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.

Comparative transcriptomics analysis of multidrug-resistant Acinetobacter baumannii in response to treatment with the terpenic compounds thymol and carvacrol.

Acinetobacter baumannii is a gram-negative opportunistic bacterium that has become a major public health concern and a substantial medical challenge due to its ability to acquire multidrug resistance (MDR), extended-drug resistance, or pan-drug resistance. In this study, we evaluated the antibacterial activity of thymol and carvacrol alone or in combination against clinical isolates of MDR A. baumannii. Additionally, we used RNA-sequency to perform a comparative transcriptomic analysis of the effects of carvacrol and thymol on the Acb35 strain under different treatment conditions. Our results demonstrated that thymol and carvacrol alone, effectively inhibited the bacterial growth of MDR A. baumannii isolates, with a minimum inhibitory concentration (MIC) lower than 500 µg/mL. Furthermore, the combination of thymol and carvacrol exhibited either synergistic (FICI ≤ 0.5) or additive effects (0.5 < FICI ≤ 4), enhancing their antibacterial activity. Importantly, these compounds were found to be non-cytotoxic to Vero cells and did not cause hemolysis in erythrocytes at concentrations that effectively inhibited bacterial growth. Transcriptomic analysis revealed the down-regulation of mRNA associated with ribosomal subunit assemblies under all experimental conditions tested. However, the up-regulation of specific genes encoding stress response proteins and transcriptional regulators varied depending on the experimental condition, particularly in response to the treatment with carvacrol and thymol in combination. Based on our findings, thymol and carvacrol demonstrate promising potential as chemotherapeutic agents for controlling MDR A. baumannii infections. These compounds exhibit strong antibacterial activity, particularly in combination and lower cytotoxicity towards mammalian cells. The observed effects on gene expression provide insights into the underlying mechanisms of action, highlighting the regulation of stress response pathways.

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.

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.

Identification and characterization of bacterial biofilm production in clinical isolates of Staphylococcus spp. against antimicrobials / Identificação e caracterização da produção de biofilme bacteriano em isolados clínicos de Staphylococcus spp. frente aos antimicrobianos / Identificación y caracterización de la producción de biopelículas bacterianas en aislamientos clínicos de Staphylococcus spp. contra los antimicrobianos

Justification and Objectives: Circulating blood is sterile and the presence of microorganisms can be of clinical interest, especially in the hospital environment, being able to cause infectious processes and substantially increase morbidity and mortality. The objective of this work was to characterize the isolates of the genus Staphylococcus spp. from bloodstream infections as to the production of bacterial biofilm and resistance to the main antimicrobials used in clinical practice. Methods: Blood cultures were collected with an indication of positivity for bacterial growth from multiple sectors of the study hospital, which were subsequently processed to identify the bacterial genus through the use of phenotypic tests for Gram positive bacteria. The verification of the resistance profile was performed following the Kirby-Bauer disk diffusion. The identification of the production and quantification of the bacterial biofilm occurred following the protocol described by O'toole (2010). Results: The most frequent clinical isolate was Coagulase negative Staphylococci 38 (54.29%), followed by Staphylococcus aureus 32 (45.71%). Resistance to erythromycin, norfloxacin, levofloxacin and azithromycin was observed in most isolates (70%). Regarding methicillin, more MRSA (59.38%) than MR-CONS (47.37%) were isolated. The ICU was the place where the formation of the biofilm showed indicative data of greater adherence, which was associated with MRSA strains. Conclusion: The bacterial isolates associated with bloodstream infections showed high resistance to antimicrobials. The presence of MRSA and MR-CONS with strong and/or moderate biofilm production capacity represents a greater risk to the health of patients affected by infections caused by these agents.(AU)

Justificativa e Objetivos: O sangue circulante é estéril e a presença de microrganismos pode ter interesse clínico, especialmente no ambiente hospitalar, sendo capaz de causar processos infecciosos e aumentar substancialmente a morbimortalidade. O objetivo deste trabalho foi caracterizar os isolados do gênero Staphylococcus spp. oriundos de infecções de corrente sanguínea quanto à produção de biofilme bacteriano e resistência aos principais antimicrobianos utilizados na prática clínica. Métodos: Foram coletadas hemoculturas com indicação de positividade para o crescimento bacteriano de múltiplos setores do hospital de estudo, as quais posteriormente foram processadas para identificação do gênero bacteriano através da utilização de testes fenotípicos para bactérias Gram positivas. A verificação do perfil de resistência foi realizada seguindo a metodologia de disco difusão de Kirby-Bauer. A identificação da produção e quantificação do biofilme bacteriano ocorreu seguindo o protocolo descrito por O'toole (2010). Resultados: O isolado clínico mais frequente foi o Staphylococcus coagulase negativo 38 (54,29%), seguido pelo Staphylococcus aureus 32 (45,71%). A resistência à eritromicina, norfloxacina, levofloxacina e azitromicina foi observada na maioria dos isolados (70%). Em relação à meticilina, foram isolados mais Staphylococcus aureus resistente à meticilina (MRSA) (59,38%) que Staphylococcus coagulase negativa resistente à meticilina (MR-CONS) (47,37%). A UTI foi o local onde a formação do biofilme apresentou dados indicativos de maior aderência, sendo essa associada às cepas MRSA. Conclusão: Os isolados bacterianos associados às infecções da corrente sanguínea apresentaram elevada resistência aos antimicrobianos. A presença de MRSA e MR-CONS com forte e/ou moderada capacidade de produção de biofilme representa maior risco à saúde dos pacientes acometidos por infecções causadas por estes agentes.(AU)

Justificación y objetivos: la sangre circulante es estéril y la presencia de microorganismos puede ser de interés clínico, especialmente en el entorno hospitalario, ya que puede causar procesos infecciosos y aumentar sustancialmente la morbilidad y la mortalidad. El objetivo de este trabajo fue caracterizar los aislamientos del género Staphylococcus spp. de infecciones del torrente sanguíneo en cuanto a la producción de biopelículas bacterianas y la resistencia a los principales antimicrobianos utilizados en la práctica clínica. Métodos: Se recogieron hemocultivos con una indicación de positividad para el crecimiento bacteriano de múltiples sectores del hospital de estudio, que posteriormente se procesaron para identificar el género bacteriano mediante el uso de pruebas fenotípicas para bacterias Gram positivas. La verificación del perfil de resistencia se realizó siguiendo la metodología de difusión de disco de Kirby-Bauer. La identificación de la producción y cuantificación de la biopelícula bacteriana se produjo siguiendo el protocolo descrito por O'toole (2010). Resultados: El aislado clínico más frecuente fue Staphylococcus coagulasa negativo 38 (54.29%), seguido de Staphylococcus aureus 32 (45.71%). Se observó resistencia a la eritromicina, norfloxacina, levofloxacina y azitromicina en la mayoría de los aislamientos (70%). Con respecto a la meticilina, se aislaron más MRSA (59,38%) que MR-CONS (47,37%). La UCI fue el lugar donde la formación de la biopelícula mostró datos indicativos de una mayor adherencia, que se asoció con las cepas de MRSA. Conclusión: los aislamientos bacterianos asociados con infecciones del torrente sanguíneo mostraron una alta resistencia a los antimicrobianos. La presencia de MRSA y MR-CONS con una capacidad de producción de biopelículas fuerte y / o moderada representa un mayor riesgo para la salud de los pacientes afectados por infecciones causadas por estos agentes.(AU)

Diverse and emerging molecular mechanisms award polymyxins resistance to Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil.

OBJECTIVE: To describe the molecular mechanisms of polymyxins resistance in five Enterobacteriaceae clinical isolates from a tertiary hospital of Recife, Brazil. METHODS: The species identification and the susceptibility to antimicrobials were firstly performed by automatized methods and polymyxin resistance was confirmed by broth microdilution methods. The genetic basis of resistance was characterized with WGS analyses to study their resistome, plasmidome and mobilome, by BLAST searches on reference databases. RESULTS: Five (5%) Enterobacteriaceae isolates, comprising Escherichia coli (n = 2), Klebsiella pneumoniae (n = 2) and Citrobacter freundii (n = 1) species, exhibited polymyxin resistance. The mcr-1.1 gene was found in identical IncX4-plasmids harbored by both K. pneumoniae C119 (PolB MIC = 512 mg/L) and E. coli C153 (PolB MIC = 8 mg/L). The remaining E. coli strain C027 harbored the mcr-5.1 gene on an undefined Inc-plasmid (PolB MIC 256 mg/L). Some amino acid substitutions in PmrA (S29G, G144S), PmrB (S202P; D283G, W350*, Y258N) and PhoP (I44L) was detected among the E. coli clinical isolates, however they were also found in colistin-susceptible strains and predicted as neutral alterations. The mgrB of the ST54 KPC-2-producing K. pneumoniae C151 (PolB MIC = 32 g/mL) was interrupted at 69 nt by the IS903 element. The ST117 C. freundii C156 (PolB MIC = 256 mg/L) showed the A91T substitution on HAMP domain of the histidine kinase sensor CrrB, predicted as deleterious and deemed the remarkable determinant to polymyxins resistance in this strain. CONCLUSIONS: Diverse mechanisms of polymyxins resistance were identified among clinical Enterobacteriaceae from a tertiary hospital of Recife, Brazil, such as plasmid-mediated MCR-1 and MCR-5; IS903-interruption of mgrB and mutation in CrrAB regulatory system. These findings highlight the involvement of the identified plasmids on mcr dissemination among Enterobacteriaceae; warn about co-selection of the polymyxin-resistant and KPC-producer K. pneumoniae ΔmgrB lineage by carbapenems usage; and demonstrate potential role of CrrAB on emerging of polymyxin resistance among Enterobacteriaceae, besides Klebsiella species.

Multidrug-resistant Acinetobacter baumannii clones persist on hospital inanimate surfaces

ABSTRACT Acinetobacter baumannii is one of the most frequent Gram-negative opportunistic pathogens associated with hospital-acquired infection worldwide. We briefly describe A. baumannii isolates that were recovered from surrounding ICU bed surfaces, exhibiting multidrug resistance phenotype and belonging to some widely spread clonal complexes of clinical A. baumannii isolates.

Multidrug-resistant Acinetobacter baumannii clones persist on hospital inanimate surfaces.

Acinetobacter baumannii is one of the most frequent Gram-negative opportunistic pathogens associated with hospital-acquired infection worldwide. We briefly describe A. baumannii isolates that were recovered from surrounding ICU bed surfaces, exhibiting multidrug resistance phenotype and belonging to some widely spread clonal complexes of clinical A. baumannii isolates.

Resistência de bactérias isoladas em equipamentos em unidade de terapia intensiva / Resistance of bacteria isolated from equipment in an intensive care unit

Objetivo: Avaliar a resistência microbiana a medicamentos de bactérias isoladas de equipamentos próximos aos pacientes da Unidade de Terapia Intensiva. Métodos Trata-se de um estudo transversal. As amostras foram coletadas com swabs umedecidos em Trypticase Soy Broth, semeados posteriormente em Ágar Sangue de Carneiro e MacConkey. A identificação fenotípica ocorreu com base na morfologia das cepas e resultados bioquímicos. A análise da resistência aos medicamentos foi baseada no método de disco-difusão de Kirby-Bauer. Resultados Apresentaram-se contaminados 94,4% dos equipamentos analisados. Os microrganismos isolados mais frequentes foram: Acinetobacter sp., Staphylococcus aureus e Pseudomonas sp. Cerca de 75% de Acinetobacter sp. apresentaram resistência à piperacilina associada a tazobactam, meropenem e levofloxacina. Da mesma forma, 36,3% de S. aureus apresentaram-se resistentes à oxacilina e 10% dos isolados de Pseudomonas sp. foram resistentes aos medicamentos testados. Conclusão A maioria dos isolados apresentaram elevadas taxas de resistência microbiana aos medicamentos.

Objective: Evaluate drug resistance of bacteria isolated from equipment placed close to patients in an Intensive Care Unit of a hospital in Caruaru/Pernambuco, Brazil. Methods This is a cross-sectional study. The samples were collected with swabs moistened with Trypticase Soy Broth, which were then cultured in sheep blood agar and MacConkey agar. The phenotypic identification performed was based on the morphology of the strains and biochemical results. The drugs resistance analysis was based on Kirby-Bauer’s Disk Diffusion protocol. Results A rate of 94.4% of the analyzed equipment was contaminated. The most frequently isolated microorganisms were: Acinetobacter sp., Staphylococcus aureus and Pseudomonas sp. Just about 75% of Acinetobacter sp. was resistant to piperacillin associated to tazobactam, meropenem and levofloxacin. Similarly, 36.3% of S. aureus showed resistance to oxacillin and 10% of Pseudomonas sp. was resistant to the drugs tested. Conclusion Most of the microorganisms presented high levels of resistance to the drugs.