Botulinum Neurotoxin F Subtypes Cleaving the VAMP-2 Q58⁻K59 Peptide Bond Exhibit Unique Catalytic Properties and Substrate Specificities.

In the recent past, about 40 botulinum neurotoxin (BoNT) subtypes belonging to serotypes A, B, E, and F pathogenic to humans were identified among hundreds of independent isolates. BoNTs are the etiological factors of botulism and represent potential bioweapons; however, they are also recognized pharmaceuticals for the efficient counteraction of hyperactive nerve terminals in a variety of human diseases. The detailed biochemical characterization of subtypes as the basis for development of suitable countermeasures and possible novel therapeutic applications is lagging behind the increase in new subtypes. Here, we report the primary structure of a ninth subtype of BoNT/F. Its amino-acid sequence diverges by at least 8.4% at the holotoxin and 13.4% at the enzymatic domain level from all other known BoNT/F subtypes. We found that BoNT/F9 shares the scissile Q58/K59 bond in its substrate vesicle associated membrane protein 2 with the prototype BoNT/F1. Comparative biochemical analyses of four BoNT/F enzymatic domains showed that the catalytic efficiencies decrease in the order F1 > F7 > F9 > F6, and vary by up to a factor of eight. KM values increase in the order F1 > F9 > F6 ≈ F7, whereas kcat decreases in the order F7 > F1 > F9 > F6. Comparative substrate scanning mutagenesis studies revealed a unique pattern of crucial substrate residues for each subtype. Based upon structural coordinates of F1 bound to an inhibitor polypeptide, the mutational analyses suggest different substrate interactions in the substrate binding channel of each subtype.

First Insight into the Genome Sequences of Two Linezolid-Resistant Nocardia farcinica Strains Isolated from Patients with Cystic Fibrosis.

The draft genome sequences of two Nocardia farcinica strains isolated from two patients with cystic fibrosis (CF), resistant to trimethoprim/sulfamethoxazole and linezolid, are reported here. The estimated genome sizes were 5.8 Mb with a 70.63% G+C content. Transposases from Tn916 were detected, but not 23S rRNA mutation (G2576T) related to linezolid resistance.

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.

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.

High genetic diversity among Stenotrophomonas maltophilia strains despite their originating at a single hospital.

The levels of genetic relatedness of 139 Stenotrophomonas maltophilia strains 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. maltophilia strain 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. maltophilia isolates 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.

Antimicrobial susceptibility profile of molecular typed cystic fibrosis Stenotrophomonas maltophilia isolates and differences with noncystic fibrosis isolates.

Multiresistance in Stenotrophomonas maltophilia limits the effectiveness of antimicrobial therapy for infections due to this organism. It can be of special concern in cystic fibrosis (CF) patients due to frequent antimicrobial administration. The in vitro activity of 41 antimicrobial agents against 76 epidemiologically defined CF S. maltophilia isolates by pulsed-field-gel electrophoresis (PFGE) technique under XbaI and SpeI restriction was compared with that obtained with 51 non-CF strains recovered from respiratory sources. Minimal inhibitory concentrations (MICs) were determined with the standard National Committee for Clinical Laboratory Standards agar dilution technique, but with 24-hr incubation. Forty-seven different PFGE profiles were observed within 76 S. maltophilia CF isolates. Minocycline (resistance rate, 0%; MIC(90), 1 microg/ml), doxycycline (6.4%; 8 microg/ml), trovafloxacin (4.2%; 2 microg/ml), moxifloxacin (6.3%; 2 microg/ml), clinafloxacin (6.3%; 2 g/ml), and moxalactam (17.0%; 64 g/ml) displayed low resistance rates. On the contrary, resistance rates were higher with ceftazidime (70.0%; 256 microg/ml), cefepime (83.0%; 128 microg/ml), piperacillin (87.2%; >1,024 microg/ml), ticarcillin (87.2%; >512 microg/ml), and aztreonam (95.7%; >1,024 microg/ml). Clavulanate reverted resistance to ticarcillin and aztreonam in 40.4% and 31.7% of strains, respectively. Aminoglycosides displayed reduced activities with susceptibility rates lower than 20% and MIC(90) higher than 128 microg/ml. With the exception of trimethoprim-sulfamethoxazole (25.4 vs. 31.3%), CF isolates were more resistant than non-CF isolates. Remarkably, resistance was enhanced in S. maltophilia isolates persistently recovered in chronically colonized patients. Susceptibility analysis demonstrated higher resistance rates among CF S. maltophilia isolates when compared with respiratory isolates from non-CF patients. Moreover, persistently recovered CF S. maltophilia isolates were more resistant than sporadic non-CF isolates.

Topoisomerase II and IV quinolone resistance-determining regions in Stenotrophomonas maltophilia clinical isolates with different levels of quinolone susceptibility.

The quinolone resistance-determining regions (QRDRs) of topoisomerase II and IV genes from Stenotrophomonas maltophilia ATCC 13637 were sequenced and compared with the corresponding regions of 32 unrelated S. maltophilia clinical strains for which ciprofloxacin MICs ranged from 0.1 to 64 microg/ml. GyrA (Leu-55 to Gln-155, Escherichia coli numbering), 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 microg/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 microg/ml) or reserpine (10 microg/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.