Enteroaggregative Escherichia coli as etiological agent of endemic diarrhea in Spain: A prospective multicenter prevalence study with molecular characterization of isolates.

Background: Enteroaggregative Escherichia coli (EAEC) is increasingly associated with domestically acquired diarrheal episodes in high-income countries, particularly among children. However, its specific role in endemic diarrhea in this setting remains under-recognized and information on molecular characteristics of such EAEC strains is limited. We aimed to investigate the occurrence of EAEC in patients with non-travel related diarrhea in Spain and molecularly characterize EAEC strains associated with illness acquired in this high-income setting. Methods: In a prospective multicenter study, stool samples from diarrheal patients with no history of recent travel abroad (n = 1,769) were collected and processed for detection of EAEC and other diarrheagenic E. coli (DEC) pathotypes by PCR. An additional case-control study was conducted among children ≤5 years old. Whole-genome sequences (WGS) of the resulting EAEC isolates were obtained. Results: Detection of DEC in the study population. DEC was detected in 23.2% of patients aged from 0 to 102 years, with EAEC being one of the most prevalent pathotypes (7.8%) and found in significantly more patients ≤5 years old (9.8% vs. 3.4%, p < 0.001). Although not statistically significant, EAEC was more frequent in cases than in controls. WGS-derived characterization of EAEC isolates. Sequence type (ST) 34, ST200, ST40, and ST10 were the predominant STs. O126:H27, O111:H21, and O92:H33 were the predominant serogenotypes. Evidence of a known variant of aggregative adherence fimbriae (AAF) was found in 89.2% of isolates, with AAF/V being the most frequent. Ten percent of isolates were additionally classified as presumptive extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), or both, and belonged to clonal lineages that could be specifically associated with extraintestinal infections. Conclusion: EAEC was the only bacterial enteric pathogen detected in a significant proportion of cases of endemic diarrhea in Spain, especially in children ≤5 years old. In particular, O126:H27-ST200, O111:H21-ST40, and O92:H33-ST34 were the most important subtypes, with all of them infecting both patients and asymptomatic individuals. Apart from this role as an enteric pathogen, a subset of these domestically acquired EAEC strains revealed an additional urinary/systemic pathogenic potential.

Infección mixta por cuatro patotipos diarreagénicos de Escherichia coli en un caso de diarrea del viajero: caracterización de los aislados obtenidos mediante secuenciación del genoma completo / Mixed infection of four diarrhoeagenic Escherichia coli pathotypes in a case of travellers' diarrhoea: Caracterisation of the isolates by whole-genome sequencing

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Evaluation of the SHIGA TOXIN QUIK CHEK after overnight enrichment as screening tool for Shiga toxin-producing Escherichia coli detection in human fecal samples.

We evaluated the SHIGA TOXIN QUIK CHEK (STQC) on its suitability for Shiga toxin-producing Escherichia coli (STEC) testing on human fecal samples after overnight enrichment. Our in-house PCR-based protocol for STEC detection was used as the standard for comparison. STQC detected all described Shiga toxin subtypes with the only exception of Stx2f. In comparison to PCR, STQC performed with an overall sensitivity of 55.4%, specificity of 100.0%, positive predictive value of 100.0%, negative predictive value of 73.0%, infinite positive likelihood ratio, and negative likelihood ratio of 0.45. We conclude that STQC may not be considered a suitable screening tool for STEC detection in human fecal samples, although it could be useful for laboratories where PCR is not a routine tool for STEC screening yet, subject to the confirmation of negative samples by a reference laboratory with full diagnostic capabilities.

Mucus-Activatable Shiga Toxin Genotype stx2d in Escherichia coli O157:H7.

We identified the mucus-activatable Shiga toxin genotype stx2d in the most common hemolytic uremic syndrome-associated Escherichia coli serotype, O157:H7. stx2d was detected in a strain isolated from a 2-year-old boy with bloody diarrhea in Spain, and whole-genome sequencing was used to confirm and fully characterize the strain.

The spread of KPC-producing Enterobacteriaceae in Spain: WGS analysis of the emerging high-risk clones of Klebsiella pneumoniae ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3.

OBJECTIVES: We analysed the microbiological traits and population structure of KPC-producing Enterobacteriaceae isolates collected in Spain between 2012 and 2014. We also performed a comparative WGS analysis of the three major KPC-producing Klebsiella pneumoniae clones detected. METHODS: Carbapenemase and ESBL genes were sequenced. The Institut Pasteur MLST scheme was used. WGS data were used to construct phylogenetic trees, to identify the determinants of resistance and to de novo assemble the genome of one representative isolate of each of the three major K. pneumoniae clones. RESULTS: Of the 2443 carbapenemase-producing Enterobacteriaceae isolates identified during the study period, 111 (4.5%) produced KPC. Of these, 81 (73.0%) were K. pneumoniae and 13 (11.7%) were Enterobacter cloacae. Three major epidemic clones of K. pneumoniae were identified: ST11/KPC-2, ST101/KPC-2 and ST512/KPC-3. ST11/KPC-2 differed from ST101/KPC-2 and ST512/KPC-3 by 27 819 and 6924 SNPs, respectively. ST101/KPC-2 differed from ST512/KPC-3 by 28 345 SNPs. Nine acquired resistance genes were found in ST11/KPC-2, 11 in ST512/KPC-3 and 13 in ST101/KPC-2. ST101/KPC-2 had the highest number of virulence genes (20). An 11 bp deletion at the end of the mgrB sequence was the cause of colistin resistance in ST512/KPC-3. CONCLUSIONS: KPC-producing Enterobacteriaceae are increasing in Spain. Most KPC-producing K. pneumoniae isolates belonged to only five clones: ST11 and ST512 caused interregional spread, ST101 caused regional spread and ST1961 and ST678 produced independent hospital outbreaks. ST101/KPC-2 had the highest number of resistance and virulence genes. ST101/KPC-2 and ST512/KPC-3 were recently implicated in the spread of KPC in Italy.

First report of Babesia divergens infection in an HIV patient.

Human babesiosis is a zoonosis primarily transmitted through Ixodes ticks and alternatively by routes such as blood transfusions from asymptomatic donors. We report the first case of human babesiosis caused by Babesia divergens in a patient with HIV. This study also focuses on elucidating the possible transmission route of infection in this patient, who received numerous blood transfusions but showed patent symptoms only after splenectomy. A battery of detection tools along with a novel Western-Blot Assay and Enzyme Linked Immunosorbent Assay using the major surface protein of B. divergens (Bd37) as a target were used to evaluate the presence of B. divergens or antibodies against the parasite in samples from the patient and the blood donors involved in this case. A retrospective study of the humoral status against the parasite revealed B. divergens IgG antibodies in one of the implicated donors, but also showed that the patient had been already exposed to the parasite before any transfusion. Thus, this analysis of natural and transfusion transmission routes suggests a pre-existing subclinical babesiosis in the patient.

The efficacy of the ultraviolet C pathogen inactivation system in the reduction of Babesia divergens in pooled buffy coat platelets.

BACKGROUND: Babesia spp. is an intraerythrocytic parasite that causes human babesiosis and its transmission by transfusion has been extensively demonstrated. The aim of this study was to ascertain the efficacy of an ultraviolet C (UVC)-based pathogen inactivation system in the reduction of Babesia divergens-infected platelet (PLT) concentrates and to determine the parasite's ability to survive in PLT concentrates stored under blood bank conditions. STUDY DESIGN AND METHODS: This study was conducted using in vitro cultures of B. divergens. The detection limit of the culture assay was established and, subsequently, 15 buffy coat-derived PLT concentrates (BC-PCs) were inoculated with 10(7) B. divergens-infected red blood cells. Infected BC-PCs were irradiated with 0.2 J/cm(2) UVC light using the THERAFLEX UV-Platelets method (Macopharma). Viability and parasite growth were evaluated before and after inactivation. Culture growth kinetics were monitored by DNA incorporation of [(3) H]thymidine. The ability of B. divergens to survive in PLT concentrates was also analyzed. RESULTS: The limit of detection in cultures was established at 0.1 × 10(-6) % parasites. The THERAFLEX UV-Platelets system inactivated B. divergens to below the limit of detection in 12 of 15 BC-PCs (log reduction, >6.0) and to the limit of detection (log reduction, 5.0) in three of 15. It was also demonstrated that B. divergens remains viable in BC-PCs stored up to 7 days. CONCLUSION: Since B. divergens can survive in PLT concentrates and given the performance of UVC, this system could be considered as an alternative to prevent B. divergens and other Babesia species from being transmitted through PLT transfusions.

Genetic variability of the 18 kDa/HP6 protective antigen in Taenia saginata and Taenia asiatica: implications for vaccine development.

Genomic characterization of the genes encoding the Taenia 18 kDa/HP6 protective antigens was carried out for Taenia saginata and T. asiatica using 42 taeniid isolates comprising 23 samples of T. saginata, 13 samples of T. asiatica and 6 samples of T. solium. The corresponding sequences from all taeniid isolates were PCR-amplified with specific primers and then sequenced. All the genes, and other described taeniid gene homologues, had the same genomic structure. Surprisingly, the T. saginata TSA18 gene showed nucleotide variability within the 23 samples analyzed. This resulted in two distinct genotypes with 96% DNA sequence similarity and deduced amino acid sequences with 21 substitutions, mainly located in the second exon which contains the fibronectin type III domain. In regards to T. asiatica, the 18 kDa gene (TASI18) was very similar to the T. saginata antigen homologues, both at the DNA and deduced amino acid sequence levels, and the TSOL18 gene was conserved among T. solium isolates as previously described. The implications of these findings on the future development of taeniid vaccines are discussed.

Blind comparison of traditional serotyping with three multiplex PCRs for the identification of Salmonella serotypes.

Salmonella serotypes are defined on the basis of somatic (O) antigens which define the serogroup and flagellar (H) factor antigens, both of which are present in the cell wall of Salmonella. Most Salmonella organisms alternatively express phase-1 or phase-2 flagellar antigens encoded by fliC and fljB genes, respectively. Our group previously published two multiplex PCRs for distinguishing the most common first- and second-phase antigens. In this paper we describe a third multiplex PCR to identify the most common serogroups (O:B; O:C1; O:C2; O:D and O:E). The combination of these three PCRs enabled us to completely serotype organisms belonging to the Salmonella species. This multiplex PCR includes 10 primers. A total of 67 Salmonella strains belonging to 32 different serotypes were tested. Each strain generated one serogroup-specific fragment ranging between 162 and 615bp. Twenty-eight strains belonging to 21 serotypes, with a serogroup different from those tested in this work, did not generate any fragments. To compare molecular serotyping with traditional serotyping, 500 strains, received according to the order of arrival in the laboratory, were serotyped using both methods. The three multiplex PCRs were able to serotype 84.6% of the tested strains. This method was found to be very helpful in our laboratory as an alternative method for typing strains causing outbreaks, and it can be used to supplement conventional serotyping, since it is also applicable to motionless and rough strains.