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ABSTRACTgyrBis used to improve the identification of the Nocardiaspecies N. brasiliensis, N. higoensis, N. ignorata, N. otitidiscaviarum, N. paucivorans, N. pneumoniae, N. puris, N. takedensis, N. veterana, and N. vinacea, but it does not improve the identification of another 12 Nocardiastudied species. gyrBprovides typing and phylogenetic markers for N. carnea, N. transvalensis, N. brasiliensis, and N. otitidiscaviarum.

ABSTRACTThis study compares the identification, typing, and phylogenetic relationships of the most prevalent clinical Nocardiaspecies in Spain, as determined via sequence analysis of their housekeeping genes gyrBand rpoB, with the results returned by the gold standard 16S rRNA method. gyrBand rpoBanalyses identified Nocardia abscessus, N. cyriacigeorgica, N. farcinica, and the N. novacomplex, species that together account for more than half of the human nocardiosis cases recorded in Spain. The individual discriminatory power of gyrBand rpoBwith respect to intraspecies typing, calculated using the Hunter-Gaston discriminatory index (HGDI), was generally high (HGDI, 0.85 to 1), except for rpoBwith respect to N. farcinica(HGDI, 0.71). Phylogenetically, different degrees of intra- and interspecies microheterogeneity were observed for gyrBand rpoBin a group of 119 clinical strains. A single 16S haplotype was obtained for each species, except for the N. novacomplex (8 types), while gyrBand rpoBwere more polymorphic: N. abscessushad 14 and 18 haplotypes, N. cyriacigeorgicahad 17 and 12, N. farcinicahad 11 and 5, and the N. novacomplex had 26 and 29 haplotypes, respectively. A diversity gradient was therefore seen, with N. farcinicaat the bottom followed by N. abscessusand N. cyriacigeorgicain the middle and N. novacomplex at the top. The complexity of the N. novacomplex is highlighted by its six variations in the GyrB 126AAAPEH motif. gyrBsequencing (with or without rpoBsequencing) offers a simple means for identifying the most prevalent Nocardiaspecies in Spanish medical laboratories and for determining the intraspecific diversity among their strains.

ABSTRACTAcinetobacter baumanniiis one of the major pathogens involved in nosocomial outbreaks. The clonal diversity of 729 epidemic strains isolated from 19 Spanish hospitals (mainly from intensive care units) was analyzed over an 11-year period. Pulsed-field gel electrophoresis (PFGE) identified 58 PFGE types that were subjected to susceptibility testing, rpoBgene sequencing, and multilocus sequence typing (MLST). All PFGE types were multidrug resistant; colistin was the only agent to which all pathogens were susceptible. The 58 PFGE types were grouped into 16 clones based on their genetic similarity (cutoff of 80%). These clones were distributed into one major cluster (cluster D), three medium clusters (clusters A, B, and C), and three minor clusters (clusters E, F, and G). The rpoBgene sequencing and MLST results reflected a clonal distribution, in agreement with the PFGE results. The MLST sequence types (STs) (and their percent distributions) were as follows: ST-2 (47.5%), ST-3 (5.1%), ST-15 (1.7%), ST-32 (1.7%), ST-79 (13.6%), ST-80 (20.3%), and ST-81 (10.2%). ST-79, ST-80, and ST-81 and the alleles cpn60-26and recA29are described for the first time. International clones I, II, and III were represented by ST-81, ST-2, and ST-3, respectively. ST-79 and ST-80 could be novel emerging clones. This work confirms PFGE and MLST to be complementary tools in clonality studies. Here PFGE was able to demonstrate the monoclonal pattern of most outbreaks, the inter- and intrahospital transmission of bacteria, and their endemic persistence in some wards. MLST allowed the temporal evolution and spatial distribution of Spanish clones to be monitored and permitted international comparisons to be made.

ABSTRACTThe severe morbidity of human brucellosis is one of the main reasons for using molecular typing in the epidemiological surveillance of this worldwide zoonosis. Multiple-locus variable-number repeat analysis (MLVA-16), hypervariable octameric oligonucleotide fingerprinting (HOOF-print), and the differences in the single nucleotide polymorphisms (SNPs) (codons 1249 and 1309) of the DNA-dependent RNA polymerase ß subunit (rpoB) were used to type a human Brucella melitensispopulation (108 strains) collected from throughout Spain over 13 years. Eighty-six MLVA types (discriminatory index, 0.99) were detected, with a wide-ranging genetic similarity coefficient (37.2 to 93.7%). The population clustered into the following groups: American, with genotypes 47 (1 strain), 48 (13 strains), 53 (12 strains), 55 (2 strains), 80 (1 strain), and a new genotype (2 strains), Western Mediterranean, with genotype 51 (9 strains), and Eastern Mediterranean, with genotypes 42 (60 strains), 43 (4 strains), and 63 (4 strains). Two profession-related and two foodborne acquisitions were confirmed. Distributed throughout Spain, Eastern Mediterranean genotype 42 was the most common (55%). The low MLVA-16 allelic polymorphism (genetic similarity range, 75 to 94%) of the genotype 42 strains suggests that they recently evolved from a common ancestor. rpoBtyping grouped the strains as rpoBtype 1 (1249-ATG/1309-CTG; 28.7%), rpoBtype 2 (1249-ATG/1309-CTA; 62.9%), and rpoBtype 3 (1249-ATA/1309-CTG; 8.3%). According to the MLVA-16 results, the population clustered by rpoBtype. Given the correlation between B. melitensisMLVA groups and rpoBtypes (American and rpoBtype 1, Eastern Mediterranean and rpoBtype 2, and Western Mediterranean and rpoBtype 3), the rpoBtype could be used as an initial marker for the epidemiological surveillance of brucellosis.

ABSTRACTThe levels of genetic relatedness of 139 Stenotrophomonas maltophiliastrains recovered from 105 hospitalized non-cystic fibrosis patients (51% from medical wards, 35% from intensive care units, and 14% from surgical wards) and 7 environmental sources in the same hospital setting during a 4-year period were typed by the pulsed-field gel electrophoresis (PFGE) technique. A total of 99 well-defined distinct XbaI PFGE patterns were identified (Simpson's discrimination index, 0.996). The dendrogram showed a Dice similarity coefficient ranging from 28 to 80%. Two major clusters (I and II), three minor clusters (III, IV, and V), and two independent branches were observed when using a 36% Dice coefficient, indicating a high diversity of genetic relatedness. It is of note that 84% of strains were grouped within two major clonal lineages. No special cluster gathering was found among strains belonging to the same sample type specimen, patients' infection or colonization status, and ward of precedence. Despite this fact, three different clones (A, B, and C) recovered from respiratory samples from six, three, and two patients, respectively, and two clones, D and E, in two bacteremic patients each, were identified. Isolation of an S. maltophiliastrain belonging to the clone A profile in a bronchoscope demonstrated a common source from this clone. This study revealed a high genetic diversity of S. maltophiliaisolates despite their origin from a single hospital, which may be related to the wide environmental distribution of this pathogen. However, few clones could be transmitted among different patients, yielding outbreak situations.

ABSTRACTThe quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophiliaATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophiliaclinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 μg/ml. GyrA (Leu-55 to Gln-155, Escherichia colinumbering), GyrB (Met-391 to Phe-513), ParC (Ile-34 to Arg-124), and ParE (Leu-396 to Leu-567) fragments from strain ATCC 13637 showed high degrees of identity to the corresponding regions from the phytopathogen Xylella fastidiosa, with the degrees of identity ranging from 85.0 to 93.5%. Lower degrees of identity to the corresponding regions from Pseudomonas aeruginosa(70.9 to 88.6%) and E. coli(73.0 to 88.6%) were observed. Amino acid changes were present in GyrA fragments from 9 of the 32 strains at positions 70, 85, 90, 103, 112, 113, 119, and 124; but there was no consistent relation to higher ciprofloxacin MICs. The absence of changes at positions 83 and 87, commonly involved in quinolone resistance in gram-negative bacteria, was unexpected. The GyrB sequences were identical in all strains, and only one strain (ciprofloxacin MIC, 16 μg/ml) showed a ParC amino acid change (Ser-80→Arg). In contrast, a high frequency (16 of 32 strains) of amino acid replacements was present in ParE. The frequencies of alterations at positions 437, 465, 477, and 485 were higher (P< 0.05) in strains from cystic fibrosis patients, but these changes were not linked with high ciprofloxacin MICs. An efflux phenotype, screened by the detection of decreases of at least twofold doubling dilutions of the ciprofloxacin MIC in the presence of carbonyl cyanide m-chlorophenylhydrazone (0.5 μg/ml) or reserpine (10 μg/ml), was suspected in seven strains. These results suggest that topoisomerases II and IV may not be the primary targets involved in quinolone resistance in S. maltophilia.

ABSTRACTSusceptibility to 41 antimicrobials was studied with 99Stenotrophomonas maltophiliastrains, and different pulsed-field gel electrophoresis profiles were identified among 130 prospectively collected isolates. Moxalactam, doxycycline, minocycline, and clinafloxacin displayed the highest activity (≥98% susceptibility). Ticarcillin resistance (75%) was reverted by clavulanate in 25% of strains. Trimethoprim-sulfamethoxazole resistance was 26.2% (≥4 [trimethoprim]/76 [sulfamethoxazole] μg/ml) and dropped to 11.1% when an 8/152-μg/ml breakpoint was applied based on its bimodal MIC distribution. Resistance was lower when unique strains were considered, because clonal organisms contribute to resistance.