René Cerritos

The genome of Bacillus coahuilensis reveals adaptations essential for survival in the relic of an ancient marine environment

The Cuatro Ciénegas Basin (CCB) in the central part of the Chihuahan desert (Coahuila, Mexico) hosts a wide diversity of microorganisms contained within springs thought to be geomorphological relics of an ancient sea. A major question remaining to be answered is whether bacteria from CCB are ancient marine bacteria that adapted to an oligotrophic system poor in NaCl, rich in sulfates, and with extremely low phosphorus levels (<0.3 μM). Here, we report the complete genome sequence of Bacillus coahuilensis, a sporulating bacterium isolated from the water column of a desiccation lagoon in CCB. At 3.35 Megabases this is the smallest genome sequenced to date of a Bacillus species and provides insights into the origin, evolution, and adaptation of B. coahuilensis to the CCB environment. We propose that the size and complexity of the B. coahuilensis genome reflects the adaptation of an ancient marine bacterium to a novel environment, providing support to a “marine isolation origin hypothesis” that is consistent with the geology of CCB. This genomic adaptation includes the acquisition through horizontal gene transfer of genes involved in phosphorous utilization efficiency and adaptation to high-light environments. The B. coahuilensis genome sequence also revealed important ecological features of the bacterial community in CCB and offers opportunities for a unique glimpse of a microbe-dominated world last seen in the Precambrian.

Bacillus coahuilensis sp. nov., a moderately halophilic species from a desiccation lagoon in the Cuatro Cienegas Valley in Coahuila, Mexico

A moderately halophilic, Gram-positive and rod-shaped bacterium, strain m4-4T, was isolated from a Chihuahuan desert lagoon in Cuatro Ciénegas, Coahuila, Mexico. Strain m4-4T was found to grow optimally at 30-37 degrees C, pH 7.0-8.0 and 5 % NaCl and to tolerate from 0.5 % to 10 % NaCl. It was shown to be aerobic. The genomic DNA G+C content was about 37 mol%. Strain m4-4T exhibited minimal or no growth on most sugars tested. Its major cellular fatty acids were C14 : 0, C16 : 0 and C18 : 1. Based on phylogenetic analysis of 16S rRNA and recA gene sequences, we observed that the closest relatives of the isolate are moderately halophilic Bacillus species, with 16S rRNA gene sequence similarity ranging from 96.6 to 97.4 % (Bacillus marisflavi, Bacillus aquimaris and Bacillus vietnamensis). Additionally, using genomic data it was determined that the type strain contains a total of nine rRNA operons with three slightly different sequences. On the basis of phenotypic and molecular properties, strain m4-4T represents a novel species within the genus Bacillus, for which the name Bacillus coahuilensis sp. nov. is proposed, with the type strain m4-4T (=NRRL B-41737T =CECT 7197T).

Human Amebiasis: Breaking the Paradigm?

For over 30 years it has been established that the Entamoeba histolytica protozoan included two biologically and genetically different species, one with a pathogenic phenotype called E. histolytica and the other with a non-pathogenic phenotype called Entamoeba dispar. Both of these amoebae species can infect humans. E. histolytica has been considered as a potential pathogen that can cause serious damage to the large intestine (colitis, dysentery) and other extraintestinal organs, mainly the liver (amebic liver abscess), whereas E. dispar is a species that interacts with humans in a commensal relationship, causing no symptoms or any tissue damage. This paradigm, however, should be reconsidered or re-evaluated. In the present work, we report the detection and genotyping of E. dispar sequences of DNA obtained from patients with amebic liver abscesses, including the genotyping of an isolate obtained from a Brazilian patient with a clinical diagnosis of intestinal amebiasis that was previously characterized as an E. dispar species. The genetic diversity and phylogenetic analysis performed by our group has shown the existence of several different genotypes of E. dispar that can be associated to, or be potentiality responsible for intestinal or liver tissue damage, similar to that observed with E. histolytica.

Diversity of culturable thermo-resistant aquatic bacteria along an environmental gradient in Cuatro Ciénegas, Coahuila, México

At the desert oasis of Cuatro Ciénegas in Coahuila, México, more than 300 oligotrophic pools can be found and a large number of endemic species of plants and animals. The most divergent taxa of diatoms, snail and fishes are located in the Churince hydrological system, where we analyzed the local diversification of cultivable Firmicutes and Actinobacteria. The Churince hydrological system is surrounded by gypsum dunes and has a strong gradient for salinity, temperature, pH and dissolved oxygen. In August 2003, surface water samples were taken in 10 sites along the Churince system together with the respective environmental measurements. 417 thermo-resistant bacteria were isolated and DNA was extracted to obtain their BOX-PCR fingerprints, revealing 55 different patterns. In order to identify similarities and differences in the diversity of the various sampling sites, an Ordination Analysis was applied using Principal Component Analysis. This analysis showed that conductivity is the environmental factor that explains the distribution of most of the microbial diversity. Phylogenetic reconstruction from their 16S rRNA sequences was performed for a sample of 150 isolates. Only 17 sequences had a 100% match in the Gene Bank (NCBI), representing 10 well known cosmopolitan taxa. The rest of the sequences cluster in 22 clades for Firmicutes and another 22 clades for Actinobacteria, supporting the idea of high diversity and differentiation for this site.

Novelties on Amoebiasis: A Neglected Tropical Disease

In accordance with the 1997 documents of the World Health Organization (WHO), amoebiasis is defined as the infection by the protozoan parasite Entamoeba histolytica with or without clinical manifestations. The only known natural host of E. histolytica is the human with the large intestine as major target organ. This parasite has a very simple life cycle in which the infective form is the cyst, considered a resistant form of parasite: The asymptomatic cyst passers and the intestinal amoebiasis patients are the transmitters; they excrete cysts in their feces, which can contaminate food and water sources. E. histolytica sensu stricto is the potentially pathogenic species and E. dispar is a commensal non-pathogenic Entamoeba. Both species are biochemical, immunological and genetically distinct. The knowledge of both species with different pathogenic phenotypes comes from a large scientific debate during the second half of the 20th century, which gave place to the rapid development of diagnostics technology based on molecular and immunological strategies. During the last ten years, knowledge of the new epidemiology of amoebiasis in different geographic endemic and non-endemic areas has been obtained by applying mostly molecular techniques. In the present work we highlight novelties on human infection and the disease that can help the general physician from both endemic and non-endemic countries in their medical practice, particularly, now that emigration is undoubtedly a global phenomenon that is modifying the previous geography of infectious diseases worldwide.

Nitrogen fixation in microbial mat and stromatolite communities from cuatro cienegas, Mexico

Nitrogen fixation (nitrogenase activity, NA) of a microbial mat and a living stromatolite from Cuatro Cienegas, Mexico, was examined over spring, summer, and winter of 2004. The goal of the study was to characterize the diazotrophic community through molecular analysis of the nifH gene and using inhibitors of sulfate reduction and oxygenic and anoxygenic photosynthesis. We also evaluated the role of ultraviolet radiation on the diazotrophic activity of the microbial communities. Both microbial communities showed patterns of NA with maximum rates during the day that decreased significantly with 3-3,4-dichlorophenyl-1′,1′-dimethylurea, suggesting the potential importance of heterocystous cyanobacteria. There is also evidence of NA by sulfur-reducing bacteria in both microbial communities suggested by the negative effect exerted by the addition of sodium molybdate. Elimination of infrared and ultraviolet radiation had no effect on NA. Both microbial communities had nifH sequences that related to group I, including cyanobacteria and purple sulfur and nonsulfur bacteria, as well as group II nitrogenases, including sulfur reducing and green sulfur bacteria.

Worldwide genealogy of Entamoeba histolytica: An overview to understand haplotype distribution and infection outcome

Although Entamoeba histolytica is one of the most prevalent intestinal parasites, how the different strains of this species are distributed all over the world and how different genotypes are associated with the infection outcome are yet to be fully understood. Recently, the use of a number of molecular markers has made the characterization of several genotypes in those regions with high incidence of amoebiasis possible. This work proposes the first genealogy of E. histolytica, with an haplotype network based on two tRNA gene-linked array of Short Tandem Repeats (STRs) reported until today, and 47 sequences from 39 new isolates of Mexican Amoebic Liver Abscesses (ALA) samples. One hundred and three sequences were obtained from D-A locus, their information about the geographic region of isolation as well as clinical diagnosis were also collected. One hundred and five sequences from N-K2 locus were also obtained as well as the region of isolation, but the information about clinical diagnosis was not available in all cases. The most abundant and widely distributed haplotype in the world is the one of E. histolytica HM1:IMSS strain. This was found in Mexico, Bangladesh, Japan, China and USA and is associated to symptomatic patients as well as asymptomatic cyst passers. Many other haplotypes were found only in a single country. Both genealogies suggest that there are no lineages within the networks that may be related to a particular geographic region or infection outcome. A concatenated analysis of the two molecular markers revealed 12 different combinations, which suggests the possibility of genetic recombination events. The present study is the first to propose a global genealogy of this species and suggests that there are still many genotypes to be discovered. The genotyping of new isolates will help to understand the great diversity and genetic structure of this parasite.

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.

Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico

BackgroundPlasmodium vivax is a protozoan parasite with an extensive worldwide distribution, being highly prevalent in Asia as well as in Mesoamerica and South America. In southern Mexico, P. vivax transmission has been endemic and recent studies suggest that these parasites have unique biological and genetic features. The msp1 gene has shown high rate of nucleotide substitutions, deletions, insertions, and its mosaic structure reveals frequent events of recombination, maybe between highly divergent parasite isolates.MethodsThe nucleotide sequence variation in the polymorphic icb5-6 fragment of the msp1 gene of Mexican and worldwide isolates was analysed. To understand how genotype diversity arises, disperses and persists in Mexico, the genetic structure and genealogical relationships of local isolates were examined. To identify new sequence hybrids and their evolutionary relationships with other P. vivax isolates circulating worldwide two haplotype networks were constructed questioning that two portions of the icb5-6 have different evolutionary history.ResultsTwelve new msp1 icb5-6 haplotypes of P. vivax from Mexico were identified. These nucleotide sequences show mosaic structure comprising three partially conserved and two variable subfragments and resulted into five different sequence types. The variable subfragment sV1 has undergone recombination events and resulted in hybrid sequences and the haplotype network allocated the Mexican haplotypes to three lineages, corresponding to the Sal I and Belem types, and other more divergent group. In contrast, the network from icb5-6 fragment but not sV1 revealed that the Mexican haplotypes belong to two separate lineages, none of which are closely related to Sal I or Belem sequences.ConclusionsThese results suggest that the new hybrid haplotypes from southern Mexico were the result of at least three different recombination events. These rearrangements likely resulted from the recombination between haplotypes of highly divergent lineages that are frequently distributed in South America and Asia and diversified rapidly.

Analysis of the Bacterial Diversity in Liver Abscess: Differences Between Pyogenic and Amebic Abscesses.

Several recent studies have demonstrated that virulence in Entamoeba histolytica is triggered in the presence of both pathogenic and nonpathogenic bacteria species using in vitro and in vivo experimental animal models. In this study, we examined samples aspirated from abscess material obtained from patients who were clinically diagnosed with amebic liver abscess (ALA) or pyogenic liver abscess (PLA). To determine the diversity of bacterial species in the abscesses, we performed partial 16S rRNA gene sequencing. In addition, the E. histolytica and Entamoeba dispar species were genotyped using tRNA-linked short tandem repeats as specific molecular markers. The association between clinical data and bacterial and parasite genotypes were examined through a correspondence analysis. The results showed the presence of numerous bacterial groups. These taxonomic groups constitute common members of the gut microbiota, although all of the detected bacterial species have a close phylogenetic relationship with bacterial pathogens. Furthermore, some patients clinically diagnosed with PLA and ALA were coinfected with E. dispar or E. histolytica, which suggests that the virulence of these parasites increased in the presence of bacteria. However, no specific bacterial groups were associated with this effect. Together, our results suggest a nonspecific mechanism of virulence modulation by bacteria in Entamoeba.