Co-occurrence of the cephalosporinase cepA and carbapenemase cfiA genes in a Bacteroides fragilis division II strain, an unexpected finding.

BACKGROUND: Bacteroides fragilis, an anaerobic gut bacterium and opportunistic pathogen, comprises two genetically divergent groups (or divisions) at the species level. Differences exist both in the core and accessory genomes and the beta-lactamase genes, with the cephalosporinase gene cepA represented only in division I and the carbapenemase gene cfiA only in division II. METHODS: Multidrug resistance in a clinical B. fragilis strain was examined by whole-genome sequencing. RESULTS: Strain CNM20200260 carried the antimicrobial resistance genes cepA, cfiA2, ant(6'), erm(F), mef(En2), est(T), tet(Q) and cat(A), along with 82-Phe mutation in gyrA (together with 47 amino acid changes in gyrA/B and parC/parE). bexA/B and other efflux pump genes were also observed. None of the detected insertion sequences was located upstream of cfiA2. The genome-based taxonomy coefficients (average nucleotide identity, DNA-DNA hybridization similarity and difference in genomic G + C%) with respect to genomes of the strains of B. fragilis division II and the novel species Bacteroides hominis (both cfiA-positive) met the criteria for CNM20200260 to belong to either species (>95%, >70% and <1%, respectively). No such similarity was seen with type strain NCTC 9343 or the representative genome FDAARGOS 1225 of B. fragilis (division I, cfiA-negative). Strain CNM20200260 harboured four out of nine Kyoto Encyclopedia of Genes and Genomes orthologues defined for division I and one of two defined for division II. CONCLUSIONS: This is the first description of the co-occurrence of cepA and cfiA in a Bacteroides strain, confirming the complexity of the taxonomy of this species.

Identification and antimicrobial susceptibility of Streptomyces and other unusual Actinobacteria clinical isolates in Spain.

Two hundred and eighty-six isolates from human clinical samples were identified between 1996 and 2019 as belonging to 8 families, 19 genera and 88 species of Actinobacteria. The most identified genera were Streptomyces (182 strains from 45 species), Actinomadura (29 strains, 5 species), Nocardiopsis (21 strains, 6 species) and Dietzia (18 strains, 5 species). The rest of the identified genera (15) contained 27 species with 36 isolates. Of the species studied, only 13/88 had been documented previously as isolates from clinical samples, and in some cases, as true pathogens. In this sense, a literature review of the species found in infections or in clinical samples without clear involvement in pathology has been carried out. Finally, the susceptibility to 8 antimicrobial agents has been studied. Streptomyces showed high resistance (80.8%) against cefotaxime and cotrimoxazole (55.5%), and no isolate resistance to amikacin and linezolid have been found. Lower percentages of resistance have been found in other genera, except in Dietzia (100% against cotrimoxazole and 44.4% against erythromycin). The greatest resistance in these genera was to cotrimoxazole (29.8) and erythromycin (27,9%), and no resistance to linezolid has been found in these genera. In Microbispora, Nonomuraea and Umezawaea, no resistant isolates have been found against any antibiotic studied. Only 3/104 isolates were resistant to amikacin in Amycolatopsis, Crossiella, and Micromonosopora. One isolate of Amycolatopsis was resistant to imipenem.

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.

Characterization and antimicrobial susceptibility of one antibiotic-sensitive and one multidrug-resistant Corynebacterium kroppenstedtii strain isolated from patients with granulomatous mastitis.

Human infections associated with Corynebacterium kroppenstedtii are rarely reported, and this organism is usually described as antibiotic sensitive. Almost all published cases of C. kroppenstedtii infections have been associated with breast pathology in women and have been described in New Zealand, France, Canada, India and Japan. Here we describe the microbiologic characteristics of two strains isolated from two women diagnosed of granulomatous mastitis in Spain. One C. kroppenstedtii isolate was antibiotic sensitive while the other was multidrug resistant. Biochemical identification was possible using a wide battery of methods including API Coryne V2.0, API Strep, API NH, API NE, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and 16S rRNA gene amplification and sequencing. Antimicrobial susceptibility to 28 antibiotics as determined by Etest showed one isolate being sensitive to benzylpenicillin, ciprofloxacin, moxifloxacin, gentamicin, vancomycin, clindamycin, tetracycline, linezolid and rifampin. The second isolate showed resistance to ciprofloxacin, moxifloxacin, clindamycin, tetracycline and rifampin. The multidrug-resistant isolate contained the erm(X), tet(W), cmx, aphA1-IAB, strAB and sul1 resistance genes known from the R plasmid pJA144188 of Corynebacterium resistens. These genes were absent in the genome of the antibiotic-sensitive isolate. This report confirms the tropism of this microorganism for women's breasts and presents the first description of a multidrug-resistant C. kroppenstedtii strain.

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.

Meningitis caused by Abiotrophia defectiva: case report and literature review.

The genus Abiotrophia comprises fastidious Gram-positive bacteria previously classified as nutritionally variant streptococci (NVS). The isolation of NVS from the central nervous system (CNS) is very rare. We describe a case of meningitis due to Abiotrophia defectiva in a patient who underwent a total hip arthroplasty 4 days previously. It is possible that the organism could be introduced through the spinal anesthesia. We also review all cases of CNS infections caused by NVS.

Two simultaneous botulism outbreaks in Barcelona: Clostridium baratii and Clostridium botulinum.

Botulism is a severe neuroparalytic disorder that can be potentially life-threatening. In Barcelona, Spain, no outbreaks had been reported in the past 25 years. However, in September 2011, two outbreaks occurred involving two different families. A rare case of Clostridium baratii which produced a neurotoxin F outbreak was detected in five family members who had shared lunch, and several days before that another family was affected by C. botulinum toxin A which was probably present in homemade pâté.

Dermabacter hominis: a usually daptomycin-resistant gram-positive organism infrequently isolated from human clinical samples.

During a 12-year period, Dermabacter hominis was isolated from 21 clinical samples belonging to 14 patients attending a tertiary hospital in León, Spain. Samples included blood cultures (14), peritoneal dialysis catheter exit sites (three), cutaneous abscesses (two), an infected vascular catheter (one) and a wound swab (one). Identification was made by API Coryne™ V2.0, Biolog™ GP2 and 16S rRNA gene amplification. Six febrile patients had positive blood cultures (one, two or three sets) and all of them were treated with teicoplanin (two patients), vancomycin, ampicillin plus gentamicin, amoxicillin/clavulanic acid and ciprofloxacin (one each). An additional patient with a single positive blood culture was not treated, the finding being considered non-significant. In the remaining seven patients the organism was isolated from a single specimen and three of them received antimicrobial treatment (ciprofloxacin, ceftriaxone plus vancomycin and amoxicillin/clavulanic acid). At least ten patients had several underlying diseases and conditions, and no direct mortality was observed in relation to the isolated organism. All isolates were susceptible to vancomycin, rifampin and linezolid. Resistance to other antibiotics varied: erythromycin (100%), clindamycin (78.5%), ciprofloxacin (21.4%) and gentamicin, quinupristin-dalfopristin, benzylpenicillin and imipenem 7.1% each. Thirteen isolates were highly resistant to daptomycin with MICs ranging from 8 to 48 (MIC90 = 32 mg/L); only one was daptomycin-sensitive (MIC = 0.19 mg/L).

Identification, molecular characterisation and antimicrobial susceptibility of genomovars of the Burkholderia cepacia complex in Spain.

Burkholderia spp. strains collected in Spain over a 13-year period from patients with cystic fibrosis (CF) (n = 148), non-CF patients (n = 103) and from environmental sources (n = 64) were characterised. One hundred and forty-one of the examined strains were involved in seven suspected nosocomial disease outbreaks. Strains were identified by their 16s rRNA and recA genes. Their genetic relatedness, the possession of cable pili and the B. cepacia epidemic strain marker (BCESM), and their susceptibility to antimicrobial agents were studied using pulsed-field gel electrophoresis (PFGE), cblA and esmR genes analysis, and by the E-test, respectively. The genomovar distribution for the 315 strains was as follows: B. stabilis 29.5 %, B. cepacia 14.9 %, B. multivorans 11.1 %, B. cenocepacia IIIA 9.5 %, B. vietnamiensis 3.8 %, B. cenocepacia IIIB 3.5 %, and B. ambifaria and B. pyrrocinia 0.3 % each. The genetic diversity of the B. cepacia complex (Bcc) was ample, with 57 different SpeI types, showing a genetic similarity of 36.4-96.6 %. No strain carried cblA, whereas 25 B. cenocepacia genotypes harboured BCESM (23 from patients with CF). Antimicrobial resistance rates to tobramycin (TOB; 86 %) and imipenem (IPM; 67 %) were high. The strains from patients with CF showed significantly greater resistance to piperacillin (PIP), levofloxacin (LVX) and co-trimoxazole (SXT) than those isolated from non-CF patients (p < 0.05). In conclusion, B. cenocepacia was the most prevalent genomovar found in patients with CF (19.1 %), whereas B. cepacia was the most common among non-CF patients (20.7 %). B. stabilis (47.6 %) was the most common environmental genomovar. Susceptibility to antimicrobial agents depended on genomovar status and strain origin.

Study of the molecular mechanisms involved in high-level macrolide resistance of Spanish Campylobacter jejuni and Campylobacter coli strains.

OBJECTIVES: To investigate the molecular mechanisms involved in the high-level erythromycin resistance of clinical Spanish Campylobacter jejuni and Campylobacter coli strains. METHODS: Overall susceptibilities of 678 C. jejuni and 119 C. coli strains, collected from 10 Spanish provinces during 2006 and 2007, were determined by Etest. In high-level erythromycin-resistant strains, molecular determinants were studied. The analysis was focused on region V of the 23S rRNA gene, the rplD and rplV ribosomal genes, and the regulatory region of the CmeABC efflux pump. RESULTS: The global resistance rate to erythromycin was 3.8%. Among the resistant strains, 93% were C. coli and 7% were C. jejuni. The A2075G mutation in the 23S rRNA gene was detected in all of the resistant strains except for two, which carried the A2074G mutation. None of the ribosomal rplD and rplV genes harboured the described mutations that confer resistance to macrolides. Different mutations affecting the regulatory region of the CmeABC efflux pump were also found. CONCLUSIONS: C. coli strains are clearly more resistant to erythromycin than C. jejuni. The mutation A2075G in the 23S rRNA gene was responsible for the resistance in most of the strains; A2074G was only found in two strains. Further studies are required to ascertain the effect of mutations in the regulatory region of cmeABC. Our data indicate that the rate of resistance was similar to that of other European countries.

Isolation of Corynebacterium ureicelerivorans from normally sterile sites in humans.

Fifteen Corynebacterium ureicelerivorans isolates were recovered in pure culture from six patients during a five-year period. Five patients had bacteremia and the other was an infection of ascitic fluid. The API Coryne numerical profile obtained corresponds to the profile for C. bovis, while Biolog GP2 identified four out of the six isolates as C. jeikeium. The organisms were molecular identified by 16S rDNA and rpoB. The present report also includes information on new phenotypic tests and, for the first time, antimicrobial susceptibility data of C. ureicelerivorans and their rpoB sequences. All macrolide-resistant isolates presented a constitutive MLS phenotype. This organism must be differentiated from other slow-growing, lipophilic, and urea-splitting corynebacteria.

Granulibacter bethesdensis isolated in a child patient with chronic granulomatous disease.

Chronic granulomatous disease was diagnosed in a 10-year-old boy who, after some weeks' decline, developed acute respiratory distress leading to multiple organ failure and death. All microbiological analyses were negative, except two blood cultures from which the novel bacterium Granulibacter bethesdensis was isolated.

The isolation of Corynebacterium coyleae from clinical samples: clinical and microbiological data.

Fourteen Corynebacterium coyleae isolates were recovered from 12 in-patients during a 5-years period. In six patients, the isolates were considered as clinically significant, three definite (sepsis), two probable (sepsis and soft tissue infection), and one possible (post-transfusional bacteremia). In the remaining 6 patients (all neonatal bacteremias), there was not enough data for considering the isolates as clinical significant. API Coryne identified all isolates as C. jeikeium, while Biolog GP2 correctly identified 7 out of the 14 isolates. Definitive identification was achieved in all isolates by the sequencing of a fragment of 724 to 1423 pb of 16S rDNA. Successive isolations from two patients presented identical random amplified polymorphic DNA (RAPD) profiles. All of the isolates were in-vitro-sensitive to beta-lactams, gentamicin, rifampin, tetracycline, vancomycin, linezolid, and resistant to clindamycin. Resistance to erythromycin occurred in 83.3% of isolates, all of them presenting phenotype cMLS and harboring the gene ermX.

Antimicrobial resistance and phage and molecular typing of Salmonella strains isolated from food for human consumption in Spain.

The aims of this study were to ascertain the population structure and antimicrobial susceptibility of Salmonella enterica serovars isolated in 2002 from food in 16 Spanish regions. Serovars were characterized by serotyping, phage typing, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) typing, and 264 nonrelated strains were selected for further analysis. The main sources were eggs and their derivatives (21.6% of strains), poultry and related products (16.6%), and seafood (16.3%). High serotype diversity was detected (51 serotypes); the most common were Enteritidis (n = 96, 36.3%) and Typhimurium (n = 53, 20.1%), followed by a miscellaneous group of 49 different serotypes (n = 115, 43.5%). A 15% increase in Salmonella Enteritidis isolation was observed. Common phage types for Salmonella Enteritidis were PT1 (41.6% of isolates), PT4 (9.4%), PT6 (9.4%), and PT6a (9.4%), and common types for Salmonella Typhimurium were DTU302 (18.8%), DT104 (15.1%), and DT104B (13.2%). Salmonella Enteritidis strains were categorized into eight PFGE types with a similarity of 81 to 96%, and 73.9% of the strains were grouped into just one cluster. Salmonella Typhimurium isolates were divided into 13 PFGE types with a similarity of 64 to 86%, and one predominant clone contained 41.5% of the strains. Resistance rates for Salmonella Enteritidis, Salmonella Typhimurium, and the miscellaneous group were, respectively, 8.3, 69.8, and 13.9% for ampicillin, 3.1, 52.8, and 59% for streptomycin, 40.6, 22.6, and 10.4% for nalidixic acid, 15.6, 71.7, and 31.1% for tetracycline, 7.3, 18.8, and 9.5% for trimethoprim-sulfamethoxazole, 0, 50.9, and 4.3% for chloramphenicol, and 6.2, 71.7, and 17.4% for multiple (at least four) antimicrobials. All the strains remained susceptible to other beta-lactams and fluoroquinolones. Surveillance of S. enterica isolated from food is strongly recommended to reduce community exposure to antimicrobial resistant strains.

Characterisation of human outbreaks of brucellosis and sporadic cases by the use of hyper-variable octameric oligonucleotide fingerprint (HOOF) variable number tandem repeats.

Hyper-variable octameric oligonucleotide fingerprints (HOOFs) enable typing of Brucella spp. by targeting the 8-bp tandem repeat in eight loci that vary in number (variable number tandem repeats; VNTRs). Brucella is one of the most important zoonotic pathogens, because of its public health and economic consequences. To assess the role of HOOFs as epidemiological markers for Brucella melitensis, which is the main species involved in human brucellosis in Spain, 87 sporadic and outbreak isolates were investigated; these originated from broad or more restricted geographical locations, including unrelated (n = 42), semi-related (n = 19) and closely related (n = 26) groups of isolates. Distinct HOOFs were detected in the entire (n = 74), unrelated (n = 42), semi-related (n = 19) and closely related (n = 13) groups. Seven of the eight VNTR markers investigated identified multiple alleles in the four groups of isolates. Using the composite data for eight VNTRs, a diversity value of 0.98 was calculated for the entire population, taking into account single- and double-locus variants. A high correlation (R = 0.98) between the maximum copy number and the number of alleles was observed. The most polymorphic markers were VNTR-1, VNTR-4, VNTR-5 and VNTR-7 (D > OR = 0.8). Characterisation of B. melitensis isolates by HOOFs enabled the recognition of related human cases and the exchange of molecular epidemiological information concerning a spreading clone, thus improving brucellosis surveillance.

Epidemiological tracing of Salmonella enterica serotype Abortusovis from Spanish ovine flocks by PFGE fingerprinting.

Salmonella enterica serotype Abortusovis, an ovine host-specific serotype, rare in most countries, is responsible for epidemic abortion episodes in Spain. With the aim of surveillance and detection of the spread of specific clones, 55 Abortusovis isolates collected during 1996-2001 from flocks in 11 provinces, were studied using XbaI-PFGE. Despite the fact that the strains were geographically and spatially related, PFGE demonstrated an epidemiologically acceptable discriminating power, identifying 20 clones (similarity, 52-96%). Clones Sabv6, 1, 5 were disseminated in seven, five and two areas respectively, while another 17 clones appeared in single places. Clones from nearby geographic regions showed a high relatedness (one band of difference in the PFGE profile) Sabv1-2-3, Sabv5-6, Sabv7-8, and Sabv13-14, suggesting a common ancestor. Co-isolation in the same flock (Sabv5-6, Sabv1-3, Sabv1-6) was detected. PFGE surveillance detected the predominance and widespread distribution of clone Sabv6 in 21 out of the 55 Abortusovis serotype episodes studied in Spain.

Prevalence and evolution of methicillin-resistant Staphylococcus aureus in Spanish hospitals between 1996 and 2002.

Pulsed-field gel electrophoretic analysis of 2,144 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients in Spanish hospitals over a 7-year period revealed 17 predominant profiles. Typing showed the replacement of Iberian clone E1 (ST247-MRSA-I) by two prevalent clones, E7 and E8, that are closely related to each other and have the same genetic background as ST125-MRSA-IV.

Outbreak of long-term intravascular catheter-related bacteremia due to Achromobacter xylosoxidans subspecies xylosoxidans in a hemodialysis unit.

Achromobacter xylosoxidans is a rare cause of bacteremia. Over a 2-week period, A. xylosoxidans subsp. xylosoxidans was isolated from blood cultures of four hemodialysis patients with long-term intravascular catheters. A culture from one atomizer that contained diluted 2.5% chlorhexidine, which had been used to disinfect the skin, yielded A. xylosoxidans subsp. xylosoxidans. No further cases were diagnosed once the use of this atomizer was discontinued. Five outbreak-related strains from the four patients and the atomizer were tested by pulsed-field gel electrophoresis (PFGE) under XbaI restriction. The isolates from the first three patients and the atomizer had identical PFGE patterns, confirming the atomizer as the source of the outbreak. The strain isolated from the fourth patient had six more bands than the outbreak strain and was considered possibly related to the outbreak strain. All patients were treated with intravenous levofloxacin. The catheter was removed in only one patient. The three patients in whom the catheter was left in place were also treated with antibiotic lock therapy with levofloxacin. All four patients were cured. This is believed to be the first reported outbreak of central venous catheter-related bacteremia due to A. xylosoxidans and the second reported outbreak with this organism associated with chlorhexidine atomizers. The use of diluted chlorhexidine via atomizers can be dangerous for the care of venous catheters and should be called into question. Patients with long-term intravascular catheter-related bacteremia due to this organism can be treated successfully with systemic antimicrobial therapy in addition to antibiotic lock therapy without catheter removal.