Rosario Morales-Espinosa

Pseudomonas aeruginosa clinical and environmental isolates constitute a single population with high phenotypic diversity

BackgroundPseudomonas aeruginosa is an opportunistic pathogen with a high incidence of hospital infections that represents a threat to immune compromised patients. Genomic studies have shown that, in contrast to other pathogenic bacteria, clinical and environmental isolates do not show particular genomic differences. In addition, genetic variability of all the P. aeruginosa strains whose genomes have been sequenced is extremely low. This low genomic variability might be explained if clinical strains constitute a subpopulation of this bacterial species present in environments that are close to human populations, which preferentially produce virulence associated traits.ResultsIn this work, we sequenced the genomes and performed phenotypic descriptions for four non-human P. aeruginosa isolates collected from a plant, the ocean, a water-spring, and from dolphin stomach. We show that the four strains are phenotypically diverse and that this is not reflected in genomic variability, since their genomes are almost identical. Furthermore, we performed a detailed comparative genomic analysis of the four strains studied in this work with the thirteen previously reported P. aeruginosa genomes by means of describing their core and pan-genomes.ConclusionsContrary to what has been described for other bacteria we have found that the P. aeruginosa core genome is constituted by a high proportion of genes and that its pan-genome is thus relatively small. Considering the high degree of genomic conservation between isolates of P. aeruginosa from diverse environments, including human tissues, some implications for the treatment of infections are discussed. This work also represents a methodological contribution for the genomic study of P. aeruginosa, since we provide a database of the comparison of all the proteins encoded by the seventeen strains analyzed.

Helicobacter pylori in the oral cavity is associated with gastroesophageal disease

BACKGROUND In Mexico, more than 80% of the population is infected with Helicobacter pylori. The frequency of H. pylori detection in the oral cavity is unknown, as its relationship with gastroesophageal pathology. AIM To detect the presence of H. pylori in the oral cavity in Mexican population by PCR and to determine its association with gastroesophageal disease. METHODS Patients were divided into two groups with different clinic conditions from whom gastric biopsy, dental plaque, and saliva samples were taken and analyzed. The first group comprised of hospitalized patients, the majority of whom were diagnosed with gastroesophageal disease, while the second group was selected from a dental clinic (ambulatory population) the majority of whom appeared to be healthy subjects. RESULTS H. pylori was detected in gastric biopsy, dental plaque and saliva samples by PCR using a set of specific primers for the signal sequence of the vacuolating cytotoxin gene; detection of H. pylori in general was higher in gastric biopsy and dental plaque samples than in saliva samples. Detection of H. pylori in the oral cavity is significantly (P = 0.0001) associated with patients presenting gastroesophageal disease, while healthy subjects and those with other non-gastric disease do not present with H. pylori in their oral cavity. CONCLUSIONS H. pylori detection in the oral cavity is associated to gastroesophageal disease. In addition, it is suggested that all patients presenting gastric symptoms and H. pylori detection in the oral cavity would begin bacterial treatment immediately.

Bleeding peptic ulcers and presence of Helicobacter pylori by various tests: a case-control study.

Background Virulence factors of Helicobacter pylori are associated with peptic ulcer disease and may be also associated with bleeding peptic ulcers (BPU). Aim To determine whether H. pylori and/or the cytotoxin-associated gene (cagA) can increase the risk of bleeding in peptic ulcers. Patients Sixty-seven patients were studied. Thirty had BPU, 20 had non-bleeding peptic ulcers (NBPU), and 17 were control subjects (NPU). Methods The prevalence of H. pylori was assessed by the urease fast test, histological examination, serology, and 16S ribosomal RNA and cagA gene amplification by polymerase chain reaction (PCR). Results Histology and PCR showed greater sensitivity for diagnosis of H. pylori under bleeding circumstances when compared with other tests. Association of H. pylori was greater in the NBPU group (odds ratio [OR] 4.91, P = 0.06) than in the BPU group (OR 1.27, P = NS) when compared with the control group. When the BPU and NBPU groups were compared, H. pylori was found more often in the NBPU group (OR 0.26, P < 0.10 ). The cagA-positive gene showed a similar distribution in the three groups. The titres for anti-CagA immunoglobulin A (IgA) antibodies were higher in NBPU patients (83%) than in BPU or control patients. Furthermore, anti-urease immunoglobulin G (IgG) was detected more frequently among BPU and NBPU patients. Conclusions NBPU patients had the highest prevalence of H. pylori by PCR. It seems unlikely that either H. pylori or the cagA-positive gene act as significant risk factors for bleeding in peptic ulcers. The lower prevalence of the microorganism among patients who bleed cannot be explained as an artificial finding.

Genetic and phenotypic characterization of a Pseudomonas aeruginosa population with high frequency of genomic islands.

Various genomic islands, PAPI-1, PAPI-2, PAGI-1, PAGI-2, PAGI-3, and PAGI-4, and the element pKLC102 have been characterized in different P. aeruginosa strains from diverse habitats and geographical locations. Chromosomal DNA macroarray of 100 P. aeruginosa strains isolated from 85 unrelated patients hospitalized in an intensive care unit was created to assess the occurrence of these genomic islands (GEIs). The macroarray was then hybridized with labeled probes derived from each genomic island. In addition, PFGE patterns with SpeI, frequency of virulence genes, and antimicrobial resistance patterns of the strains were studied. Our results showed that almost all P. aeruginosa strains presented up to eight virulence genes. By SpeI macrorestriction fragment analysis we were able to identify 49 restriction patterns; 35 patterns correspond to single strains and the remaining 14 to strains subgroup (a–n). Most of the strains showed variation in number or composition of GEIs and a specific antimicrobial pattern indicating that each strain was an unrelated isolate. In terms of the number of genomic islands per strain, 7 GEIs were found in 34% of the strains, 6 in 18%, 5 in 12%, 4 in 14%, 3 in 10%, 2 in 7%, and 1 in 4%; only one isolate did not present any GEI. The genomic islands PAPI-1 and PAPI-2 and the element pKLC102 were the most frequently detected. The analysis of the location of each GEI in the chromosome of two strains show that the islands PAGI-3, PAPI-1, PAPI-2 and pKLC102 are present in the insertion site previously reported, but that PAGI-2 and PAGI-4 are inserted in another chromosome place in a site not characterized yet. In conclusion our data show that P. aeruginosa strains exhibited an epidemic population structure with horizontal transfer of DNA resulting in a high frequency of GEIs.

Occurrence in Mexico, 1998–2008, of Vibrio cholerae CTX+ El Tor carrying an additional truncated CTX prophage

Significance Vibrio cholerae classical (CL) biotype was isolated, along with biotype El Tor (ET) and altered ET carrying the cholera toxin (CTX) gene of CL biotype, during the 1991 cholera epidemic in Mexico, subsequently becoming endemic until 1997. Microbiological, molecular, and phylogenetic analyses of V. cholerae isolated from both clinical and environmental samples during 1998–2008 confirm important genetic events, namely predominance of ET over CL and altered ET in Mexico. Although altered ET is predominantly associated with cholera globally, progression of CTX+ V. cholerae ET with truncated CTX prophage to the predominant pathogen causing endemic cholera in Mexico may prove to be yet another key historical point in the global epidemiology of cholera. The seventh cholera pandemic caused by Vibrio cholerae O1 El Tor (ET) has been superseded in Asia and Africa by altered ET possessing the cholera toxin (CTX) gene of classical (CL) biotype. The CL biotype of V. cholerae was isolated, along with prototypic and altered ET, during the 1991 cholera epidemic in Mexico and subsequently remained endemic until 1997. Microbiological, molecular, and phylogenetic analyses of clinical and environmental V. cholerae isolated in Mexico between 1998 and 2008 revealed important genetic events favoring predominance of ET over CL and altered ET. V. cholerae altered ET was predominant after 1991 but not after 2000. V. cholerae strains isolated between 2001 and 2003 and a majority isolated in 2004 lacked CTX prophage (Φ) genes encoding CTX subunits A and B and repeat sequence transcriptional regulators of ET and CL biotypes: i.e., CTXΦ−. Most CTXΦ− V. cholerae isolated in Mexico between 2001 and 2003 also lacked toxin coregulated pili tcpA whereas some carried either tcpAET or a variant tcpA with noticeable sequence dissimilarity from tcpACL. The tcpA variants were not detected in 2005 after CTXΦ+ ET became dominant. All clinical and environmental V. cholerae O1 strains isolated during 2005–2008 in Mexico were CTXΦ+ ET, carrying an additional truncated CTXΦ instead of RS1 satellite phage. Despite V. cholerae CTXΦ− ET exhibiting heterogeneity in pulsed-field gel electrophoresis patterns, CTXΦ+ ET isolated during 2004–2008 displayed homogeneity and clonal relationship with V. cholerae ET N16961 and V. cholerae ET isolated in Peru.

Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.

Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.

Lewis Antigen Expression by Helicobacter pylori Strains Colonizing Different Regions of the Stomach of Individual Patients

ABSTRACT The diversity in the expression of Lewis antigens (Le) of 226 single colonies of Helicobacter pylori isolated from four regions of the stomach of eight adults is shown. Ley was expressed more in strains colonizing antrum than in strains colonizing fundus, whereas Lex was more common in fundus strains. cagA+ strains were more associated with Le-negative strains.

Integrated view of Vibrio cholerae in the Americas

Wave upon wave of disease The cholera pathogen, Vibrio cholerae, is considered to be ubiquitous in water systems, making the design of eradication measures apparently fruitless. Nevertheless, local and global Vibrio populations remain distinct. Now, Weill et al. and Domman et al. show that a surprising diversity between continents has been established. Latin America and Africa bear different variants of cholera toxin with different transmission dynamics and ecological niches. The data are not consistent with the establishment of long-term reservoirs of pandemic cholera or with a relationship to climate events. Science, this issue p. 785, p. 789 Multiple waves of local outbreaks and pandemic cholera indicate independence from climate change and marine reservoirs Latin America has experienced two of the largest cholera epidemics in modern history; one in 1991 and the other in 2010. However, confusion still surrounds the relationships between globally circulating pandemic Vibrio cholerae clones and local bacterial populations. We used whole-genome sequencing to characterize cholera across the Americas over a 40-year time span. We found that both epidemics were the result of intercontinental introductions of seventh pandemic El Tor V. cholerae and that at least seven lineages local to the Americas are associated with disease that differs epidemiologically from epidemic cholera. Our results consolidate historical accounts of pandemic cholera with data to show the importance of local lineages, presenting an integrated view of cholera that is important to the design of future disease control strategies.

New enterovirulent Escherichia coli serogroup 64474 showing antigenic and genotypic relationships to Shigella boydii 16.

Studies based on the analysis of housekeeping genes indicate that Escherichia coli and all Shigella species, except for Shigella boydii type 13, belong to a single species. This study analysed the phenotypic and genotypic characteristics of 23 E. coli strains isolated in different countries from faecal specimens taken from children with diarrhoea. Strains were identified using the VITEK system and typed with rabbit sera obtained against 186 somatic and 53 flagellar E. coli antigens and against 45 Shigella somatic antigens. Biochemical analysis of these strains showed a typical E. coli profile with a defined reaction against both E. coli O179 and S. boydii 16 somatic antisera. Agglutination assays for flagellar antigens showed a response against H2 in 7 (30 %) strains, H10 in 2 (9 %) strains, H32 in 12 (52 %) strains and H34 in 2 (9 %) strains, demonstrating 4 serotypes associated with this new somatic antigen 64474. A serum against one of these E. coli strains (64474) was prepared. Absorption assays of S. boydii 16 and E. coli 64474 antisera with E. coli O179 antigen removed the agglutination response against this O179 antigen completely, while the agglutination titres against both S. boydii 16 and E. coli 64474 remained the same. Four (17 %) E. coli strains showed antimicrobial resistance to piperacillin only, one (4 %) to piperacillin and trimethoprim/sulfamethoxazole, one (4 %) to ciprofloxacin, nitrofurantoin and piperacillin, and two (9 %) strains were resistant to ciprofloxacin, norfloxacin, ofloxacin, piperacillin and trimethoprim/sulfamethoxazole. With regards to PCR assays, one (4 %) of the strains was positive for Shigella gene ipaH, one (4 %) for ipaA, two (9 %) for ipaB, one (4 %) for ipaD, two (9 %) for sepA and three (13 %) for ospF. The rfb gene cluster in the E. coli strains was analysed by RFLP and compared with the gene cluster obtained from S. boydii 16. The rfb-RFLP patterns for all 23 E. coli strains were similar to those obtained for S. boydii 16. The results from PCR tests to detect rfb genes wzx (encoding O unit flippase) and wzy (encoding polymerase) belonging to a cluster related to the biosynthesis of the S. boydii 16-specific O antigen were positive in 21 (91 %) and 22 (96 %) of the strains, respectively. PCR assays to detect E. coli virulence genes were also performed. These assays detected enterotoxigenic E. coli genes ltA1 in 12 of the strains (52 %), st1a in 4 (17 %), cfa1 in 6 (26 %), cs1 in 1 (4 %), cs3 in 3 (13 %), cs13 in 9 (39 %) and cs14 in 5 (22 %) of the strains. Results from the PFGE analyses confirmed the wide geographical distribution of these strains suggesting that 64474 : H2, 64474 : H10, 64474 : H32 and 64474 : H34 are new serotypes of E. coli strains with a defined virulence capacity, and share a common O antigen with S. boydii 16.

Fingerprint Analysis and Identification of Strains ST309 as a Potential High Risk Clone in a Pseudomonas aeruginosa Population Isolated from Children with Bacteremia in Mexico City

Pseudomonas aeruginosa is an opportunistic pathogen and is associated with nosocomial infections. Its ability to thrive in a broad range of environments is due to a large and diverse genome of which its accessory genome is part. The objective of this study was to characterize P. aeruginosa strains isolated from children who developed bacteremia, using pulse-field gel electrophoresis, and in terms of its genomic islands, virulence genes, multilocus sequence type, and antimicrobial susceptibility. Our results showed that P. aeruginosa strains presented the seven virulence genes: toxA, lasB, lecA, algR, plcH, phzA1, and toxR, a type IV pilin alleles (TFP) group I or II. Additionally, we detected a novel pilin and accessory gene, expanding the number of TFP alleles to group VI. All strains presented the PAPI-2 Island and the majority were exoU+ and exoS+ genotype. Ten percent of the strains were multi-drug resistant phenotype, 18% extensively drug-resistant, 68% moderately resistant and only 3% were susceptible to all the antimicrobial tested. The most prevalent acquired β-Lactamase was KPC. We identified a group of ST309 strains, as a potential high risk clone. Our finding also showed that the strains isolated from patients with bacteremia have important virulence factors involved in colonization and dissemination as: a TFP group I or II; the presence of the exoU gene within the PAPI-2 island and the presence of the exoS gene.