Pilar Villalón

Clonal Diversity of Nosocomial Epidemic Acinetobacter baumannii Strains Isolated in Spain

ABSTRACT Acinetobacter baumannii is one of the major pathogens involved in nosocomial outbreaks. The clonal diversity of 729 epidemic strains isolated from 19 Spanish hospitals (mainly from intensive care units) was analyzed over an 11-year period. Pulsed-field gel electrophoresis (PFGE) identified 58 PFGE types that were subjected to susceptibility testing, rpoB gene sequencing, and multilocus sequence typing (MLST). All PFGE types were multidrug resistant; colistin was the only agent to which all pathogens were susceptible. The 58 PFGE types were grouped into 16 clones based on their genetic similarity (cutoff of 80%). These clones were distributed into one major cluster (cluster D), three medium clusters (clusters A, B, and C), and three minor clusters (clusters E, F, and G). The rpoB gene sequencing and MLST results reflected a clonal distribution, in agreement with the PFGE results. The MLST sequence types (STs) (and their percent distributions) were as follows: ST-2 (47.5%), ST-3 (5.1%), ST-15 (1.7%), ST-32 (1.7%), ST-79 (13.6%), ST-80 (20.3%), and ST-81 (10.2%). ST-79, ST-80, and ST-81 and the alleles cpn60-26 and recA29 are described for the first time. International clones I, II, and III were represented by ST-81, ST-2, and ST-3, respectively. ST-79 and ST-80 could be novel emerging clones. This work confirms PFGE and MLST to be complementary tools in clonality studies. Here PFGE was able to demonstrate the monoclonal pattern of most outbreaks, the inter- and intrahospital transmission of bacteria, and their endemic persistence in some wards. MLST allowed the temporal evolution and spatial distribution of Spanish clones to be monitored and permitted international comparisons to be made.

Emergence in Spain of a Multidrug-Resistant Enterobacter cloacae Clinical Isolate Producing SFO-1 Extended-Spectrum β-Lactamase

ABSTRACT Between February 2006 and October 2009, 38 patients in different wards at the A Coruña University Hospital (northwest Spain) were either infected with or colonized by an epidemic, multidrug-resistant (MDR), and extended-spectrum-β-lactamase (ESBL)-producing strain of Enterobacter cloacae (EbSF), which was susceptible only to carbapenems. Semiautomated repetitive extragenic palindromic sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE) analysis revealed that all of the E. cloacae isolates belonged to the same clone. Cloning and sequencing enabled the detection of the SFO-1 ESBL in the epidemic strain and the description of its genetic environment. The presence of the ampR gene was detected upstream of bla SFO-1, and two complete sequences of IS26 surrounding ampR and ampA were detected. These IS26 sequences are bordered by complete left and right inverted repeats (IRL and IRR, respectively), which suggested that they were functional. The whole segment flanked by two IS26 copies may be considered a putative large composite transposon. A gene coding for aminoglycoside acetyltransferase (gentamicin resistance gene [aac3]) was found downstream of the 3′ IS26. Despite the implementation of strict infection control measures, strain EbSF spread through different areas of the hospital. A case-control study was performed to assess risk factors for EbSF acquisition. A multivariate analysis revealed that the prior administration of β-lactam antibiotics, chronic renal failure, tracheostomy, and prior hospitalization were statistically associated with SFO-1-producing E. cloacae acquisition. This study describes for the first time an outbreak in which an SFO-1-producing E. cloacae strain was involved. Note that so far, this β-lactamase has previously been isolated in only a single case of E. cloacae infection in Japan.

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.

Molecular epidemiology, antimicrobial susceptibilities and resistance mechanisms of Streptococcus pyogenes isolates resistant to erythromycin and tetracycline in Spain (1994–2006)

BackgroundGroup A Streptococcus (GAS) causes human diseases ranging in severity from uncomplicated pharyngitis to life-threatening necrotizing fasciitis and shows high rates of macrolide resistance in several countries. Our goal is to identify antimicrobial resistance in Spanish GAS isolates collected between 1994 and 2006 and to determine the molecular epidemiology (emm/T typing and PFGE) and resistance mechanisms of those resistant to erythromycin and tetracycline.ResultsTwo hundred ninety-five out of 898 isolates (32.8%) were erythromycin resistant, with the predominance of emm 4T4, emm 75T25, and emm 28T28, accounting the 67.1% of the 21 emm/T types. Spread of emm 4T4, emm 75T25 and emm 28T28 resistant clones caused high rates of macrolide resistance. The distribution of the phenotypes was M (76.9%), cMLSB (20.3%), iMLSB (2.7%) with the involvement of the erythromycin resistance genes mef(A) (89.5%), msr(D) (81.7%), erm(B) (37.3%) and erm(A) (35.9%).Sixty-one isolates were tetracycline resistant, with the main representation of the emm 77T28 among 20 emm/T types. To note, the combination of tet(M) and tet(O) tetracycline resistance genes were similar to tet(M) alone reaching values close to 40%. Resistance to both antibiotics was detected in 19 isolates of 7 emm/T types, being emm 11T11 and the cMLSB phenotype the most frequent ones. erm(B) and tet(M) were present in almost all the strains, while erm(A), mef(A), msr(D) and tet(O) appeared in less than half of them.ConclusionsSpanish GAS were highly resistant to macrolides meanwhile showed minor resistance rate to tetracycline. A remarkable correlation between antimicrobial resistance and emm/T type was noticed. Clonal spread of emm 4T4, emm 75T25 and emm 28T28 was the main responsable for macrolide resistance where as that emm 77T28 clones were it to tetraclycline resistance. A wide variety of macrolide resistance genes were responsible for three macrolide resistance phenotypes.

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.

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.

Endemic and epidemic Acinetobacter baumannii clones: a twelve-year study in a tertiary care hospital

BackgroundNosocomial outbreaks of multidrug-resistant Acinetobacter baumannii are of worldwide concern. Using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and multiple locus variable number tandem repeat sequence (VNTR) analysis (MLVA), the present work examines the genetic diversity of the endemic and epidemic A. baumannii clones isolated in a single hospital over a twelve-year period.ResultsPFGE analysis of 405 A. baumannii-calcoaceticus complex isolates detected 15 A. baumannii endemic/epidemic PFGE types (EE1 to EE15) that grouped into five clusters: EE1-EE8, EE9, EE10, EE11 and EE12-EE15. The MLST sequence type (ST) distributions were: international clone II (ST-2) 60%, international clone III (ST-3) 26.7%, ST-15 6.7%, and ST-80 6.7%. MLVA-8Orsay returned 17 allelic profiles. The large (L) VNTR marker profiles were fully concordant with the detected STs, and concordant with 14 up to 15 PFGE types. Imipenem resistance was detected in five PFGE types; the prevalence of the blaOXA-58-like and blaOXA-40-like genes was 60% and 40% respectively.ConclusionsPFGE proved to be a vital tool for analysis of the temporal and spatial distribution of the clones. MLST and the VNTR L-markers grouped the isolates into clonal clusters. The wide diversity of MLVA small (S)-markers, however, did not permit clustering. The present results demonstrate the persistence of several endemic PFGE types in the hospital, the involvement of some of them in outbreaks, and the inter hospital transmission of extensively drug-resistant ST-15 and ST-80.

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.