Fiocruz in Antarctica - health and environmental surveillance facing the challenges of the 21st century.

FioAntar, FIOCRUZ's research project in Antarctica, is based on the One Health approach. FioAntar aims to generate relevant information that will help reduce the risk of future pandemics and improve the search for chemical compounds and new biological molecules. After four expeditions to Antarctica under the scope of PROANTAR, Fiocruz has identified Influenza H11N2 virus in environmental fecal samples, as well as Histoplasma capsulatum and Bacillus cereus in soil samples. In addition, in a prospective virome analysis from different lakes in the South Shetland Islands, six viral orders were described, supporting future research related to the biodiversity and viral ecology in this extreme ecosystem. Our findings of environmental pathogens of public health importance are a warning about the urgency of establishing a surveillance agenda on zoonoses in Antarctica due to the imminent risks that ongoing environmental and climate changes impose on human health across the planet. FioAntar strives to establish a comprehensive surveillance program across Antarctica, monitoring circulation of pathogens with the potential to transcend continent boundaries, thereby mitigating potential spread. For Fiocruz, Antarctica signifies a new frontier, teeming with opportunities to explore novel techniques, refine established methodologies, and cultivate invaluable knowledge.

Molecular characterization and toxigenic profiles of Bacillus cereus isolates from foodstuff and food poisoning outbreaks in Brazil.

Bacillus cereus sensu stricto (s.s.) is a well-known foodborne pathogen that produces a range of enterotoxins and is able to cause two different types of foodborne illnesses-the emetic and the diarrheal syndromes. In this study, 54 B. cereus s.s. strains isolated from foodstuff and foods involved in food poisoning outbreaks were characterized according to the presence of toxin-encoding genes, virulence-encoding genes, and panC typing. Most isolates were assigned to panC groups IV (61.1%) and III (25.9%), but members of groups II and V could also be found. Investigation of specific alleles revealed high numbers of isolates carrying toxin and other virulence genes including nheA (100%), nheB (100%), hblA (79.6%), hblC (79.6%), hblD (74.1%), cytK-2 (61.1%), clo (100%), pc-plc (75.9%), sph (68.5%), pi-plc (66.6%), hlyIII (62.9%), and hlyII (24.1%). All isolates were negative for ces and cytK-1. In summary, we detected various enterotoxin and other virulence factor genes associated with diarrheal syndrome in strains analyzed, implicated or not with food poisoning. Furthermore, the most isolates analyzed belong to high-risk phylogenetic groups' panC types III and IV. Our study provides a convenient molecular scheme for characterization of B. cereus s.s. strains responsible for food poisoning outbreaks in order to improve the monitoring and investigation and assess emerging clusters and diversity of strains.

Molecular Detection of Histoplasma capsulatum in Antarctica.

We detected Histoplasma capsulatum in soil and penguin excreta in the Antarctic Peninsula by sequencing after performing species-specific PCR, confirming previous observations that this pathogen occurs more broadly than suspected. This finding highlights the need for surveillance of emerging agents of systemic mycoses and their transmission among regions, animals, and humans in Antarctica.

Subacute infective endocarditis caused by Bacillus cereus in a patient with Systemic Lupus Erythematosus.

A rare and difficult to diagnose case of subacute infective endocarditis caused by Bacillus cereus in a patient with systemic lupus erythematosus and Libman-Sacks endocarditis has been reported. Our aim is to highlight the importance of molecular methods such as MALDI-TOF and PCR to explain clinical and epidemiological issues about infections caused by unusual pathogen.

Detection of Bacillus anthracis and Bacillus anthracis-like spores in soil from state of Rio de Janeiro, Brazil.

BACKGROUND: Bacillus anthracis is the aetiologic agent of anthrax, a re-emerging, septicaemic, haemorrhagic and lethal disease that affects humans, domestic ruminants and wildlife. Plasmids pXO1 and pXO2 are attributes that confer pathogenicity to B. anthracis strains. This bacterium was used as biological weapon in the World Wars and in the biological attack in the United States of America at 2001. B. anthracis is classified as a Tier 1 bioterrorism agent by the Centers for Diseases Control and Prevention. Anthrax is recognised as a re-emerging disease. Several studies concerning the dynamics of B. anthracis cycle in soil revealed that nonpathogenic B. anthracis strains due to lack of pXO2 plasmid are commonly found in some types of soil. OBJECTIVES: This study aimed isolation and identification of B. anthracis spores in soil samples of the state of Rio de Janeiro, Brazil. METHODS: Phenotypic and genotypic approaches were used to identify isolates including MALDI-TOF/MS, motility test, susceptibility to gamma phage and penicillin, survey for pag and cap genes as surrogates of pXO1 and pXO2 plasmids, respectively, and sequencing of 16SrRNA-encoding gene. Physicochemical analysis of the soil samples were carried out to describe soil characteristics. FINDINGS: We observed the presence of one B. anthracis pXO1+ and pXO2- isolated from clay loam soil; one B. anthracis-like strain pXO1+ and pXO2-isolated from loamy sand; and 10 Bacillus spp. strains sensitive to phage-gamma that need better characterisation to define which their species were recovered from loamy sand. MAIN CONCLUSIONS: This work showed promising results and it was the first study to report results from an active surveillance for B. anthracis in Brazil.

Molecular Characterization and Risk Assessment of Bacillus cereus Sensu Lato Isolated from Ultrahigh-Temperature and Pasteurized Milk Marketed in Rio de Janeiro, Brazil.

The presence of Bacillus cereus in milk is a major concern in the dairy industry. In this study 27 Bacillus cereus sensu lato isolates from pasteurized and ultrahigh-temperature (UHT) milk (24 whole UHT and 4 pasteurized samples) collected at supermarket chains in Rio de Janeiro, Brazil, were evaluated to assess the potential risk for food poisoning. Toxigenic and virulence profiles were defined by gene-specific PCR. Affiliation to phylogenetic groups was assigned by panC sequencing. Microbiological analysis revealed the presence of B. cereus s.l. in eight (33.3%) brands (six brands of UHT and two brands of pasteurized milk). Twenty-seven isolates were recovered (13 B. cereus and 14 Bacillus thuringiensis ). Predominant toxigenic patterns were type I (contains all toxin genes except ces) and type II (does not contain cytK and ces), with seven (25.9%) isolates each. Predominant virulence patterns were type 2 (does not contain hlyII or shp) and type 3 (contains all virulence genes), with five (18.5%) isolates each. All isolates belonged to phylogenetic groups III and IV. Presence of hbl, piplc, and sph was associated with group IV isolates. Our results suggest that B. thuringiensis and B. cereus sensu stricto should be considered potential foodborne pathogens. Because the majority of the milk isolates studied have the potential to cause food poisoning because of the high prevalence of toxin and virulence genes and the specific phylogenetic group affiliations, these milk products can be potentially hazardous for human consumption.

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee.

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.

Genetic diversity, antimicrobial resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over three decades.

Bacillus cereus is an ever-present problem. It is widely distributed in several environments such as soil and plants and is commonly isolated from food and additives. In this study we analyzed 97 foodborne B. cereus sensu stricto strains isolated in Brazil in the 1980's, 1990's and 2000's in order to investigate the genetic diversity (assessed by Rep-PCR), antimicrobial resistance and toxigenic profiles (presence of hblA, hblC and hblD; nheA, nheB and nheC as well as cytK, ces and entFM genes) of such strains. The majority of the strains (79, 81.4%) were ß-hemolytic. The NHE complex was found in 82 strains (84.5%) and HBL complex was found in 61 (62.9%) strains. All strains were negative to ces. The cytK-2 gene was found in 44 (45.4%) strains. The predominant toxigenic pattern was type I (32, 33%) which included strains positive for all toxin genes but ces. Computer assisted cluster analysis of Rep-PCR profiles showed a high genetic diversity. Seven major clusters comprising two or more strains were found and cluster 1 was predominant (ten strains, nine of them showing 100% similarity). This cluster included strains isolated in the 1980's and the 1990's. Cluster analysis of Rep-PCR profiles based on decade of isolation, source, hemolytic pattern, toxigenic and antibiotic resistance patterns revealed a similar clustering pattern as found in the analysis including all strains. The inability to observe a predominant band pattern when Rep-PCR cluster analysis was based on decade of isolation suggests that this diversity has been maintained over time. All strains were susceptible to gentamicin. We detected resistance to tetracycline (11 strains showing intermediate resistance and nine completely resistant strains), clindamycin (ten intermediate strains) and vancomycin (one strain). Clindamycin resistance showed statistical association with strains isolated in 2000's. The predominant resistance pattern was type A (72, 72.2%) which included strains susceptible to all drugs tested. Our results suggest that the majority of the strains present in several types of food in Brazil pose a potential risk to cause food poisoning due to the high prevalence of toxin genes found in these strains. However, additional studies involving cytotoxicity tests and affiliation of these strains to phylogenetic groups based on molecular data would be useful to better evaluate this potential and could provide a more accurate indication of the risk.

Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages.

In only a few instances has the clonal composition of Staphylococcus aureus collections that include methicillin-susceptible S. aureus (MSSA) been extensively characterized. In order to investigate the clonal composition of MSSA and methicillin-resistant S. aureus (MRSA) and examine whether the infections diagnosed at our hospital were related to internationally distributed S. aureus lineages, we collected 89 clinical S. aureus isolates from patients at a public university hospital in Rio de Janeiro, Brazil, from September 1999 to June 2000. All S. aureus isolates were genotyped by pulsed-field gel electrophoresis and multilocus restriction fragment typing (MLRFT), and a subset (n = 17) was further characterized by multilocus sequence typing (MLST). The 34 MRSA isolates were additionally characterized by SCCmec typing. The MSSA population (n = 55) was grouped into 18 restriction fragment types (RFTs); of these, five RFTs accounted for 67% (37) of the MSSA isolates. MRSA isolates were clustered into only three RFTs (P = 0.02). The majority of MSSA RFTs were related to sequence type 30 (ST30) (12 isolates, 22%), ST1, ST188, and ST432 (6 isolates, 11% each). The predominant MRSA RFT comprised 31 (91%) of 34 isolates; four randomly selected isolates of this RFT were ST239, the previously described widely disseminated Brazilian clone. However, a fifth isolate belonging to this RFT was the ST644, a new single locus variant of ST239. By applying MLRFT and MLST, we found evidence for a clonal structure in MSSA isolates and detected the dissemination of MSSA clonal complexes 1, 5, 8, 30, and 45.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution - A Review

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.

Mupirocin for controlling methicillin-resistant Staphylococcus aureus: lessons from a decade of use at a university hospital.

BACKGROUND: From 1990 to 1995 at Hospital Universitário Clementino Fraga Filho, patients colonized or infected with methicillin-resistant Staphylococcus aureus (MRSA) were treated with mupirocin to eliminate MRSA carriage. In 1995, 65% of MRSA patients at this hospital had mupirocin-resistant isolates. Starting in 1996, mupirocin use was restricted to patients colonized, but not infected, with MRSA. OBJECTIVES: To describe the use of mupirocin for controlling MRSA over a decade and to analyze the molecular epidemiology of mupirocin-resistant MRSA infections at this hospital. SETTING: A 490-bed, tertiary-care university hospital. METHODS: The incidence densities of patients with MRSA and acquisition of mupirocin by the hospital were calculated for the period 1992-2001. S. aureus isolates from 1999-2000 were analyzed by pulsed-field gel electrophoresis. Mupirocin-resistant MRSA isolates from 1994-1995 and 1999-2000 were analyzed for ileS-2 gene background polymorphisms. RESULTS: The incidence density of MRSA patients increased slightly over time, whereas the purchase of mupirocin decreased dramatically. Mupirocin-resistant MRSA infections decreased from 65% in 1994-1995 to 15% in 1999-2000. The MRSA Brazilian clone, detected in 1992, was still highly prevalent. The same ileS-2 encoding plasmid found in 1994-1995 persisted in three identical MRSA isolates from 1999-2000 belonging to the Brazilian clone. CONCLUSIONS: After mupirocin use decreased, the ileS-2 encoding plasmid persisted in only a few Brazilian clone isolates. Our data on mupirocin-resistant MRSA incidence and mupirocin use strongly suggested that restricted use was related to decreased rates of mupirocin resistance at our hospital.

Application of molecular techniques in the study of Staphylococcus aureus clonal evolution--a review.

Staphylococcus aureus is an important agent of healthcare-associated and community-acquired infections. A major characteristic of this microorganism is the ability to develop resistance to antimicrobial agents. Several molecular techniques have been applied for the characterization of S. aureus in epidemiological studies. In the present review, we discuss the application of molecular techniques for typing S. aureus strains and describe the nomenclature and evolution of epidemic clones of this important pathogen.