Juan Marzoa

Four Main Virotypes among Extended-Spectrum-β-Lactamase-Producing Isolates of Escherichia coli O25b:H4-B2-ST131: Bacterial, Epidemiological, and Clinical Characteristics

ABSTRACT A total of 1,021 extended-spectrum-β-lactamase-producing Escherichia coli (ESBLEC) isolates obtained in 2006 during a Spanish national survey conducted in 44 hospitals were analyzed for the presence of the O25b:H4-B2-ST131 (sequence type 131) clonal group. Overall, 195 (19%) O25b-ST131 isolates were detected, with prevalence rates ranging from 0% to 52% per hospital. Molecular characterization of 130 representative O25b-ST131 isolates showed that 96 (74%) were positive for CTX-M-15, 15 (12%) for CTX-M-14, 9 (7%) for SHV-12, 6 (5%) for CTX-M-9, 5 (4%) for CTX-M-32, and 1 (0.7%) each for CTX-M-3 and the new ESBL enzyme CTX-M-103. The 130 O25b-ST131 isolates exhibited relatively high virulence scores (mean, 14.4 virulence genes). Although the virulence profiles of the O25b-ST131 isolates were fairly homogeneous, they could be classified into four main virotypes based on the presence or absence of four distinctive virulence genes: virotypes A (22%) (afa FM955459 positive, iroN negative, ibeA negative, sat positive or negative), B (31%) (afa FM955459 negative, iroN positive, ibeA negative, sat positive or negative), C (32%) (afa FM955459 negative, iroN negative, ibeA negative, sat positive), and D (13%) (afa FM955459 negative, iroN positive or negative, ibeA positive, sat positive or negative). The four virotypes were also identified in other countries, with virotype C being overrepresented internationally. Correspondingly, an analysis of XbaI macrorestriction profiles revealed four major clusters, which were largely virotype specific. Certain epidemiological and clinical features corresponded with the virotype. Statistically significant virotype-specific associations included, for virotype B, older age and a lower frequency of infection (versus colonization), for virotype C, a higher frequency of infection, and for virotype D, younger age and community-acquired infections. In isolates of the O25b:H4-B2-ST131 clonal group, these findings uniquely define four main virotypes, which are internationally distributed, correspond with pulsed-field gel electrophoresis (PFGE) profiles, and exhibit distinctive clinical-epidemiological associations.

Poultry as reservoir for extraintestinal pathogenic Escherichia coli O45:K1:H7-B2-ST95 in humans

Escherichia coli strains O45:K1:H7 are implicated in severe human infections such as meningitis. Since an increasing prevalence of serogroup O45 among avian pathogenic (APEC) and human extraintestinal pathogenic (ExPEC) E. coli strains isolated in Spain have been noticed, the aims of the present study were to investigate similarities between poultry and human O45 isolates, and to investigate the evolutionary relationship of ST95 types. The genetic relatedness and virulence gene profiles of 55 O45 APEC obtained from an avian colibacillosis collection (1991-2011) and 19 human O45 ExPEC from a human septicemic/uropathogenic (UPEC) E. coli collection (1989-2010) were determined by multilocus sequence typing (MLST), pulsed-field-gel-electrophoresis (PFGE), ECOR phylogrouping, and PCR-based genotyping. Two main clonal groups were established. The most prevalent and highly pathogenic O45:K1:H7-B2-ST95 shows a successful persistence since the 90s to the present, with parallel evolution both in human and poultry, on the basis of their PFGE and virulence gene profile similarities (9 human strains and 15 avian strains showed ≥85% PFGE identity). Comparison of this group with other ST95 closely related members (O1:K1:H7 and O18:K1:H7 isolates from our collections) shows pathogenic specialization through conserved virulence genotypes. The other prevalent O45 clonal group characterized in this study, the O45:HNM/H19-D-ST371/ST2676 was only detected in APEC strains suggesting host specificity. In conclusion, poultry could be acting as a reservoir of O45:K1:H7-B2-ST95 and other pathogenic ST95 serotypes in humans. Further studies would be necessary to clarify if pathogenic mechanisms used by ST95 strains are the same in avian and human hosts.

Detection of quinolone-resistant Escherichia coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona, Spain

OBJECTIVES The present study was carried out to evaluate the prevalence of clonal group O25b:H4-B2-ST131 in water environments with faecal pollution (urban sewage and river water) in the north-east of Spain and to study the virulence gene content of environmental isolates and to compare them with isolates causing human extraintestinal infections in Spain. METHODS This study was performed with 10 sewage samples (collected in Catalonia, north-eastern Spain, in autumn 2009 from the influent raw urban sewage of a wastewater treatment plant that serves a large urban area) and 6 river water samples (collected monthly from February to April 2010 in the Llobregat river catchment area, near Barcelona, a watercourse subjected to heavy anthropogenic pressure). Escherichia coli colonies were screened by PCR for the rfbO25b gene associated with the clonal group O25b:H4-B2-ST131. Sequence types (STs), serotypes, virulence genes, PFGE profiles, antimicrobial resistance and extended-spectrum β-lactamase (ESBL) enzymes were determined in 75 E. coli isolates positive for the O25b molecular subtype. RESULTS Of the 75 O25b-positive isolates, 51 belonged to the O25b:H4-B2-ST131 clonal group and the remaining 24 belonged to clonal group O25b:H4-D-ST69. The majority of ST69 isolates (23 of 24) were isolated from urban sewage, whereas ST131 isolates were isolated from urban sewage (25 isolates) as well as from river water (26 isolates). ST131 and ST69 isolates carried 4-13 virulence genes, the majority (82%) being quinolone resistant. CONCLUSIONS We showed the presence of E. coli isolates belonging to clonal groups O25b:H4-B2-ST131 and O25b:H4-D-ST69 in raw sewage and river water in Barcelona. Furthermore, we observed that the environmental O25b:H4-B2-ST131 isolates showed similar virulence and macrorestriction profiles to clinical human isolates. To our knowledge, this is the first study describing the O25b:H4-D-ST69 clonal group.

Emergence of new variants of ST131 clonal group among extraintestinal pathogenic Escherichia coli producing extended-spectrum β-lactamases

Having shown that Lucus Augusti Hospital in Lugo, Spain, has been affected by Escherichia coli clone O25:H4-ST131 producing CTX-M-15, the present study was carried out to evaluate the prevalence of this clone among the extended-spectrum β-lactamase (ESBL)-producing E. coli isolates and to identify novel variants of this clone. Of the 77 ESBL-producing E. coli isolated between January and April 2012, 47 (61%) were identified as belonging to the ST131 clonal group, comprising 38 O25b:H4-B2-ST131 (34 CTX-M-15, 2 CTX-M-14, 1 CTX-M-1 and 1 CTX-M-27), 7 O-non-typeable:H4-B2-ST131 (all CTX-M-15) and 2 O16:H5-B2-ST131 (both CTX-M-14). The 47 isolates of ST131 exhibited a significantly higher virulence score (mean of 9.1 virulence genes) compared with the 30 non-ST131 isolates (mean of 4.3 virulence genes). A new virulence profile (fimH, papG II, sat, cnf1, hlyA, iucD, kpsM II-K5, traT, malX, usp) was detected among O25b:H4-B2-ST131 isolates belonging to the new Pasteur sequence type PST621. To our knowledge, this is the first study to report the O-non-typeable:H4-B2-ST131 and O16:H5-B2-ST131 variants in Europe.

Virulence Patterns in a Murine Sepsis Model of ST131 Escherichia coli Clinical Isolates Belonging to Serotypes O25b:H4 and O16:H5 Are Associated to Specific Virotypes

Escherichia coli sequence type (ST)131 is an emerging disseminated public health threat implicated in multidrug-resistant extraintestinal infections worldwide. Although the majority of ST131 isolates belong to O25b:H4 serotype, new variants with different serotypes, STs using the discriminative multilocus sequence typing scheme of Pasteur Institute, and virulence-gene profiles (virotypes) have been reported with unknown implications on the pattern of spread, persistence and virulence. The aim of the present study was to compare virulence in a mouse subcutaneous sepsis model of representative ST131 clinical isolates belonging to 2 serotypes (O25b:H4, O16:H5) and nine virotypes and subtypes (A, B, C, D1, D2, D3, D4, D5 and E). Fourteen out of the 23 ST131 isolates tested (61%) killed 90 to 100% of mice challenged, and 18 of 23 (78%) at least 50%. Interestingly, different virulence patterns in association with virotypes were observed, from highly rapid lethality (death in less than 24 h) to low final lethality (death at 7 days) but with presence of an acute inflammation. This is the first study to assess virulence of ST131 isolates belonging to serotype O16:H5, which exhibited virotype C. In spite of their low virulence-gene score, O16:H5 isolates did not show significant differences in final lethality compared with highly virulent O25b:H4 isolates of virotypes A, B and C, but killed mice less rapidly. Significant differences were found, however, between virotypes A, B, C (final lethality ≥80% of mice challenged) and virotypes D, E. Particularly unexpected was the low lethality of the newly assigned virotype E taking into account that it exhibited high virulence-gene score, and the same clonotype H30 as highly virulent O25b:H4 isolates of virotypes A, B and C. In vivo virulence diversity reported in this study would reflect the genetic variability within ST131 clonal group evidenced by molecular typing.

Molecular epidemiology and virulence of Escherichia coli O16: H5-ST131: Comparison with H30 and H30-Rx subclones of O25b: H4-ST131

The present study was carried out to evaluate the prevalence of the clonal subgroup O16:H5-ST131 and the H30 and H30-Rx subclones among E. coli isolates causing extraintestinal infections and to know their virulence potential. The ST131 clonal group accounted for 490 (16%) of the 2995 isolates obtained from clinical samples in five Spanish hospitals during the study period (2005-2012). Among those 490 ST131 isolates, 456 belonged to serotype O25b:H4, 27 to O16:H5 and seven were O-non-typeable:H4 (ONT:H4). All 27 O16:H5 isolates showed fimH41, whereas fimH30 and fimH22 alleles were the most frequently detected among O25b:H4 isolates. The majority (381/490; 78%) of ST131 isolates belonged to H30 subclone, and 302 of 381 (79%) H30 isolates belonged to the H30-Rx subclone. Of the 27 O16:H5 isolates, 48% produced CTX-M-14; however, none produced CTX-M-15. In contrast, 46% of O25b:H4 isolates produced CTX-M-15 while only 2% produced CTX-M-14. More than a half of the O16:H5 isolates (56%) showed the ExPEC status which was significantly more prevalent within O25b:H4 isolates (81%) (P<0.01), especially among H30-Rx (97%) isolates. In the present study, a modified virotype scheme was applied within which approximately half (52%) of the O16:H5 isolates showed the C1 specific virotype. Despite their low virulence-gene score (mean of virulence genes 6.4 versus 8.5 in O25b:H4 isolates), six out of the 10 O16:H5 isolates assayed showed high virulence in the mouse model of sepsis (killed 90-100% of mice challenged). Furthermore, four O16:H5 isolates of virotypes A and C1, carrying K2 variant of group II capsule, showed lethality at 24h. Thus, certain O16:H5 fimH41 isolates show a similar in vivo virulence to that reported with the highly virulent O25b:H4 H30-Rx isolates (Mora et al., PLOS ONE 2014, e87025), supporting their potential virulence for humans.

Induction of immune responses by purified outer membrane protein complexes from Neisseria meningitidis

A broad-spectrum vaccine against disease caused by serogroup B of Neisseria meningitidis is still a challenge due to antigenic variability. In the present study outer membrane protein complexes and their components were analysed using non-denaturing 2D electrophoresis and identified using LC/MS-MS and MALDI-TOF. Outer membrane protein complexes were purified from both the wild-type strain H44/76 and their knock-out mutants lacking PorA, PorB, RmpM or FetA. The immune responses elicited by the whole outer membrane vesicles (OMV) and the purified complexes were analysed for bactericidal activity, antibody surface binding, antibody-mediated C3b/iC3b deposition, membrane attack complex (MAC) deposition and induction of opsonophagocytosis, both on the homologous and several heterologous strains. The main antigenic complexes found were homomeric, formed by the 60 kDa chaperonin (MSP63) or PorB, or heteromeric, formed by different combinations of PorA, PorB and/or RmpM. The lack of some of these proteins in the OMVs from the knock-out mutants did not affect significantly the immune responses analysed except MAC, which was significantly reduced in the anti-PorA- and anti-PorB- sera, and bactericidal activity, which was absent in the anti-PorA- serum. The sera against purified native complexes showed variable activities against the homologous strain, with greatest responses observed for anti-chaperonin and anti-PorA/PorB/RmpM sera. When tested against heterologous strains, the only anti-complex serum showing consistent responses was that against the 60 kDa chaperonin. The comparison of the responses elicited by the different sera suggests an important role of conformational epitopes, present only in native complexes, in the induction of more effective responses against N. meningitidis.

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1‐D blue native (BN) electrophoresis, 2‐D BN/SDS‐PAGE and 2‐D diagonal electrophoresis, followed by LC/MS‐MS (for 1‐D gels) or MALDI‐TOF (for 2‐D gels) revealed at least six porin complexes in the wild‐type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild‐type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (≈︂800 kDa). Our results suggest that PorA and PorB could form a ‘basic’ template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.

Diversity of Shiga toxin-producing Escherichia coli in sheep flocks of Paraná State, southern Brazil

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep.

Characterisation and immune responses to meningococcal recombinant porin complexes incorporated into liposomes

We have analysed the structure of meningococcal outer membrane complexes and found that the main complexes are formed by different combinations of PorA and/or PorB molecules, associated to other proteins such as RmpM. In view of the growing knowledge of the importance of conformational epitopes in the immune responses to many pathogens, our aim in this study was to analyse the interactions of PorA and PorB by reconstitution of both recombinant porins into liposomes and determine the relevance of these interactions for the immune response. Recombinant PorA and PorB incorporated into liposomes associate forming complexes that are homomeric when only one of the porins is present, but heteromeric when both neisserial porins are present, mimicking those found previously in native outer membrane vesicles (OMVs). Association of PorA and PorB to form heterocomplexes modifies the immunogenicity of at least PorB, allowing the production of antibodies that recognise conformational epitopes, and produces new epitopes that react with a 50 kDa outer membrane protein not yet identified.