Gema Carrasco

Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-β-lactamase genes, and of common insertion sequences, in epidemic clones of Acinetobacter baumannii from Spain

OBJECTIVES To study the distribution, diversity and activity of Acinetobacter-derived cephalosporinase (ADC)-, carbapenem-hydrolysing oxacillinase (CHO)- and metallo-β-lactamase (MBL)-encoding genes, and of the most common insertion sequences (ISs), in the genome of nosocomial, epidemic, multidrug-resistant Acinetobacter baumannii (MDRAB) clones from Spain. METHODS The studied population included 59 MDRAB strains previously genotyped by PFGE and multilocus sequence typing. The search for the ADC (bla(ADC)), CHO (bla(OXA-51-like), bla(OXA-23-like), bla(OXA-40-like) and bla(OXA-58-like)) and MBL (bla(IMP), bla(VIM), bla(SIM-1), bla(GIM-1), bla(SPM-1) and bla(NDM-1)) genes, and for the ISs (ISAba1, ISAba2, ISAba3, ISAba4 and IS18) was done by PCR assays. The phenotypic presence of MBL enzymes was examined using imipenem/imipenem + EDTA strips. RESULTS The most prevalent IS, ISAba1 (93.2%), was detected upstream of bla(ADC) and bla(OXA-51-like). These genes showed ample diversity (10 and 8 alleles, respectively). Four ADC sequences (ADC-1-like(P240S), ADC-2-like(N260H/T264N), ADC-11-like(Q163K) and ADC-11-like(G342R)) are described here for the first time. bla(OXA-58-like) was carried by 20.3% of strains, in association with ISAba2, ISAba3 or IS18. bla(OXA-40-like) was the most prevalent acquired CHO gene (57.6%), and was associated with none of the studied ISs. Neither bla(OXA-23-like) nor ISAba4 was detected in any strain. Some 67.8% of strains with MBL activity showed no corresponding gene in PCR; these results were more common in strains with a highly active CHO, such as OXA-40. CONCLUSIONS All the studied genes and their related ISs showed a clonal distribution. Imipenem resistance was probably provided by OXA-40 for the most part, while MBL- and OXA-23-encoding genes were absent in the studied population.

Epidemiological and Phylogenetic Analysis of Spanish Human Brucella melitensis Strains by Multiple-Locus Variable-Number Tandem-Repeat Typing, Hypervariable Octameric Oligonucleotide Fingerprinting, and rpoB Typing

ABSTRACT The 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 β subunit (rpoB) were used to type a human Brucella melitensis population (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. rpoB typing grouped the strains as rpoB type 1 (1249-ATG/1309-CTG; 28.7%), rpoB type 2 (1249-ATG/1309-CTA; 62.9%), and rpoB type 3 (1249-ATA/1309-CTG; 8.3%). According to the MLVA-16 results, the population clustered by rpoB type. Given the correlation between B. melitensis MLVA groups and rpoB types (American and rpoB type 1, Eastern Mediterranean and rpoB type 2, and Western Mediterranean and rpoB type 3), the rpoB type could be used as an initial marker for the epidemiological surveillance of brucellosis.

Identification, Typing, and Phylogenetic Relationships of the Main Clinical Nocardia Species in Spain According to Their gyrB and rpoB Genes

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

Increase in isolation of Burkholderia contaminans from Spanish patients with cystic fibrosis

Species of the Burkholderia cepacia complex are associated with opportunistic infection in patients with cystic fibrosis. For years now, B. multivorans and B. cenocepacia have been the most frequently isolated species within the complex in such patients. However, between 2008 and 2012, the overall incidence of these species in Spain (17.7% and 12.5% respectively) was overtaken by that of B. contaminans (36.5%). The population structure of B. contaminans isolates from Spanish patients with cystic fibrosis was analysed using multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). Three major known sequence types (ST102, ST404 and ST482) and a new one (ST771) were identified among 59 isolates. In addition, PFGE detected 17 pulsotypes. Susceptibility to antibiotics was examined using the Etest. Cotrimoxazole and ceftazidime were the most active antibiotics against B. contaminans, inhibiting growth in 88% and 86% of the isolates, respectively. In addition, this species showed less resistance to most of the antibiotics tested than did either B. multivorans or B. cenocepacia isolates recovered from similar Spanish patients.

Molecular screening for rifampicin and fluoroquinolone resistance in a clinical population of Brucella melitensis

OBJECTIVES The aim of this study was to determine, using molecular methods, whether rifampicin and fluoroquinolone resistance was present in a clinical Brucella melitensis population. METHODS Sixty-two B. melitensis strains, isolated from humans-most experiencing their first brucellosis episode-over an 11 year period in Spain, were genotyped by multiple locus variable analysis (MLVA-16) for future studies. In the present work, molecular screening was undertaken to detect the presence of rpoB and gyrA/gyrB/parC/parE mutations (previously described in in vitro Brucella spp. mutants) related to resistance to rifampicin and fluoroquinolones, respectively. RESULTS Sixty-two MLVA-16 genotypes were identified among the B. melitensis population, with genetic similarity values ranging from 32% to 94%. rpoB mutations related to rifampicin resistance (positions 154, 526, 536, 539, 541, 574) were not detected. Neither were changes in GyrA described in in vitro mutants (67, 71, 87, 91 and an insertion at 340) detected in these strains. All showed identical GyrA, GyrB, ParC and ParE sequences with respect to B. melitensis 16M, except for one strain (ciprofloxacin and moxifloxacin MICs 0.25-0.50 mg/L) that harboured the Val264Ala replacement outside the GyrA quinolone resistance-determining region (QRDR); no differences were seen, however, in the NorMI/II efflux pump genes. CONCLUSIONS The absence of rpoB mutations clearly related to rifampicin resistance in clinical B. melitensis strains reinforces the first-choice status of this antibiotic in the treatment of first brucellosis episodes, and demonstrates the usefulness of molecular screening for resistant genotypes. The absence of topoisomerase II-IV mutations, however, cannot rule out fluoroquinolone resistance due to the interplay of different mechanisms.

Shortcomings of the Commercial MALDI-TOF MS Database and Use of MLSA as an Arbiter in the Identification of Nocardia Species

Nocardia species are difficult to identify, a consequence of the ever increasing number of species known and their homogeneous genetic characteristics. 16S rRNA analysis has been the gold standard for identifying these organisms, but proteomic techniques such as matrix-assisted laser desorption ionization-time of flight (MALDI-TOF MS) and housekeeping gene analysis, have also been explored. One hundred high (n = 25), intermediate (n = 20), and low (n = 55) prevalence (for Spain) Nocardia strains belonging to 30 species were identified via 16S rRNA and MALDI-TOF MS analysis. The manufacturer-provided database MALDI Biotyper library v4.0 (5.627 entries, Bruker Daltonik) was employed. In the high prevalence group (Nocardia farcinica, N. abscessus, N. cyriacigeorgica and N. nova), the 16S rRNA and MALDI-TOF MS methods provided the same identification for 76% of the strains examined. For the intermediate prevalence group (N. brasiliensis, N. carnea, N. otitidiscaviarum and N. transvalensis complex), this figure fell to 45%. In the low-prevalence group (22 species), these two methods were concordant only in six strains at the species level. Tetra-gene multi-locus sequencing analysis (MLSA) involving the concatemer gyrB-16S rRNA-hsp65-secA1 was used to arbitrate between discrepant identifications (n = 67). Overall, the MLSA confirmed the results provided at species level by 16S rRNA analysis in 34.3% of discrepancies, and those provided by MALDI-TOF MS in 13.4%. MALDI-TOF MS could be a strong candidate for the identification of Nocardia species, but only if its reference spectrum database improves, especially with respect to unusual, recently described species and species included in the described Nocardia complexes.

Resistance gene pool to co-trimoxazole in non-susceptible Nocardia strains.

The soil-borne pathogen Nocardia sp. causes severe cutaneous, pulmonary, and central nervous system infections. Against them, co-trimoxazole (SXT) constitutes the mainstay of antimicrobial therapy. However, some Nocardia strains show resistance to SXT, but the underlying genetic basis is unknown. We investigated the presence of genetic resistance determinants and class 1–3 integrons in 76 SXT-resistant Nocardia strains by PCR and sequencing. By E test, these clinical strains showed SXT minimum inhibitory concentrations of ≥32:608 mg/L (ratio of 1:19 for trimethoprim: sulfamethoxazole). They belonged to 12 species, being the main representatives Nocardia farcinica (32%), followed by N. flavorosea (6.5%), N. nova (11.8%), N. carnea (10.5%), N. transvalensis (10.5%), and Nocardia sp. (6.5%). The prevalence of resistance genes in the SXT-resistant strains was as follows: sul1 and sul2 93.4 and 78.9%, respectively, dfrA(S1) 14.7%, blaTEM-1 and blaZ 2.6 and 2.6%, respectively, VIM-2 1.3%, aph(3′)-IIIa 40.8%, ermA, ermB, mefA, and msrD 2.6, 77.6, 14.4, and 5.2%, respectively, and tet(O), tet(M), and tet(L) 48.6, 25.0, and 3.9%, respectively. Detected amino acid changes in GyrA were not related to fluoroquinolone resistance, but probably linked to species polymorphism. Class 1 and 3 integrons were found in 93.42 and 56.57% strains, respectively. Class 2 integrons and sul3 genes were not detected. Other mechanisms, different than dfrA(S1), dfrD, dfrF, dfrG, and dfrK, could explain the strong trimethoprim resistance shown by the other 64 strains. For first time, resistance determinants commonly found in clinically important bacteria were detected in Nocardia sp. sul1, sul2, erm(B), and tet(O) were the most prevalent in the SXT-resistant strains. The similarity in their resistome could be due to a common genetic platform, in which these determinants are co-transferred.

Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain

Objectives: The aims of this study were to explore the clinical distribution, by species, of the genus Nocardia and to assess the antimicrobial susceptibilities of the 10 most prevalent species identified in Spain. Methods: Over a 10 year period (2005–14), 1119 Nocardia strains were molecularly identified and subjected to the Etest. The distribution and resistance trends over the sub-periods 2005–09 and 2010–14 were also examined. Results: Of the strains examined, 82.9% belonged to the following species: Nocardia cyriacigeorgica (25.3%), Nocardia nova (15.0%), Nocardia abscessus (12.7%), Nocardia farcinica (11.4%), Nocardia carnea (4.3%), Nocardia brasiliensis (3.5%), Nocardia otitidiscaviarum (3.1%), Nocardia flavorosea (2.6%), Nocardia rhamnosiphila (2.6%) and Nocardia transvalensis (2.4%). Their prevalence values were similar during 2005–09 and 2010–14, except for those of N. abscessus, N. farcinica and N. transvalensis, which fell significantly in the second sub-period (P ⩽ 0.05). The major location of isolation was the respiratory tract (∼86%). Half (13/27) of all strains from the CNS were N. farcinica. Significant differences in MIC results were recorded for some species between the two sub-periods. According to the CLSI’s breakpoints, low resistance rates (⩽15%) were recorded for seven species with respect to cefotaxime, imipenem and tobramycin; five species showed similar rates with respect to trimethoprim/sulfamethoxazole. Linezolid and amikacin were the most frequently active agents. Conclusion: The accurate identification of the infecting species and the determination of its susceptibility to antimicrobial agents, given the large number of strains with atypical patterns, are crucial if patients with nocardiosis are to be successfully treated.

Paenibacillus spp. isolated from human and environmental samples in Spain: detection of 11 new species

One hundred thirty-six isolates, 88 human and 48 environmental, that met the requirements to belong to the genus Paenibacillus were identified using a polyphasic taxonomic approach known as 16S rRNA plus phenotypic traits. Thirty-seven Paenibacillus species were identified; some had not been previously reported from clinical samples. The main species were P. pabuli (13 isolates), P. provencensis (11), P. phoenicis (9) and P. lautus (8). P. pabuli (11/13) and P. provencensis (8/11) were mainly environmental isolates, while P. phoenicis (9/9) and P. lautus (6/8) were mainly human isolates. Despite the difficulties in assigning to human Paenibacillus isolates a role as a pathogen or contaminant, here 25% of the isolates were involved in true infections, especially in those cases that affected abscesses, wound exudates, ocular infections and diverse fluids. In addition, 15 isolates were identified as 11 ‘Candidatus’ to a new species, all of them from human specimens except one that was obtained from laboratory air. The antimicrobial susceptibility testing showed 95.6% of isolates were resistant to ampicillin, 44% were resistant to cotrimoxazole, 20 to 30% were resistant to cefotaxime and vancomycin and 13% were resistant to rifampicin and erythromycin.

gyrB Analysis as a Tool for Identifying Nocardia Species and Exploring Their Phylogeny

ABSTRACT gyrB is used to improve the identification of the Nocardia species 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 Nocardia studied species. gyrB provides typing and phylogenetic markers for N. carnea, N. transvalensis, N. brasiliensis, and N. otitidiscaviarum.