Jose Luis Mendez

Cholera between 1991 and 1997 in Mexico Was Associated with Infection by Classical, El Tor, and El Tor Variants of Vibrio cholerae

ABSTRACT Vibrio cholerae O1 biotype El Tor (ET), the cause of the current 7th pandemic, has recently been replaced in Asia and Africa by an altered ET biotype possessing cholera toxin (CTX) of the classical (CL) biotype that originally caused the first six pandemics before becoming extinct in the 1980s. Until recently, the ET prototype was the biotype circulating in Peru; a detailed understanding of the evolutionary trend of V. cholerae causing endemic cholera in Latin America is lacking. The present retrospective microbiological, molecular, and phylogenetic study of V. cholerae isolates recovered in Mexico (n = 91; 1983 to 1997) shows the existence of the pre-1991 CL biotype and the ET and CL biotypes together with the altered ET biotype in both epidemic and endemic cholera between 1991 and 1997. According to sero- and biotyping data, the altered ET, which has shown predominance in Mexico since 1991, emerged locally from ET and CL progenitors that were found coexisting until 1997. In Latin America, ET and CL variants shared a variable number of phenotypic markers, while the altered ET strains had genes encoding the CL CTX (CTXCL) prophage, ctxBCL and rstR CL, in addition to resident rstR ET, as the underlying regional signature. The distinct regional fingerprints for ET in Mexico and Peru and their divergence from ET in Asia and Africa, as confirmed by subclustering patterns in a pulsed-field gel electrophoresis (NotI)-based dendrogram, suggest that the Mexico epidemic in 1991 may have been a local event and not an extension of the epidemics occurring in Asia and South America. Finally, the CL biotype reservoir in Mexico is unprecedented and must have contributed to the changing epidemiology of global cholera in ways that need to be understood.

Vibrio cholerae Classical Biotype Strains Reveal Distinct Signatures in Mexico

ABSTRACT Vibrio cholerae O1 classical (CL) biotype caused the fifth and sixth pandemics, and probably the earlier cholera pandemics, before the El Tor (ET) biotype initiated the seventh pandemic in Asia in the 1970s by completely displacing the CL biotype. Although the CL biotype was thought to be extinct in Asia and although it had never been reported from Latin America, V. cholerae CL and ET biotypes, including a hybrid ET, were found associated with areas of cholera endemicity in Mexico between 1991 and 1997. In this study, CL biotype strains isolated from areas of cholera endemicity in Mexico between 1983 and 1997 were characterized in terms of major phenotypic and genetic traits and compared with CL biotype strains isolated in Bangladesh between 1962 and 1989. According to sero- and biotyping data, all V. cholerae strains tested had the major phenotypic and genotypic characteristics specific for the CL biotype. Antibiograms revealed the majority of the Bangladeshi strains to be resistant to trimethoprim-sulfamethoxazole, furazolidone, ampicillin, and gentamicin, while the Mexican strains were sensitive to all of these drugs, as well as to ciprofloxacin, erythromycin, and tetracycline. Pulsed-field gel electrophoresis (PFGE) of NotI-digested genomic DNA revealed characteristic banding patterns for all of the CL biotype strains although the Mexican strains differed from the Bangladeshi strains in 1 to 2 DNA bands. The difference was subtle but consistent, as confirmed by the subclustering patterns in the PFGE-based dendrogram, and can serve as a regional signature, suggesting the pre-1991 existence and evolution of the CL biotype strains in the Americas, independent from Asia.

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.

Genetic characterization of atypical Citrobacter freundii.

The ability of a bacterial population to survive in different niches, as well as in stressful and rapidly changing environmental conditions, depends greatly on its genetic content. To survive such fluctuating conditions, bacteria have evolved different mechanisms to modulate phenotypic variations and related strategies to produce high levels of genetic diversity. Laboratories working in microbiological diagnosis have shown that Citrobacter freundii is very versatile in its colony morphology, as well as in its biochemical, antigenic and pathogenic behaviours. This phenotypic versatility has made C. freundii difficult to identify and it is frequently confused with both Salmonella enterica and Escherichia coli. In order to determine the genomic events and to explain the mechanisms involved in this plasticity, six C. freundii isolates were selected from a phenotypic variation study. An I-CeuI genomic cleavage map was created and eight housekeeping genes, including 16S rRNA, were sequenced. In general, the results showed a range of both phenotypes and genotypes among the isolates with some revealing a greater similarity to C. freundii and some to S. enterica, while others were identified as phenotypic and genotypic intermediary states between the two species. The occurrence of these events in natural populations may have important implications for genomic diversification in bacterial evolution, especially when considering bacterial species boundaries. In addition, such events may have a profound impact on medical science in terms of treatment, course and outcomes of infectious diseases, evading the immune response, and understanding host-pathogen interactions.

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.

The Re-emergence of Cholera in the Americas

In the history of cholera, seven cholera pandemics occurred during the nineteenth and twentieth centuries and five of these affected the American continent. Ships transporting European immigrants in the nineteenth century probably brought the disease to the American continent during the second pandemic. Between 1973 and 1991, infrequent indigenous cholera cases were reported in different parts of the USA including Louisiana, Florida, Georgia, Maryland, which have been linked to the Gulf Coast reservoir. In 1991, cholera resurfaced in Latin America accounting for 66% (396,536 cases) of all cholera cases reported worldwide that year. Unexpectedly, cholera re-emerged in Peru during early 1991 and quickly turned into an epidemic that extended to Bolivia, Brazil, Chile, Colombia, Ecuador, El Salvador, Guatemala, Honduras, Panama, Venezuela, and Mexico. During this time, several important environmental factors including El Nino were attributed to the increased incidence of cholera. During and after the cholera outbreaks in South America, intensive measures were taken that include epidemiological surveillance, setting up of a laboratory network, proper patient care, improvements in basic sanitation, and clean water supply systems. Such timely measures helped to control cholera and other diarrheal diseases. Several molecular techniques have allowed the study of clonal variations in Vibrio cholerae for the first time. Molecular tools helped in identifying the epidemiological links with import of cholera by travelers as well as of the environmental origin. Several serotypes of V. cholerae exist in costal environments and seroconversion may play an important role in the epidemiology of cholera.