Lyophilization for bacteria preservation: a promising approach for Yersinia pestis strains from an unique collection in Brazil (Fiocruz-CYP).

Yersinia pestis, the causative agent of plague, is a highly virulent bacterium that poses a significant threat to human health. Preserving this bacterium in a viable state is crucial for research and diagnostic purposes. This paper presents and evaluates a simple lyophilization protocol for the long-term storage of Y. pestis strains from Fiocruz-CYP, aiming to explore its impact on viability and long-term stability, while replacing the currently used methodologies. The lyophilization tests were conducted using the non-virulent Y. pestis strain EV76, subjected to the lyophilization process under vacuum conditions. Viability assessment was performed to evaluate the effects of lyophilization and storage conditions on Y. pestis under multiple temperature conditions (- 80 °C, - 20 °C, 4-8 °C and room temperature). The lyophilization protocol employed in this study consistently demonstrated its efficacy in maintaining high viability rates for Y. pestis samples in a up to one year follow-up. The storage temperature that consistently exhibited the highest recovery rates was - 80 °C, followed by - 20 °C and 4-8 °C. Microscopic analysis of the post-lyophilized cultures revealed preserved morphological features, consistent with viable bacteria. The high viability rates observed in the preserved samples indicate the successful preservation of Y. pestis using this protocol. Overall, the presented lyophilization protocol provides a valuable tool for the long-term storage of Y. pestis, offering stability, viability, and functionality. By refining the currently used methods of lyophilization, this protocol can improve long-term preservation for Y. pestis strains collections, facilitating research efforts, diagnostic procedures, and the development of preventive and therapeutic strategies against plague.

CYP broth: a tool for Yersinia pestis isolation in ancient culture collections and field samples.

We developed a simple new selective LB-based medium, named CYP broth, suitable for recovering long-term stored Y. pestis subcultures and for isolation of Y. pestis strains from field-caught samples for the Plague surveillance. It aimed to inhibit the growth contaminating microorganisms and enrich Y. pestis growth through iron supplementation. The performance of CYP broth on microbial growth from different gram-negative and gram-positive strains from American Type Culture Collection (ATCC®) and other clinical isolates, field-caught rodent samples, and more importantly, on several vials of ancient Y. pestis subcultures was evaluated. Additionally, other pathogenic Yersinia species such as Y. pseudotuberculosis and Y. enterocolitica were also successfully isolated with CYP broth. Selectivity tests and bacterial growth performance on CYP broth (LB broth supplemented with Cefsulodine, Irgasan, Novobiocin, nystatin and ferrioxamine E) were evaluated in comparison with LB broth without additive; LB broth/CIN, LB broth/nystatin and with traditional agar media including LB agar without additive, and LB agar and Cefsulodin-Irgasan-Novobiocin Agar (CIN agar) supplemented with 50 µg/mL of nystatin. Of note, the CYP broth had a recovery twofold higher than those of the CIN supplemented media or other regular media. Additionally, selectivity tests and bacterial growth performance were also evaluated on CYP broth in the absence of ferrioxamine E. The cultures were incubated at 28 °C and visually inspected for microbiological growth analysis and O.D.625 nm measurement between 0 and 120 h. The presence and purity of Y. pestis growth were confirmed by bacteriophage and multiplex PCR tests. Altogether, CYP broth provides an enhanced growth of Y. pestis at 28 °C, while inhibiting contaminant microorganisms. The media is a simple, but powerful tool to improve the reactivation and decontamination of ancient Y. pestis culture collections and for the isolation of Y. pestis strains for the Plague surveillance from various backgrounds. KEY POINTS: • The newly described CYP broth improves the recuperation of ancient/contaminated Yersinia pestis culture collections • CYP broth was also efficient in reducing environmental contamination in field-capture samples, improving Y. pestis isolation • CYP broth can also be used for the isolation of Y. enterocolitica and Y. pseudotuberculosis.

[Have you eaten any DNA today? Science communication during Science and Technology Week in Brazil]. / Você já comeu DNA hoje? Divulgação científica durante a Semana da Ciência e Tecnologia no Brasil.

During the first National Science and Technology Week held in 2004, science centers and museums, universities and schools engaged in activities with the idea of divulging science to the people. Demonstrations of the extraction of DNA from fruits were conducted in supermarkets in 11 Brazilian cities by two institutions, DNA Vai à Escola and Conselho de Informação e Biotecnologia. This article describes the formation of a national network of people interested in communicating information about genetics to the lay public and the implementation of a low-cost science communication activity in different parts of the country simultaneously. It also analyzes the impact caused by this initiative and the perceptions of those involved in its organization.

Performance assessment of a new indirect rapid diagnostic test for plague detection in humans and other mammalian hosts.

Plague is a flea-borne zoonosis that affects a wide range of mammals and still causes outbreaks in human populations yearly across several countries. While crucial for proper treatment, early diagnosis is still a major challenge in low- and middle-income countries due to poor access to laboratory infrastructure in rural areas. To tackle this issue, we developed and evaluated a new Fraction 1 capsular antigen (F1)-based rapid diagnostic test (RDT) as an alternative method for plague serological diagnosis and surveillance in humans and other mammals. In this study, 187 serum samples from humans, dogs, rodents and rabbits were retrospectively assessed using the plague RDT method. To calculate its performance, results were compared to those obtained by traditional hemagglutination (HA) and ELISA, which are well-established methods in the plague routine serodiagnosis. Remarkably, the results from RDT were in full agreement with those from the ELISA and HA assays, resulting in 100% (CI 95% = 95.5-100%) of sensitivity and 100% (CI 95% = 96.6-100%) of specificity. Accordingly, the Cohen's Kappa test coefficient was 1.0 (almost perfect agreement). Moreover, the RDT showed no cross-reaction when tested with sera from individuals positive to other pathogens, such as Y. pseudotuberculosis, Yersinia enterocolitica, Anaplasma platys, Ehrlichia canis and Leishmania infantum. Although preliminary, this study brings consistent proof-of-concept results with high performance of the Plague RDT when compared to HA and ELISA. Although further human and animal population-based studies will be necessary to validate these findings, the data presented here show that the plague RDT is highly sensitive and specific, polyvalent to several mammal species and simple to use in field surveillance or point-of-care situations with instant results.

A single introduction of Yersinia pestis to Brazil during the 3rd plague pandemic.

Yersinia pestis was introduced to Brazil during the third plague pandemic and currently exists in several recognized foci. There is currently limited available phylogeographic data regarding Y. pestis in Brazil. We generated whole genome sequences for 411 Y. pestis strains from six Brazilian foci to investigate the phylogeography of Y. pestis in Brazil; these strains were isolated from 1966 to 1997. All 411 strains were assigned to a single monophyletic clade within the 1.ORI population, indicating a single Y. pestis introduction was responsible for the successful establishment of endemic foci in Brazil. There was a moderate level of genomic diversity but little population structure among the 411 Brazilian Y. pestis strains, consistent with a radial expansion wherein Y. pestis spread rapidly from the coast to the interior of Brazil and became ecologically established. Overall, there were no strong spatial or temporal patterns among the Brazilian strains. However, strains from the same focus tended to be more closely related and strains isolated from foci closer to the coast tended to fall in more basal positions in the whole genome phylogeny than strains from more interior foci. Overall, the patterns observed in Brazil are similar to other locations affected during the 3rd plague pandemic such as in North America and Madagascar.

Rodents and other small mammal reservoirs in plague foci in northeastern Brazil.

INTRODUCTION: Plague is an acute, infectious zoonotic disease, primarily of wild rodents and their fleas, that affects humans and other mammals. In Brazil, several plague foci are located in the northeast region. Plague surveillance based on monitoring of rodents was discontinued in 2007, and the current information on rodent populations is unsatisfactory. Our purpose was to update the information on rodents and other small mammals in plague foci in northeastern Brazil. METHODOLOGY: Nine surveys in the historically most important northeastern plague areas were conducted in 2013-2015. RESULTS: In this study, 393 animals (13 rodent and four marsupial species) were entrapped. The plague bacterium Yersinia pestis was not detected in tissue sample cultures from the 225 animals that were analyzed. Eighty sera samples were analyzed for anti-F1 antibodies by hemagglutination (HA) and protein A ELISA tests, and all were negative, except for one marsupial, Monodelphis domestica, which was HA positive. CONCLUSIONS: Qualitative and quantitative differences in the animal populations were observed in the areas surveyed, and the antibody positive marsupial indicated that plague continues to circulate in the wild.

Molecular analysis of clinical isolates of Providencia alcalifaciens.

In an attempt to elucidate the virulence factors and the pathogenic mechanisms of Providencia alcalifaciens, 36 isolates identified in 1994-1995 in Recife city, Brazil were analysed by PCR to investigate the presence of DNA sequences homologous to virulence genes described in other invasive enterobacteria, as well as their ability to invade HeLa cells, their plasmid profiles and antibiotic resistance patterns. The genetic diversity of the isolates was also analysed by RAPD-PCR. No homologous sequences of virulence genes were observed with any of the P. alcalifaciens isolates studied. Ten isolates had no plasmid and 26 harboured one-to-five plasmids of 147-<6.9 kb. Invasion of HeLa cells was observed in only 10 isolates. No correlation between the plasmid content of the strains, their invasion of HeLa cells or their resistance to antimicrobial drugs could be established. The isolates could be distributed into 10 genotypic groups by RAPD-PCR. Considering the genotypic profile and ability to invade HeLa cells, 7 of the 10 invasive isolates belonged to the same genotypic group. The presence of invasive isolates in the same or a related genotypic group suggests the existence of a clonal lineage responsible for the invasiveness.

Seroprevalence of hantavirus and Yersinia pestis antibodies in professionals from the Plague control program.

INTRODUCTION: Professionals who handle rodents in the field and in the laboratory are at risk of infection by the microorganis mharbored by these animals. METHODS: Serum samples from professionals involved in rodent and Yersinia pestis handling in field or laboratory work were analyzed to determine hantavirus and plague seroprevalence and to establish a relationship between these activities and reports of illnesses. RESULTS: Two individuals had antibodies against hantavirus, and two harbored antibodie against the plague; none of the individuals had experienced an illness related to their duties. CONCLUSIONS: These results confirm the risks of hantavirus- and plague-related field and laboratory activities and the importance of protective measures for such work.

Voce ja comeu DNA hoje? Divulgacao cientifica durante a Semana da Ciencia e Tecnologia no Brasil / Have you eaten any DNA today? Science communication during Science and Technology Week in Brazil

Durante a Primeira Semana Nacional de Ciências e Tecnologia, em 2004, centros e museus de ciência, universidades e escolas implementaram atividades com o objetivo de divulgar ciência para a população. O DNA Vai à Escola se juntou ao Conselho de Informação e Biotecnologia e praticou a extração de DNA de frutas em supermercados de 11 cidades brasileiras. Este artigo descreve a formação da rede nacional de pessoas interessadas em transmitir informações sobre genética para o público leigo e a implementação de uma atividade de divulgação científica de baixo orçamento em vários pontos do país, simultaneamente. Apresenta ainda o impacto causado pela atividade e as percepções daqueles envolvidos na sua organização.

During the first National Science and Technology Week held in 2004, science centers and museums, universities and schools engaged in activities with the idea of divulging science to the people. Demonstrations of the extraction of DNA from fruits were conducted in supermarkets in 11 Brazilian cities by two institutions, DNA Vai à Escola and Conselho de Informação e Biotecnologia. This article describes the formation of a national network of people interested in communicating information about genetics to the lay public and the implementation of a low-cost science communication activity in different parts of the country simultaneously. It also analyzes the impact caused by this initiative and the perceptions of those involved in its organization.

Soroprevalência da infecção por hantavírus em roedores do estado do Ceará, Brasil / Seroprevalence of hantavirus infection in rodents from the State of Ceara, Brazil

A síndrome pulmonar cardiovascular por hantavírus (SPCVH) e a peste (infecção pela Yersinia pestis) são zoonoses que ocorrem no Brasil. As áreas de distribuição dos dois agravos poderão se sobrepor, pois eles compartilham os mesmos reservatórios. Nosso objetivo foi analisar a prevalência de anticorpos contra hantavírus em roedores de áreas focais de peste no estado do Ceará. A amostra foi composta por 239 animais: 136 Necromys lasiurus, 31 Oligoryzomys nigripes, 33 Oryzomys subflavus, 4 Holochilus sciureus, 24 Rattus rattus, 1 Mus musculus, 8 Trichomys apereoides e 2 Galea spixii. Os soros de dois N. lasiurus capturados na serra da Ibiapaba apresentaram anticorpos anti-hantavirus, evidenciando que as populações humanas dessas áreas estão sob risco de contrair uma infecção potencialmente letal, o que requer a estruturação de um programa de pesquisas com o objetivo de estudar sistematicamente a hantavirose.

The hantavirus cardiovascular pulmonary syndrome (HCVPS) and the plague (Yersinia pestis infection) are zoonoses that occur in Brazil, having in common rodent reservoirs. Therefore their distribution areas may overlap. The prevalence of hantavirus antibodies in animals caught in the routine activities of the Plague ControlProgram (PCP) in the State of Ceará in the period from 2002 to 2005 was analyzed in the rodent population in the plague areas. The sample consisted of 239 animals: 136 Necromys lasiurus, 31 Oligoryzomys nigripes, 33 Oryzomys subflavus, 4 Holochilus sciureus, 24 Rattus rattus, 1 Mus musculus, 8, Trichomys apereoides and 2 Galea spixii. Two N. lasiurus captured in the Serra da Ibiapaba were positive forhantavirus antibodies, indicating that humans in these areas are at risk of contracting a potentially lethal infection what justifies the implementation of a research program to study systematically the hantaviruses and rodent hosts in this area.

Homology among extra-cryptic DNA bands and the typical plasmids in brazilian Yersina pestis strains

Yersina pestis, the etiologic agent of plague, harbors three well-characterized plasmids: pFra (90-110kb), pYV (70kb) and pPst (9.5kb). Furthermore, some extra-cryptic DNA bands have been observed in a number of wild strains from several foci of the world. Additional bands have also been reported in Brazilian strains. Looking for any relationship among these cryptic DNA bands and the three-prototypical plasmids, we analyzed twelve strains displaying different plasmid content. The DNA bands were hybridized by southern blot with probes directed at the genes cafl, lcrV and 'pla' located respectively on the plasmids pFra, pYV and pPst. The probes were constructed by PCR amplification and labeled with digoxigenin. The Pla probe hybridized with its target (pPst) and with bands of about 35 kb suggesting some homology among them. The Cafl probe hybridized with the target (pFra) as well as with higher bands. The LcrV also hybridized with the target (pYV) and both with the bands higher than pFra and the bands between pFra and pYV. These results suggest that the large-cryptic bands could represent some rearrangement, open circular or linearized forms of the pFras and pYV plasmids.