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ABSTRACTMultilocus sequence typing and nrdAsequence analysis identified 6 different species or genogroups and 13 sequence types (STs) among 15 Achromobacterisolates from cystic fibrosis (CF) patients and 7 species or genogroups and 11 STs among 11 isolates from non-CF patients. Achromobacter xylosoxidanswas the most frequently isolated species among CF patients.

ABSTRACTMost Staphylococcus aureussmall-colony variants (SCVs) are auxotrophs for menadione, hemin, or thymidine but rarely for CO2. We conducted a prospective investigation of all clinical cases of CO2-dependent S. aureusduring a 3-year period. We found 14 CO2-dependent isolates of S. aureusfrom 14 patients that fulfilled all requirements to be considered SCVs, 9 of which were methicillin resistant. The clinical presentations included four cases of catheter-related bacteremia, one complicated by endocarditis; two deep infections (mediastinitis and spondylodiscitis); four wound infections; two respiratory infections; and two cases of nasal colonization. Pulsed-field gel electrophoresis typing showed that the 14 isolates were distributed into 4 types corresponding to sequence types ST125-agrgroup II (agrII), ST30-agrIII, ST34-agrIII, and ST45-agrI. An array hybridization technique performed on the 14 CO2-dependent isolates and 20 S. aureusisolates with normal phenotype and representing the same sequence types showed that all possessed the enterotoxin gene cluster egc, as well as the genes for a-hemolysin and d-hemolysin; biofilm genes icaA, icaC, and icaD; several microbial surface components recognizing adhesive matrix molecules (MSCRAMM) genes (clfA, clfB, ebh, eno, fib, ebpS, sdrC, and vw); and the isaBgene. Our study confirms the importance of CO2-dependent SCVs of S. aureusas significant pathogens. Clinical microbiologists should be aware of this kind of auxotrophy because recovery and identification are challenging and not routine. Further studies are necessary to determine the incidence of CO2auxotrophs of S. aureus, the factors that select these strains in the host, and the genetic basis of this type of auxotrophy.

ABSTRACTMolecular epidemiology of Staphylococcus aureusstrains causing bacteremia in neonates during 2002 to 2005 revealed seven clones, with four MSSA clones responsible for 80% of the cases. Some clones persisted or reappeared throughout the study. Three bacteremic clones were found colonizing health care workers (HCWs), particularly clone C, which was harbored by at least 15% of HCWs.

ABSTRACTWe conducted a population-based molecular epidemiological study of tuberculosis (TB) in Madrid, Spain (2002 to 2004), to define transmission patterns and factors associated with clustering. We particularly focused on examining how the increase in TB cases among immigrants in recent years (2.8% in 1997 to 1999 to 36.2% during the current study) was modifying transmission patterns. Mycobacterium tuberculosisisolates obtained from patients living in nine districts of Madrid (1,459,232 inhabitants) were genotyped. The TB case rate among foreign-born people was three to four times that of Spanish-born people, and the median time from arrival to the onset of treatment was 22.4 months. During the study period, 227 (36.3%) patients were grouped in 64 clusters, and 115 (50.7%) of them were in 21 clusters with mixed Spanish-born and foreign-born patients. Three of the 21 mixed clusters accounted for 21.1% of clustered patients. Twenty-two of 38 (57.9%) immigrants in mixed clusters were infected with TB strains that had already been identified in the native population in 1997 to 1999, including the three most prevalent strains. Factors identified as independent predictors of clustering were homelessness (odds ratio [OR], 2.3; 95% confidence interval [95% CI], 1.2 to 4.5; P= 0.011) and to be born in Spain (OR, 1.8; 95% CI, 1.2 to 2.6; P= 0.002). The results indicated that (i) TB transmission was higher in Spanish-born people, associated mainly with homelessness, (ii) that foreign-born people were much less likely to be clustered, suggesting a higher percentage of infection before arriving in Spain, and (iii) that an extensive transmission between Spanish- and foreign-born populations, caused mainly by autochthonous strains, was taking place in Madrid.

ABSTRACTThe emergence of resistance to antituberculosis drugs is a relevant matter worldwide, but the retrieval of antibiograms for Mycobacterium tuberculosisis severely delayed when phenotypic methods are used. Genotypic methods allow earlier detection of resistance, although conventional approaches are cumbersome or lack sensitivity or specificity. We aimed to design a new real-time PCR method to detect rifampin (RIF)- and isoniazid (INH)-resistant M. tuberculosisstrains in a single reaction tube. First, we characterized the resistant isolates in our area of Spain by DNA sequencing. Some mutation was found within the rpoBcore region in all the RIF-resistant (RIFr) strains. Forty-six percent of the INH-resistant (INHr) strains showed a mutation in katGcodon 315, and most of these were associated with high MICs. Eighteen of the RIFr, INHr, and multidrug-resistant strains sequenced were tested by our real-time PCR assay; and full concordance of the results of the PCR with the sequencing data was obtained. In addition, a blind test was performed with a panel of 15 different susceptible and resistant strains from throughout Spain, and our results were also in 100% agreement with the sequencing data. Ours is the first assay based on rapid-cycle PCR able to simultaneously detect in a single reaction tube a large variety of mutations associated with RIF resistance (12 different mutations affecting 8 independent codons, including the most prevalent mutations at positions 526 and 531) and the most frequent INH resistance mutations. Our design could be a model for new, rapid genotypic methods able to simultaneously detect a wide variety of antibiotic resistance mutations.

ABSTRACTGenetic alterations in the rpoBgene were characterized in 50 rifampin-resistant (Rifr) clinical isolates of Mycobacterium tuberculosiscomplex from Spain. A rapid PCR–enzyme-linked immunosorbent assay (ELISA) technique for the identification of rpoBmutations was evaluated with isolates of the M. tuberculosiscomplex and clinical specimens from tuberculosis patients that were positive for acid-fast bacilli (AFB). Sequence analysis demonstrated 11 differentrpoBmutations among the Rifrisolates in the study. The most frequent mutations were those associated with codon 531 (24 of 50; 48%) and codon 526 (11 of 50; 22%). Although the PCR-ELISA does not permit characterization of the specific Rifrallele within each strain, 10 of the 11 Rifrgenotypes were correctly identified by this method. We used the PCR-ELISA to predict the rifampin susceptibility of M. tuberculosiscomplex organisms from 30 AFB-positive sputum specimens. For 28 samples, of which 9 contained Rifrorganisms and 19 contained susceptible strains, results were concordant with those based on culture-based drug susceptibility testing and sequencing. Results from the remaining two samples could not be interpreted because of low bacillary load (microscopy score of 1+ for 1 to 9 microorganisms/100 fields). Our results suggest that the PCR-ELISA is an easy technique to implement and could be used as a rapid procedure for detecting rifampin resistance to complement conventional culture-based methods.

ABSTRACTTo investigate the diversity of IS6110fingerprints ofMycobacterium tuberculosisisolates in the United States and to determine if matching IS6110fingerprints represent recent interstate tuberculosis transmission, we performed restriction fragment length polymorphism analysis of M. tuberculosisisolates from 1,326 patients in three geographically separated states. Seven hundred ninety-five different IS6110fingerprint patterns were generated, and pattern diversity was similar in each state. Ninety-six percent of the fingerprint patterns were observed in only one state, demonstrating that most IS6110fingerprint patterns are confined to a single geographic location. Of the IS6110fingerprint patterns that were shared by isolates from more than one state, most isolates with 1 to 5 IS6110copies were separable by pTBN12 fingerprinting whereas those with >15 copies were not. One high-copy-number M. tuberculosisstrain had identical IS6110and pTBN12 fingerprints and included 57 isolates from three states. Epidemiological data demonstrated significant recent transmission of tuberculosis within each city but not among the states. This suggests that identical fingerprints of isolates from geographically separate locations most likely reflect interstate tuberculosis transmission in the past, with subsequent intrastate spread of disease. Further evaluation of M. tuberculosisstrains that cause outbreaks in different geographic locations will provide insight into the epidemiological and bacteriological factors that facilitate the spread of tuberculosis.

ABSTRACTFrom 2002 to 2003, 32 isolates of Staphylococcus hominissubsp. novobiosepticus(SHN) were recovered from 21 patients, 18 of whom were neonates, with 13 considered to have late-onset SHN sepsis. All isolates from neonates had an indistinguishable pulsed-field gel electrophoresis pattern. Our data support SHN as an important nosocomial pathogen in neonates.

ABSTRACTA total of 15 of 101 (14.8%) nasal methicillin-resistant Staphylococcus aureus(MRSA) isolates exhibited mupirocin resistance (Mupr) compared with 1 of 154 (0.6%) methicillin-susceptible Staphylococcus aureusisolates. A total of 14 (93%) isolates exhibiting high-level Muprbelonged to a single clone. Horizontal plasmid transfer and transmission of Muprstrains contribute to a high incidence of MuprMRSA at our institution.

ABSTRACTWe report the case of a teenager with chronic lymphocytic meningitis for whom Tropheryma whipplei16S rRNA PCR results were positive in two cerebrospinal fluid samples and one duodenal biopsy specimen. PCR targeting another specific sequence of Tropheryma whippleiand sequencing of the initially amplified 16S rRNA fragment did not confirm the results.