7 9 April, 2009 Beirut (Lebanon) 1 Small ruminants are very important for: both the subsistence and economic
Multiple drug resistance pattern in Urinary Tract Infection patients in Aligarh Author(s): Asad U Khan and Mohd S Zaman Vol. 17, No. 3 (2006-09 - 2006-12) Biomedical Research 2006; 17 (3): 179-181 Asad
Isolation and identification of major causing bacteria from bovinemastitis R. Lakshmi 1 and K.K. Jayavardhanan 2 1 PhD Scholar, Department of Veterinary Biochemistry, College of Veterinary and Animal Sciences,
Guidelines for Laboratory Verification of Performance of the FilmArray BCID System Purpose The Clinical Laboratory Improvement Amendments (CLIA), passed in 1988, establishes quality standards for all laboratory
USE OF ANTIBIOTIC RESIDUE TEST KITS FOR GOAT MILK E. N. Escobar E (Kika) de la Garza Institute for Goat Research Langston University Langston, Oklahoma 73050 Background and Purpose Mastitis is known as
Antibiotics: Antil susceptibility testing are natural chemical substances produced by certain groups of microorganisms (fungi, ) that inhibit the growth of or kill the other that cause infection. Several
Journal of Antimicrobial Chemotherapy (1997) 40, 105 108 Brief reports JAC Decreased susceptibility to imipenem among penicillin-resistant Streptococcus pneumoniae Andreas Pikis a *, Jacob A. Donkersloot
Project Summary Emerging Pathogens in US Cattle Principal Investigators: Jeffrey LeJeune and Gireesh Rajashekara Food Animal Health Research Program The Ohio Agricultural Research and Development Center
General Q&A New EU Regulation on transmissible animal diseases ("Animal Health Law") March 2016 Table of Contents Scope of the Regulation on transmissible animal diseases (Animal Health Law)... 2 Entry
Biotyping and Genotyping (MLVA16) of Brucella abortus Isolated from Cattle in Brazil, 1977 to 2008 Sílvia Minharro 1,2 1 1 1 3, Juliana P. Silva Mol, Elaine M. S. Dorneles, Rebeca B. Pauletti, Heinrich
Understanding the Hospital Antibiogram Sharon Erdman, PharmD Clinical Professor Purdue University College of Pharmacy Infectious Diseases Clinical Pharmacist Eskenazi Health 5 Understanding the Hospital
12.12.2003 L 325/31 DIRECTIVE 2003/99/EC OF THE EUROPEAN PARLIAMT AND OF THE COUNCIL of 17 November 2003 on the monitoring of zoonoses and zoonotic agents, amending Council Decision 90/424/EEC and repealing
Biology 120 Lab Exam 2 Review Student Learning Services and Biology 120 Peer Mentors Sunday, November 26 th, 2017 4:00 pm Arts 263 Important note: This review was written by your Biology Peer Mentors (not
Brucella melitensis in France: Persistence in Wildlife and Probable Spillover from Alpine Ibex to Domestic Animals Virginie Mick 1 *, Gilles Le Carrou 1, Yannick Corde 1, Yvette Game 2, Maryne Jay 1, Bruno
OIE Reference Laboratory Reports Activities Activities in 2016 This report has been submitted : 2017-01-04 14:57:02 Name of disease (or topic) for which you are a designated OIE Reference Laboratory: Contagious
Chapter 2. Disk diffusion method Tendencia, Eleonor A. Date published: 2004 To cite this document : Tendencia, E. A. (2004). Chapter 2. Disk diffusion method. In Laboratory manual of standardized methods
PROTOCOL For antimicrobial susceptibility testing of Salmonella, Campylobacter and optional genotypic characterisation of AmpC-, ESBL- and carbapenemase-producing test strains 1 INTRODUCTION... 1 2 OBJECTIVES...
Burn Infection & Laboratory Diagnosis Introduction Burns are one the most common forms of trauma. 2 million fires each years 1.2 million people with burn injuries 100000 hospitalization 5000 patients die
EUCAST Workshop: Antimicrobial susceptibility testing with EUCAST breakpoints and methods Susceptibility testing of infrequently isolated fastidious organisms Luis Martinez-Martínez Service of Microbiology