Evaluation of CHROMagar™ B. cepacia agar for the detection of Burkholderia cepacia complex species from sputum samples of patients with cystic fibrosis.

INTRODUCTION: Burkholderia cepacia complex (BCC) are non-fermenting Gram-negative bacteria that can chronically colonize the lungs of people with cystic fibrosis (pwCF), causing a severe and progressive respiratory failure, post-transplant complications and epidemic outbreaks. Therefore, rapid and accurate identification of these bacteria is relevant for pwCF, in order to facilitate early eradication and prevent chronic colonization. However, BCCs are often quite difficult to detect on culture media as they have a slow growth rate and can be hidden by other fast-growing microorganisms, including Pseudomonas aeruginosa and filamentous fungi. MATERIAL AND METHODS: We evaluated the sensitivity of CHROMagar™ B. cepacia agar using 11 isolates from a well-characterized BCC collection, using BCA agar (Oxoid, UK) as a gold standard. We also studied 180 clinical sputum samples to calculate positive (PPV) and negative (NPV) predictive values. Furthermore, we used three of the well-characterized BCC isolates to determine the limit of detection (LOD). RESULTS: Eleven isolates grew on CHROMagar™ B. cepacia at 37ºC after 48 h. The NPV and PPV of CHROMagar™ B. cepacia were 100% and 87.5%, respectively. The LOD of CHROMagar™ B. cepacia was around 1 × 103 CFU/ml, requiring a ten-fold dilution lower bacterial load than BCA for BCC detection. CONCLUSION: CHROMagar™ B. cepacia agar proved to have a very good sensitivity and specificity for the detection of clinical BCCs. Moreover, the chromogenic nature of the medium allowed us to clearly differentiate BCC from other Gram-negative species, filamentous fungi and yeasts, thereby facilitating the identification of contaminants.

Focusing on Gordonia Infections: Distribution, Antimicrobial Susceptibilities and Phylogeny.

The immunosuppression conditions and the presence of medical devices in patients favor the Gordonia infections. However, the features of this aerobic actinomycete have been little explored. Strains (n = 164) were characterized with 16S rDNA and secA1 genes to define their phylogenetic relationships, and subjected to broth microdilution to profile the antimicrobial susceptibilities of Gordonia species that caused infections in Spain during the 2005-2021 period. Four out of the eleven identified species were responsible for 86.0% of the infections: Gordonia sputi (53.0%), Gordonia bronchialis (18.3%), Gordonia terrae (8.5%) and Gordonia otitidis (6.1%). Respiratory tract infections (61.6%) and bacteremia (21.9%) were the most common infections. The secA1 gene resolved the inconclusive identification, and two major clonal lineages were observed for G. sputi and G. bronchialis. Species showed a wide antimicrobial susceptibility profile. Cefoxitin resistance varies depending on the species, reaching 94.2% for G. sputi and 36.0% for G. terrae. What is noteworthy is the minocycline resistance in G. sputi (11.5%), the clarithromycin resistance in G. bronchialis secA1 lineage II (30.0%) and the amoxicillin-clavulanate and cefepime resistance in G. terrae (21.4% and 42.8%, respectively). G. sputi and G. bronchialis stand out as the prevalent species causing infections in Spain. Resistance against cefoxitin and other antimicrobials should be considered.

Exploring the genetic background of the botulism neurotoxin BoNT/B2 in Spain.

To determine whether the neurotoxin BoNT/B2 causing botulism in Spain is clonal, the genetic diversity and phylogenetic relationships of Clostridium botulinum from food-borne episodes and infant cases of the condition were explored. The botulinum toxin gene (bont) subtype, the variable region of the flagellin gene (flaVR), and a seven-gene multi-locus sequence type were examined by sequencing 37 BoNT-positive cultures obtained over the period 2010 to 2022. Out of 37 botulism events, 16 food-borne episodes and 16 infant cases were associated with bont/b2. Eight bont/b2 alleles were detected [nucleotide distance range 0.0259-0.415%, Hunter and Gaston discrimination index (HGDI) 0.71]. The most common bont/b2 allele corresponded to that of strain Prevot 25 NCASE and its single and double locus variations (87.5%). Four known flaVR types were identified (HGDI 0.79), along with one previously unknown (flaVR-15). Sixteen sequence types (STs) (HGDI 0.89) were recorded including seven new STs (ST164-ST170; 10 new alleles) and five new STs (ST171-ST175; with new allele combinations) were also noted. Correlations among some STs and flaVR types were seen. Overall, the present results show that the combined analysis of bont/b2-flaVR-ST at the nucleotide level could be used to track botulism events in Spain. The neurotoxin BoNT/B2 has largely been responsible for human botulism in Spain. The polymorphism analysis of bont/b2, flaVR typing, and sequence type determinations, revealed a wide variety of clones to be responsible for human botulism, ruling out a common source of acquisition. IMPORTANCE Botulism, a potentially fatal disease, is classically characterized by a symmetrical descending flaccid paralysis, which if left untreated can lead to respiratory failure and death. Botulinum neurotoxin (BoNT), produced by certain species of Clostridium, is the most potent biological toxin known, and the direct cause of botulism. This study characterizes the acquisition in Spain of two forms of botulism, i.e., food-borne and infant botulism, which are largely caused by the main neurotoxin BoNT/B2. Polymorphism analysis of the bont/b2 gene, typing of the flagellin variable region sequence (flaVR), and multilocus sequence typing, were used to explore the genetic background of Clostridium botulinum group I. To our knowledge, this is the first phylogenetic and typing study of botulism undertaken in Spain.

National Surveillance of Tetracycline, Erythromycin, and Clindamycin Resistance in Invasive Streptococcus pyogenes: A Retrospective Study of the Situation in Spain, 2007-2020.

BACKGROUND: This work reports on antimicrobial resistance data for invasive Streptococcus pyogenes in Spain, collected by the 'Surveillance Program for Invasive Group A Streptococcus', in 2007-2020. METHODS: emm typing was determined by sequencing. Susceptibility to penicillin, tetracycline, erythromycin, and clindamycin was determined via the E-test. tetM, tetO, msrD, mefA, ermB, ermTR, and ermT were sought by PCR. Macrolide-resistant phenotypes (M, cMLSB, and iMLSB) were detected using the erythromycin-clindamycin double-disk test. Resistant clones were identified via their emm type, multilocus sequence type (ST), resistance genotype, and macrolide resistance phenotype. RESULTS: Penicillin susceptibility was universal. Tetracycline resistance was recorded for 237/1983 isolates (12.0%) (152 carried only tetM, 48 carried only tetO, and 33 carried both). Erythromycin resistance was detected in 172/1983 isolates (8.7%); ermB was present in 83, mefA in 58, msrD in 51, ermTR in 46, and ermT in 36. Clindamycin resistance (methylase-mediated) was present in 78/1983 isolates (3.9%). Eight main resistant clones were identified: two that were tetracycline-resistant only (emm22/ST46/tetM and emm77/ST63/tetO), three that were erythromycin-resistant only (emm4/ST39/mefA-msrD/M, emm12/ST36/mefA-msrD/M, and emm28/ST52/ermB/cMLSB), and three that were tetracycline-erythromycin co-resistant (emm11/ST403/tetM-ermB/cMLSB, emm77/ST63/tetO-ermTR/iMLSB, and emm77/ST63/tetM-tetO-ermTR/iMLSB). CONCLUSIONS: Tetracycline, erythromycin, and clindamycin resistance rates declined between 2007 and 2020. Temporal variations in the proportion of resistant clones determined the change in resistance rates.

Botulism in Spain: Epidemiology and Outcomes of Antitoxin Treatment, 1997-2019.

BACKGROUND: Botulism is a low incidence but potentially fatal infectious disease caused by neurotoxins produced mainly by Clostridium botulinum. There are different routes of acquisition, food-borne and infant/intestinal being the most frequent presentation, and antitoxin is the treatment of choice in all cases. In Spain, botulism is under surveillance, and case reporting is mandatory. METHODS: This retrospective study attempts to provide a more complete picture of the epidemiology of botulism in Spain from 1997 to 2019 and an assessment of the treatment, including the relationship between a delay in antitoxin administration and the length of hospitalization using the Cox proportional hazards test and Kruskal-Wallis test, and an approach to the frequency of adverse events, issues for which no previous national data have been published. RESULTS: Eight of the 44 outbreaks were associated with contaminated commercial foods involving ≤7 cases/outbreak; preserved vegetables were the main source of infection, followed by fish products; early antitoxin administration significantly reduces the hospital stay, and adverse reactions to the antitoxin affect around 3% of treated cases.

Invasive Streptococcus pyogenes disease in Spain: a microbiological and epidemiological study covering the period 2007-2019.

The aim of this study is to present the first nationwide microbiological and epidemiological study of invasive group A Streptococcus (iGAS) disease in Spain. One thousand eight hundred ninety-three iGAS isolates were analyzed over 2007-2019. emm typing was performed by sequencing the gene's variable 5' end, exotoxin genes were identified by PCR, and antimicrobial susceptibility explored via the E test and disk diffusion. Five hundred twenty-three isolates were associated with sepsis, 292 with cellulitis, 232 with scarlet fever, 153 with pneumonia, 141 with streptococcal toxic shock syndrome, and 94 with necrotizing fasciitis. The most prevalent emm types were emm1 (449/1893 isolates), emm89 (210/1893), emm3 (208/1893), emm4 (150/1893), emm12 (112/1893) emm6 (107/1893), emm87 (89/1893), emm28 (88/1893), emm75 (78/1893), emm77 (78/1893), emm11 (58/1893), and emm22 (35/1893). emm1, emm3, emm4, and emm6 were the predominant types affecting children (mostly respiratory infections), while emm11, emm77, and emm89 prevailed in the elderly (mostly skin infections). Each emm type was associated with one or more exotoxin gene (spe, sme, and ssa) profiles. speA was detected in 660 isolates, speB in 1829, speC in 1014, speF in 1826, speG in 1651, speJ in 716, speH in 331, smeZ in 720, and ssa in 512. Isolates with speA were associated with the most severe infections. Penicillin susceptibility was universal. Two hundred twenty-four isolates were resistant to tetracycline, 169 to erythromycin, and 81 to clindamycin. Tetracycline, erythromycin, and clindamycin resistance rates declined over the study period. The above information could serve as the basis for continued surveillance efforts designed to control disease cause by this bacterium.

Molecular Characterization and Antimicrobial Susceptibilities of Nocardia Species Isolated from the Soil; A Comparison with Species Isolated from Humans.

Nocardia species, one of the most predominant Actinobacteria of the soil microbiota, cause infection in humans following traumatic inoculation or inhalation. The identification, typing, phylogenetic relationship and antimicrobial susceptibilities of 38 soil Nocardia strains from Lara State, Venezuela, were studied by 16S rRNA and gyrB (subunit B of topoisomerase II) genes, multilocus sequence analysis (MLSA), whole-genome sequencing (WGS), and microdilution. The results were compared with those for human strains. Just seven Nocardia species with one or two strains each, except for Nocardia cyriacigeorgica with 29, were identified. MLSA confirmed the species assignments made by 16S rRNA and gyrB analyses (89.5% and 71.0% respectively), and grouped each soil strain with its corresponding reference and clinical strains, except for 19 N. cyriacigeorgica strains found at five locations which grouped into a soil-only cluster. The soil strains of N. cyriacigeorgica showed fewer gyrB haplotypes than the examined human strains (13 vs. 17) but did show a larger number of gyrB SNPs (212 vs. 77). Their susceptibilities to antimicrobials were similar except for beta-lactams, fluoroquinolones, minocycline, and clarithromycin, with the soil strains more susceptible to the first three (p ≤ 0.05). WGS was performed on four strains belonging to the soil-only cluster and on two outside it, and the results compared with public N. cyriacigeorgica genomes. The average nucleotide/amino acid identity, in silico genome-to-genome hybridization similarity, and the difference in the genomic GC content, suggest that some strains of the soil-only cluster may belong to a novel subspecies or even a new species (proposed name Nocardia venezuelensis).

Dynamics of a Sporadic Nosocomial Acinetobacter calcoaceticus - Acinetobacter baumannii Complex Population.

Our objective was to improve current knowledge of sporadic (Spo) nosocomial Acinetobacter calcoaceticus-Acinetobacter baumannii (Acb) complex populations, and thus better understand the epidemiology of Spo and endemoepidemic (EE) strains. Between 1999 and 2010, 133 isolates of Spo Acb complex were obtained from a single hospital. Species were identified by gyrB-PCR, and via gyrB- and rpoB-sequencing. Clonal analysis was undertaken using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. Susceptibility to antimicrobial agents was determined by microdilution and E-tests. Carbapenemase genes were detected by PCR. One hundred and one PFGE types were detected. A. baumannii was the most common (67/101 PFGE types), followed by Acinetobacter pittii (22/101), Acinetobacter lactucae (6/101), and Acinetobacter calcoaceticus (2/101). gyrB, rpoB1, and rpoB2 sequencing returned 49, 13, and 16 novel sequences, respectively. Sixty-three sequence types (STs) (38 new STs and 66 new alleles) were detected; the most common were ST2 (29/133 isolates) and ST132 (14/133). Twenty-six OXA-51 allelic variants were detected, nine of which were novel. The PFGE types were generally susceptible (88/101) to all the tested antimicrobials; 3/101 were carbapenem-resistant due to the presence of the genetic structure ISAba2-bla OXA-58-like-ISAba3, and 2/101 were multidrug-resistant. It can be concluded that the examined Spo Acb complex population was mainly composed of A. baumannii. Many different clones were detected (with ST2 clearly dominant), all largely susceptible to antimicrobials; multidrug resistance was rare. In contrast, a previously examined EE Acb population was composed of just four expanding, multidrug-resistant A. baumannii clones -ST2, ST3, ST15, and ST80-.

First Draft Genome Sequence of a Clinical Strain of Nocardia cerradoensis.

This paper reports the first draft genome sequence for a strain of Nocardia cerradoensis obtained from an immunocompetent patient with a knee infection. The 8.2-Mb genome has 8,329 coding sequences, including intrinsic resistance genes, biosynthetic gene clusters for polyketide synthase and nonribosomal peptide synthase, virulence genes, and prophages.

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.

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.

Traumatic endophthalmitis caused by Nocardia kruczakiae in a patient with traumatic eye injury.

BACKGROUND: We describe a case of traumatic ocular endophthalmitis caused by Nocardia kruczakiae after vegetable trauma in an immunocompetent child. FINDINGS: A 5-year-old boy suffered from a trauma with a palm tree leaflet. Two months later, he was diagnosed with traumatic infectious uveitis and intumescent cataract with anterior capsule rupture. Intensive treatment with systemic and topical vancomycin, ceftazidime and methylprednisolone began. After 1 month, he underwent phacoemulsification with intraocular lens implantation (IOL). After some episodes of reactivation, he was diagnosed with traumatic nocardial endophthalmitis from aqueous humour samples. Several operations and specific antibiotic therapy resolved the infection. CONCLUSIONS: In cases of traumatic endophthalmitis and several recurrences, it is extremely useful to make an etiologic diagnosis in order to treat the patient with specific antibiotics.

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.

gyrB analysis as a tool for identifying Nocardia species and exploring their phylogeny.

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.

Identification, typing, and phylogenetic relationships of the main clinical Nocardia species in spain according to their gyrB and rpoB genes.

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 (126)AAAPEH 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.

Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-ß-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-ß-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.

Clonal outbreak of ST17 multidrug-resistant Enterococcus faecium harbouring an Inc18-like::Tn1546 plasmid in a haemo-oncology ward of a Spanish hospital.

OBJECTIVES: To report a clonal outbreak of ST17 vancomycin-resistant Enterococcus faecium (VREfm) carrying Tn1546 (vanA) in a haemo-oncology ward of a tertiary teaching hospital in the south of Spain (January-September 2009). PATIENTS AND METHODS: Twenty-two VREfm strains from 13 patients were characterized by PFGE, multiple-locus variable-number tandem-repeat analysis (MLVA) and multilocus sequence typing (MLST). Genes encoding antibiotic resistance and putative virulence traits and the Tn1546 backbone were investigated by PCR. Plasmid characterization included determination of size (S1-PFGE) and replication modules (PCR, hybridization and sequencing). Patient clinical records were analysed retrospectively. RESULTS: A single ST17 E. faecium clone (MT-7 MLVA type) carrying esp and hyl plus a 30 kb Inc18-like::Tn1546 (IS1216) plasmid was identified. Ampicillin resistance was linked to PBP5 showing mutations at positions 24, 27, 34, 66, 68, 85, 100, 144, 172, 177, 204, 216, 324, 462, 466', 470, 485, 496, 499, 525, 546, 558, 582, 586, 629, 632, 642 and 667. Other resistance genes identified were erm(B), ant(6')-Ia and aph(3')-IIIa. Fluoroquinolone resistance was attributable to ParC (Arg-61 → Gly and Ser-80 → Arg) and GyrA (Ser-83 → Arg) mutations. CONCLUSIONS: A nosocomial outbreak caused by an ST17 (CC17) E. faecium clone harbouring Esp and Hyl and a 30 kb Inc18-like::Tn1546 plasmid among haemo-oncology patients is reported. The failure of early infection control practices indicates an undetected reservoir and the ability of this strain to persist over long periods. The potential spread of epidemic clones and broad host plasmids carrying vancomycin resistance in Spain is of concern since it might contribute towards a higher rate of VREfm infection.

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.

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 beta 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.

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.