New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

Epidemiological analysis of bacterial strains involved in hospital infection in a University Hospital from Brazil

Hospital infections cause an increase in morbidity and mortality of hospitalized patients with significant rise in hospital costs. The aim of this work was an epidemiological analysis of hospital infection cases occurred in a public University Hospital in Rio de Janeiro. Hence, 238 strains were isolated from 14 different clinical materials of 166 patients hospitalized in the period between August 1995 and July 1997. The average age of the patients was 33.4 years, 72.9% used antimicrobials before having a positive culture. The most common risk conditions were surgery (19.3%), positive HIV or AIDS (18.1%) and lung disease (16.9%). 24 different bacterial species were identified, S. aureus (21%) and P. aeruginosa (18.5%) were predominant. Among 50 S. aureus isolated strains 36% were classified as MRSA (Methicillin Resistant S. aureus). The Gram negative bacteria presented high resistance to aminoglycosides and cephalosporins. A diarrhea outbreak, detected in high-risk neonatology ward, was caused by Salmonella serovar Infantis strain, with high antimicrobial resistance and a plasmid of high molecular weight (98Mda) containing virulence genes and positive for R factor.

Frequency, diversity, and productivity study on the Aedes aegypti most preferred containers in the city of Manaus, Amazonas, Brazil

The most preferred containers by Aedes aegypti were studied April and July (rainy and dry periods) in two Manaus neighbourhoods. In all, 2,700 premises and 13,912 containers were examined, most (87%) recorded outdoors. Out of the 13,100 inspected premises, only 1.6% showed to be positive for Aedes aegypti, summing up to 7,916 collected samples. Most frequently found containers outdoors in either neighbourhood regardless of rain or dry period were Bottles flasks and Storage, and indoors, Fixed, Flowerpots, and buckets. Productivity was estimated according to the number of premises and positive containers investigated, showing the actual container groups productivity. Considering both rainy and dry periods outdoors at Praça 14 the groups of Tyre, Flask, Bottle, Construction Equipment and Fixed, had the highest averages respectively. Construction Equipment and Flask groups were the most productive in Coroado in April. Flask, Construction Equipment and Storage groups stood out in July.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.

Affinity and diversity indices for anopheline immature forms

As for the entire Amazon Region, malaria continues to be a major health public problem in Roraima that presented an Annual Parasitic Index of 85.4 in 2005, the highest in Brazil. Information on anopheline breeding sites is an essential component in malaria control strategies. Aiming to contribute to the limited knowledge on anopheline immature forms in Roraima, collections and breeding site observations were performed in 10 breeding sites around the capital city Boa Vista. Collections were carried out in the rainy and dry season periods between April 2004 and January 2005. Breeding sites comprised natural and artificial water reservoirs. A total of 623 immature forms were collected belonging to Anopheles albitarsis s.l., An.triannulatus s.l., An. nuneztovari/dunhami, An. braziliensis, An. evansae, An. oswaldoi s.l., An. strodei and An. darlingi. An. albitarsis and An. braziliensis were the most frequently found species. Eight larvae of An. darlingi were found in only one breeding site located in the forest. An. triannulatus/An. nuneztovari and An. albitarsis/An. braziliensis were the pairs of species that mostly occurred together. Both pair of species displayed the highest affinity index what might indicate a high compatibility for the same breeding conditions and/or a synergistic co-occurrence. Species diversity index was higher for the dry season.

Seroprevalence of selected viral, bacterial and parasitic infections among inpatients of a public psychiatric hospital of Mexico

We sought to determine the frequency of serological markers of selected infections in a population of psychiatric patients in Durango City, Mexico, and to determine whether there are any epidemiological characteristics of the subjects associated with the infections. One hundred and five inpatients of a public psychiatric hospital of Durango were examined for HBsAg, anti-HCV antibodies, anti-HIV antibodies, anti-Brucella antibodies, rapid plasma reagin and anti-Cysticercus antibodies by commercially available assays. Anti-Cysticercus antibodies were confirmed by Western blot and HBsAg by neutralization assay. Epidemiological data from each participant were also obtained. Seroprevalences of HBsAg, anti-HCV, anti-HIV, anti-Brucella, rapid plasma reagin and anti-Cysticercus antibodies found were 0.0%, 4.8%, 0.9%, 0.0%, 1.9%, and 0.9%, respectively. Overall, 9 (8.6%) inpatients showed seropositivity to any infection marker. We concluded that our psychiatric inpatients have serological evidence of a number of infections. HCV is an important pathogen among our psychiatric inpatients. Health care strategies for prevention and control of infections in Mexican psychiatric patients should be considered.

Microbiological contamination of a hemodialysis center water distribution system

The microbiological monitoring of the water used for hemodialysis is extremely important, especially because of the debilitated immune system of patients suffering from chronic renal insufficiency. To investigate the occurrence and species diversity of bacteria in waters, water samples were collected monthly from a hemodialysis center in upstate São Paulo and tap water samples at the terminal sites of the distribution system was sampled repeatedly (22 times) at each of five points in the distribution system; a further 36 samples were taken from cannulae in 19 hemodialysis machines that were ready for the next patient, four samples from the reuse system and 13 from the water storage system. To identify bacteria, samples were filtered through 0.22 µm-pore membranes; for mycobacteria, 0.45 µm pores were used. Conventional microbiological and molecular methods were used in the analysis. Bacteria were isolated from the distribution system (128 isolates), kidney machine water (43) and reuse system (3). Among these isolates, 32 were Gram-positive rods, 120 Gram-negative rods, 20 Gram-positive cocci and 11 mycobacteria. We propose the continual monitoring of the water supplies in hemodialysis centers and the adoption of effective prophylactic measures that minimize the exposure of these immunodeficient patients to contaminated sources of water.

Genetic diversity and primary resistance among HIV-1-positive patients from Maringá, Paraná, Brazil

The objective of this study is to identify subtypes of Human Immunodeficiency Virus type 1 (HIV-1) and to analyze the presence of mutations associated to antiretroviral resistance in the protease (PR) and reverse transcriptase (RT) regions from 48 HIV-1 positive treatment naïve patients from an outpatient clinic in Maringá, Paraná, Brazil. Sequencing was conducted using PR, partial RT and group-specific antigen gene (gag) nested PCR products from retrotranscribed RNA. Transmitted resistance was determined according to the Surveillance Drug Resistance Mutation List (SDRM) algorithm. Phylogenetic and SimPlot analysis of concatenated genetic segments classified sequences as subtype B 19/48 (39.6%), subtype C 12/48 (25%), subtype F 4/48 (8.3%), with 13/48 (27.1%) recombinant forms. Most recombinant forms were B mosaics (B/F 12.5%, B/C 10.4%), with one C/F (2.1%) and one complex B/C/F mosaic (2.1%). Low levels of transmitted resistance were found in this study, 2/48 (2.1% to NRTIs and 2.1% for PI). This preliminary data may subsidize the monitoring of the HIV evolution in the region.

Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina

Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

PREVALENCE OF BACTERIAL RESISTANCE IN HOSPITALIZED CIRRHOTIC PATIENTS IN SOUTHERN BRAZIL: A NEW CHALLENGE

Background & Aims: An increased frequency of infections by multiresistant bacteria has been described in hospitalized patients. The aim of this study was to evaluate the bacterial resistance profile in cirrhotic patients. Methods: This is a retrospective observational study. We assessed the antimicrobial susceptibility of 5,839 bacterial isolates from patients with and without cirrhosis. Regarding the multidrug resistance, we evaluated 4,505 bacterial isolates from 2,180 patients. Results: Two hundred and fifty-one patients had cirrhosis (mean age 57.6 ± 11 years; 61.8% were male, 47.8% of cases associated with hepatitis C virus). Of the isolates of patients with and without cirrhosis, 174/464 (37.5%) and 1,783/4,041 (44.1%) were multiresistant, respectively (p = 0.007). E. coli was the most common multiresistant bacteria in both groups. Approximately 20% of E. coli and Klebsiella sp. isolates were ESBL-producers and 44% of S. aureus isolates were methicillin-resistant in cirrhotic patients. In cirrhotic patients admitted to the emergency department, hospital ward, and intensive care unit, 28.3%, 50% and 40% had multiresistant isolates, respectively. In patients with and without cirrhosis, 36.2% and 33.5% of isolates were resistant to third-generation cephalosporins, respectively. Conclusions: The empirical treatment of infections in hospitalized patients using broad-spectrum antibiotics should consider the observed pattern of bacterial resistance.