Jesús Agüero

Characterization of the Urease Operon of Brucella abortus and Assessment of Its Role in Virulence of the Bacterium

ABSTRACT Most members of the genus Brucella show strong urease activity. However, the role of this enzyme in the pathogenesis of Brucella infections is poorly understood. We isolated several Tn5 insertion mutants deficient in urease activity from Brucella abortus strain 2308. The mutations of most of these mutants mapped to a 5.7-kbp DNA region essential for urease activity. Sequencing of this region, designated ure1, revealed the presence of seven open reading frames corresponding to the urease structural proteins (UreA, UreB, and UreC) and the accessory proteins (UreD, UreE, UreF, and UreG). In addition to the urease genes, another gene (cobT) was identified, and inactivation of this gene affected urease activity in Brucella. Subsequent analysis of the previously described sequences of the genomes of Brucella spp. revealed the presence of a second urease cluster, ure2, in all them. The ure2 locus was apparently inactive in B. abortus 2308. Urease-deficient mutants were used to evaluate the role of urease in Brucella pathogenesis. The urease-producing strains were found to be resistant in vitro to strong acid conditions in the presence of urea, while urease-negative mutants were susceptible to acid treatment. Similarly, the urease-negative mutants were killed more efficiently than the urease-producing strains during transit through the stomach. These results suggested that urease protects brucellae during their passage through the stomach when the bacteria are acquired by the oral route, which is the major route of infection in human brucellosis.

Resistance to Nonnucleoside Reverse-Transcriptase Inhibitors and Prevalence of HIV Type 1 Non-B Subtypes Are Increasing among Persons with Recent Infection in Spain

The prevalence of drug resistance mutations was 12.1% among 198 persons who experienced human immunodeficiency virus (HIV) seroconversion identified in Spain during 1997-2004. There was a significant increase of K103N and of non-B subtypes over time. Transmission of HIV infection around the time of seroconversion was shown in 8 couples and in 2 clusters of 3 individuals.

Rapid Identification of Rhodococcus equi by a PCR Assay Targeting the choE Gene

ABSTRACT The actinomycete Rhodococcus equi is an important pathogen of horses and an emerging opportunistic pathogen of humans. Identification of R. equi by classical bacteriological techniques is sometimes difficult, and misclassification of an isolate is not uncommon. We report here on a specific PCR assay for the rapid and reliable identification of R. equi. It is based on the amplification of a fragment of the choE gene encoding cholesterol oxidase. The choE-based PCR was assessed by using a panel of strains comprising 132 isolates from different sources and of different geographical origins, all initially identified biochemically as R. equi, and 30 isolates of representative non-R. equi actinomycete species, including cholesterol oxidase producers. The expected 959-bp amplicon was observed only with R. equi isolates, as confirmed by sequencing of a variable region of the 16S RNA gene from a random sample of 20 PCR-positive isolates. All R. equi isolates gave a positive choE-based PCR result, which correlated with a high degree of conservation of the choE gene. Three of the 132 strains originally identified as R. equi were negative for the choE gene, and subsequent analysis of their 16S RNA gene sequences confirmed that they belonged to other bacterial species (Dietzia maris, Mycobacterium peregrinum, and Staphylococcus epidermidis). All non-R. equi isolates were negative by the choE-based PCR. ATCC 21387, the only known isolate of Brevibacterium sterolicum, gave a 959-bp amplicon whose DNA sequence was virtually identical to that of R. equi choE. Comparison of the 16S RNA genes indicated that ATCC 21387 should be considered an R. equi isolate.

Clinical and Microbiological Characteristics of 28 Patients with Staphylococcus schleiferi Infection

Abstract The aim of this study was to analyse the clinical and microbiological characteristics of a series of patients with infection by Staphylococcus schleiferi. Seventy-one isolates were recovered from 36 patients between January 1993 and June 1999 at a tertiary care centre in northern Spain. There were 28 patients with well-documented clinical data. Infection was more frequent in men (89.3%), and more than half of the patients had some degree of immunosuppression, mainly malignant neoplasms. Infection was nosocomial in 22 cases and community-acquired in the remaining cases. Staphylococcus schleiferi was frequently associated with wound infections, mainly surgical-site infections, although unusual types of infections were detected. Infection-related mortality was low. This study highlights the importance of careful identification of Staphylococcus schleiferi in the clinical microbiology laboratory. Due to the documented association of Staphylococcus schleiferi with clinical infections in humans, any isolates of this organism should be assumed to be pathogenic, unless proven otherwise.

The defect in the metabolism of erythritol of the Brucella abortus B19 vaccine strain is unrelated with its attenuated virulence in mice.

The role of the defect in erythritol catabolism in the attenuated virulence of Brucella abortus B19 vaccine strain in mice was investigated by means of five different strains: (i) the erythritol sensitive B19 vaccine strain; (ii) a natural erythritol tolerant (NET) mutant obtained spontaneously from B19; (iii) an erythritol resistant derivative from B19 (FJS19) obtained by gene replacement of the deleted ery region; (iv) the erythritol resistant B. abortus 2308 reference virulent strain; and (v) an erythritol sensitive mutant (227 strain) obtained from strain 2308 by transposon insertion in the chromosomal ery region. Besides virulence for mice, erythritol oxidation as well as other phenotypic markers were tested in all the strains. The 2308 and FJS19 strains grew in the presence of erythritol and oxidized the sugar, whereas the B19 and 227 strains did not. The NET strain grew in presence of erythritol but was unable to oxidize it. The B19 vaccine strain and its two erythritol resistant derivatives, NET and FJS19, showed similar residual virulence and splenic time courses in mice. Moreover, the virulent strain 2308 and its erythritol sensitive derivative (227 strain) exhibited similar levels of splenic infection. Altogether, these results demonstrate that the genetic region implicated in erythritol catabolism is not related to the low virulence exhibited by B19 in mice.

Colonisation and infection due to Enterobacteriaceae producing plasmid-mediated AmpC β-lactamases

OBJECTIVES To investigate the epidemiology and clinical features of infections caused by Enterobacteria producing plasmid-mediated AmpC β-lactamases (pAmpC), which are emerging as a cause of resistance to extended-spectrum cephalosporins. METHODS A prospective multicentre cohort of patients with infection/colonisation due to pAmpC-producing Enterobacteriaceae was performed in 7 Spanish hospitals from February throughout July 2009. pAmpCs were characterised by PCR and sequencing. RESULTS 140 patients were included; organisms isolated were Escherichia coli (n = 100), Proteus mirabilis (n = 20), Klebsiella pneumoniae (n = 17), and others (n = 3). Overall, 90% had a chronic underlying condition. The acquisition was nosocomial in 43%, healthcare-associated in 41% (14% of those were nursing home residents), and community in 16%. Only 5% of patients had no predisposing feature for infection with multidrug-resistant bacteria. Nineteen percent of patients were bacteraemic. Inappropriate empirical therapy was administered to 81% of bacteraemic patients, who had a crude mortality rate of 48%. The most frequent enzyme was CMY-2 (70%, predominantly in E. coli and P. mirabilis) followed by DHA-1 (19%, predominantly in K. pneumoniae). CONCLUSION pAmpC-producing Enterobacteriaceae caused nosocomial, healthcare-associated and community infections mainly in predisposed patients. Invasive infections were associated with high mortality which might be partly related to inappropriate empirical therapy.

Brucella abortus strain 2308 produces brucebactin, a highly efficient catecholic siderophore

Brucella abortus is known to produce 2,3-dihydroxybenzoate (2,3-DHBA) and to use this catechol as a siderophore to grow under iron-limited conditions. In this study a mutant (BAM41) is described that is deficient in siderophore production by insertion of Tn5 in the virulent B. abortus strain 2308. This mutant was unable to grow on iron-deprived medium and its growth could not be restored by addition of 2,3-DHBA. Production of catecholic compounds by both the Brucella mutant and parental strains under iron-deprivation conditions was assayed by TLC. Two catecholic substances were identified in the supernatant of the parental strain 2308. The faster migrating spot showed the same retention factor (R(f)) as that of purified 2,3-DHBA. The mutant BAM41 overproduced 2,3-DHBA, but failed to form the slower migrating catechol. This defect could only be complemented by the addition of the slow-migrating catechol from strain 2308. The genomic region containing Tn5 in BAM41 was cloned and the position of the transposon was determined by nucleotide sequencing. The sequence revealed that the insertion had occurred at a gene with homology to Escherichia coli entF, a locus involved in the late steps of the biosynthesis of the complex catecholic siderophore enterobactin. Intracellular survival and growth rates of the B. abortus wild-type and entF mutant strains in mouse-derived J774 macrophages were similar, indicating that production of this siderophore was not essential in this model of infection. It is concluded that B. abortus synthesizes a previously unknown and highly efficient catecholic siderophore, different from 2,3-DHBA, for which the name brucebactin is proposed.

Identification of Clinically Relevant Corynebacterium spp., Arcanobacterium haemolyticum, and Rhodococcus equi by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT The identification of 83 Corynebacterium, 13 Arcanobacterium haemolyticum, and 10 Rhodococcus equi strains by conventional methods (API Coryne complemented with 16S rRNA gene sequence analysis) was compared with matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry identification. The correlation between API and MALDI-TOF results was 89%. MALDI-TOF is a rapid and accurate system for identification of the above-mentioned microorganisms.

Identification of Brucella abortus B19 vaccine strain by the detection of DNA polymorphism at the ery locus

As sensitivity to erythritol is the main feature of the Brucella abortus B19 vaccine strain, an attempt to define the genetic basis of this phenotypic trait was made in order to explore its usefulness as a means of molecular identification of strain B19. With this purpose, an erythritol-sensitive derivative of B. abortus strain 2308 was generated by Tn5 transposition methods. Restriction fragments from a cloned 13 kb Tn5-containing EcoRI fragment from the ery locus of strain 2308 were used to probe DNA from B. abortus B19 and other Brucella reference strains. Genetic rearrangement in strain B19 was revealed by the display of restriction fragment length polymorphisms at the ery locus. No pattern differences were detected among all other Brucella strains tested. The polymorphism exhibited by B19 represents the first characterization of this vaccine strain at a molecular level, and can be used to distinguish it from all other Brucella species.

Identification of Atypical Rhodococcus-Like Clinical Isolates as Dietzia spp. by 16S rRNA Gene Sequencing

ABSTRACT Rhodococcus equi and Dietzia spp. are closely related actinomycetes that show similar phenotypic properties. In humans, R. equi is an opportunistic pathogen associated with severe immunodeficiency. Dietzia spp. are environmental bacteria that have been isolated recently from clinical material and are presumptively associated with human infections. During the last 5 years, 15 bacterial isolates from human clinical samples collected at the Hospital Marqués de Valdecilla, Santander, Spain, were identified as R. equi by the API Coryne test. 16S rRNA gene sequencing confirmed seven isolates to be true R. equi strains, whereas the other eight were identified as members of the genus Dietzia, including Dietzia maris (four isolates), Dietzia natronolimnaea (two isolates), and Dietzia timorensis and Dietzia sp. (one isolate each). The eight Dietzia isolates were highly sensitive to 12 antimicrobial compounds.