Assessment of cadmium tolerance and biosorptive potential of Bacillus Cereus GCFSD01 isolated from cadmium contaminated soil / Avaliação da tolerância ao cádmio e do potencial biossorptivo de Bacillus Cereus GCFSD01 isolado de solo contaminado com cádmio

Continuous occurrence of heavy metals is a major cause of environmental pollution due to its toxic effects. At minimum concentrations, these metals are highly reactive and can gather in the food chains and food web, causing major dangers to public health concerns. Soil samples were collected from Paharang drain, Faisalabad. Cadmium tolerant bacteria were isolated and evaluated for their MIC against Cd. The isolated bacterial strain GCFSD01 showed MIC value upto 30 mM/L. The bacterial strain with the highest resistance against Cd was selected for further study. Molecular characterization of bacterial isolate GCFSD01 was performed by 16S rRNA which confirmed it as Bacillus cereus. Optimum growth conditions of bacterial strain were also evaluated. Strain GCFSD01 showed optimum growth at pH 7 and 37 °C temperature. Our result revealed that B. cereus strain GCFSD01 reduced 61.3% Cd after 48 hrs. Multiple metal tolerance and Cd reduction by B. cereus indicate its potential for further use for decontamination of polluted soil.

A ocorrência contínua de metais pesados é uma das principais causas de poluição ambiental devido aos seus efeitos tóxicos. A contaminação por metais pesados representa um grande risco para todas as formas de vida encontradas no meio ambiente. Em concentrações mínimas, esses metais são altamente reativos e podem se acumular nas cadeias alimentares e na cadeia alimentar, causando grandes perigos às preocupações com a saúde pública. Amostras de solo foram coletadas no esgoto de Paharang, Faisalabad. Bactérias tolerantes ao cádmio foram isoladas da amostra coletada pelo método da placa de ágar. As colônias separadas individuais selecionadas foram avaliadas quanto às suas concentrações inibitórias mínimas contra Cd. A cepa bacteriana isolada GCFSD01 apresentou valores de CIM de 30 mM/L. A colônia bacteriana que apresentou maior resistência contra o Cd foi selecionada para identificação. Após seleção da maior colônia bacteriana resistente ao Cd, coloração de Gram e diferentes testes bioquímicos foram realizados para a caracterização da bactéria isolada. A caracterização molecular do isolado bacteriano GCFSD01 foi realizada por PCR 16S rRNA confirmando a presença de Bacillus cereus. Após a identificação molecular, as condições ótimas de crescimento da cepa bacteriana também foram verificadas. A cepa GCFSD01 apresentou crescimento ótimo em pH 7 e temperatura de 37 °C. Nosso resultado revelou que a cepa de B. cereus GCFSD01 reduziu 61,3% de Cd após 48 horas. A tolerância a múltiplos metais e a redução de Cd por B. cereus indicam seu potencial para uso posterior na descontaminação do solo poluído.

Presencia de los genes de toxigenicidad nheA, nheBy nheC en cepas de Bacillus cereus aisladas de leches deshidratadas en Costa Rica / Detection of toxigenic genes nheA, nheB and nheC in Bacillus cereus strains isolated from powdered milk samples in Costa Rica

La leche en polvo es un producto de alto consumo humano que no precisa de ser conservado en frío, no obstante, diversos microorganismos pueden deteriorarlo. En la población costarricense, también se observa este alto consumo, por la facilidad del alimento para transporte, preparación y su costo competitivo. Bacillus cereus es una bacteria potencialmente patógena asociada a este tipo de producto, capaz de desarrollar toxinas dependiendo de la presencia o ausencia de los respectivos genes codificantes. En este estudio se determinó la presencia de los genes toxigénicos nheA, nheB y nheC en cepas de B. cereus aisladas de leche deshidratada vendida en el mercado nacional costarricense.Se examinaron cinco lotes diferentes, de diez marcas comerciales de leche en polvo distribuidos en el área metropolitana de San José Costa Rica. Se procedió a cuantificar B. cereus en las muestras de leche en polvo mediante la técnica de Número Más Probable (NMP) e identificar los aislamientos utilizando el equipo automatizado Vitek®. Adicionalmente, se determinó la presencia de los genes nheA, nheB y nheC mediante la técnica de PCR. La frecuencia de aislamiento de Bacillus cereus en las muestras de leche en polvo analizadas alcanzó un 50%, con cantidades que oscilaron entre 3 y >100 NMP/g. Se recuperaron 19 cepas de B. cereus aisladas, cinco fueron positivas para los tres genes toxigénicos, lo cual revela la presencia de B. cereus potencialmente toxigénico en leches deshidratadas del mercado nacional, lo que representa un riesgo para la salud pública.

Powdered milk is a frequently consumed product that does not need to be kept under cold conditions. Nevertheless, different microorganisms may contaminate it. Powdered milk is a highly consumed product by Costa Rican population, and Bacillus cereus is a potentially pathogenic bacteria associated to it, with the ability to develop toxins depending on the presence of the respective codifying genes. The aim of this study was to determine the presence of the toxigenic genes nheA, nheB and nheC from B. cereus strains, found in powdered milk sold at the Costa Rican national market. Five different lots of ten brands of powdered milk, distributed in the metropolitan area of San José, Costa Rica were analyzed. B cereus load was quantified using the Most Probable Number technique and identified using the Vitek® system. The presence of the toxigenic genes was determined using the PCR technique. The isolation frequency of this bacteria in the powdered milk samples analyzed reached 50%, with populations ranging from 3 to >100 MPN/g. Five out from nineteen strains were found positive for the three toxigenic genes, indicating contamination with potentially toxigenic B. cereus in powdered milk distributed in the national market, and an important risk for public health.

Gene detection and toxin production evaluation of hemolysin BL of Bacillus cereus isolated from milk and dairy products marketed in Brazil

Bacillus cereusis an ubiquitous, spore-forming bacteria that can survive pasteurization and the majority of the heating processes used in the dairy industry. Besides, it is a pathogen responsible for different types of food poisoning. One type of foodborne disease caused by B.cereusis the diarrheal syndrome, which is caused by the ingestion of vegetative cells producing toxins in the small intestine. One virulence factor for the diarrheal syndrome is the toxin hemolysin BL (HBL), a three-component protein formed by the L1, L2 and B components. In order to evaluate the presence of diarrheal strains isolated from milk and dairy products, 63 B. cereus isolates were obtained from 260 samples of UHT milk, pasteurized milk and powdered milk, sold in commercial establishments and from different brands. The isolates were subjected to the Polymerase Chain Reaction (PCR) for the detection of the encoding genes for the L1, L2 and B components and the toxin production capacity were evaluated with an immunoassay. A total of 23 [36.5%] isolates were identified carrying simultaneously the three tested genes, from which, 20 [86.9%] showed toxigenic capacity. 26 [41.3%] isolates did not carry any of genes tested and the other 14 [22.2%] were positive for one or two of them. The results showed a high toxigenic capacity among the B. cereus isolates able to produce the HBL, indicating a potential risk for consumers.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Phenotypic & genetic characterization of Bacillus cereus isolated from the acute diarrhoeal patients.

Background & objectives: Bacillus cereus is one of the pathogens responsible for human diarrhoea, mainly due to consumption of contaminated food. The present study was undertaken to determine the occurrence of B. cereus among diarrhoeal patients and its phenotypic and genetic characteristics that determine the virulence and clonal features. Methods: Stool specimens were collected for two years from acute diarrhoeal patients attending the two referral hospitals in Kolkata. Presence of virulence genes in B. cereus was determined by PCR. Clonality was assessed by pulsed-field gel analysis (PFGE) by restriction digestion with SmaI and NotI enzymes. Enterotoxins were detected by haemolysin assay and using BCET-RPLA kit. Invasion assay was done on Hep-2 cell line. Antimicrobial susceptibility was tested by disc diffusion method. Results: B. cereus was identified in 54 (3.5%) of the 1536 diarrhoeal cases studied. Majority of the isolates were susceptible to many antibiotics but showed resistant to amoxyclav and cephalosporins. Six genes covering the two different enterotoxic complexes determining the pathogenicity of B. cereus have been characterized by PCR. The nhe genes were detected in a higher proportion than hbl. Except in two, clonal diversity was noticed among 21 B. cereus isolates. Haemolytic enterotoxin was detected in 76 per cent of the isolates. Majority of the isolates (67%) produced in vitro enterotoxin (BCET) confirming its involvement in the infection. Interpretation & conclusions: Though the presence of B. cereus was not high in patients with diarrhoea, several virulence factors confirm their association with diarrhoea. Distinct clonality was identified in majority of the isolates indicating their origin from different sources.

Enterotoxigenic gene profiles of Bacillus cereus and Bacillus megaterium isolates recovered from honey / Búsqueda de factores de virulencia en cepas de Bacillus cereus y de Bacillus megaterium aisladas de miel

One hundred and thirty two Bacillus cereus and 52 Bacillus megaterium isolates from honeys were evaluated for the presence of genes encoding enterotoxin HBL, enterotoxin-T, cytotoxin K and the NHE complex, respectively. The relationship between hemolytic and coagulase activity and its correlation with the presence of the four mentioned enterotoxins was determined by principal component analysis (PCA). PCA in B. cereus revealed a positive correlation among free coagulase, hemolysis and the presence of genes hblA, hblB, hblC, hblD (HBL complex) and bceT (enterotoxin-T), but no correlation with the clumping factor (bound coagulase) and the presence of sequences of the NHE complex. On the other hand, PCA in B. megaterium showed a high positive correlation between coagulase (bound and free) and the haemolytic activity but no correlation in relation to the presence of genes of the HBL complex, cytotoxin K, enterotoxin T and the NHE complex. To our knowledge, this is the first report of the detection of cytotoxin K and of the NHE complex genes in B. megaterium. The relationship between the coagulase activity and the presence of virulence factors has not been described before in the genus Bacillus, being this work the first report of this correlation. Interestingly, the presence of the cytK gene was almost independent of the presence of the rest of virulence factors herein analyzed both in B. cereus and B. megaterium populations. Our results suggest that honey could be a possible vehicle for foodborne illness due to the presence of toxigenic B. cereus and B. megaterium strains containing different virulence factors.

Se evaluaron 132 aislamientos de Bacillus cereus y 52 de Bacillus megaterium provenientes de mieles de distintos orígenes geográficos para investigar la presencia de secuencias de ADN relacionadas con genes de virulencia y su posible correlación con la actividad hemolítica y coagulasa. Con respecto a los genes de virulencia, se analizaron por PCR secuencias de ADN de los genes nhe (A, B y C), HBL (A, B, C, D), cytK y bceT. La relación entre las variables fue evaluada mediante un análisis de componentes principales, donde se encontró que los aislamientos de B. cereus mostraron una correlación positiva entre actividad de coagulasa (coagulasa libre) y presencia de los genes del complejo HBL y bceT, mientras que en B. megaterium se halló una alta correlación positiva entre actividad de coagulasa (libre y fija) y actividad hemolítica, pero no se observó correlación significativa entre la presencia de genes de virulencia y dichas actividades. Este estudio constituye el primer registro de la presencia de los genes cyt K y NHE en cepas de B. megaterium y el primer trabajo que analiza la relación entre la actividad de coagulasa y la presencia de genes de virulencia en B. cereus y B. megaterium. La presencia del gen cytK en ambas especies resultó totalmente independiente del resto de los factores de virulencia analizados. Nuestros hallazgos sugieren que la miel podría vehiculizar enfermedades transmisibles por alimentos debido a la presencia de cepas de B. cereus y B. megaterium potencialmente tóxicas.

Antibacterial & antiplasmid activities of Helicteres isora L.

Background & objectives: The multiple drug resistance (MDR) is a serious health problem and major challenge to the global drug discovery programmes. Most of the genetic determinants that confer resistance to antibiotics are located on R-plasmids in bacteria. The present investigation was undertaken to investigate the ability of organic extract of the fruits of Helicteres isora to cure R-plasmids from certain clinical isolates. Methods: Active fractions demonstrating antibacterial and antiplasmid activities were isolated from the acetone extracts of shade dried fruits of H. isora by bioassay guided fractionation. Minimal inhibitory concentration (MIC) of antibiotics and organic extracts was determined by agar dilution method. Plasmid curing activity of organic fractions was determined by evaluating the ability of bacterial colonies (pre treated with organic fraction for 18 h) to grow in the presence of antibiotics. The physical loss of plasmid DNA in the cured derivatives was further confirmed by agarose gel electrophoresis. Results: The active fraction did not inhibit the growth of either the clinical isolates or the strains harbouring reference plasmids even at a concentration of 400 μg/ml. However, the same fraction could cure plasmids from Enterococcus faecalis, Escherichia coli, Bacillus cereus and E. coli (RP4) at curing efficiencies of 14, 26, 22 and 2 per cent respectively. The active fraction mediated plasmid curing resulted in the subsequent loss of antibiotic resistance encoded in the plasmids as revealed by antibiotic resistance profile of cured strains. The physical loss of plasmid was also confirmed by agarose gel electrophoresis. Interpretation & conclusions: The active fraction of acetone extract of H. isora fruits cured R-plasmids from Gram-positive and Gram-negative clinical isolates as well as reference strains. Such plasmid loss reversed the multiple antibiotic resistance in cured derivatives making them sensitive to low concentrations of antibiotics. Acetone fractions of H. isora may be a source to develop antiplasmid agents of natural origin to contain the development and spread of plasmid borne multiple antibiotic resistance.

PCR-based detection of enterotoxigenic isolates of Bacillus cereus from tropical seafood.

Background & objectives: Bacillus cereus is an important enterotoxigenic food borne pathogen. The present study was undertaken to assess the occurrence of B. cereus in tropical fish and evaluation of virulent gene specific PCR for differentiation of diarrhoeal enterotoxin producing isolates of B. cereus from non enterotoxigenic isolates. Methods: Selective plating on polymixin-pyruvate-egg yolk-mannitol-bromocresol purple agar (PEMPA) was used for isolation of B. cereus from finfish, prawn and clams. Enterotoxin producing ability of all 42 isolates obtained from the samples was judged by reverse passive latex agglutination (RPLA) test and the presence of different virulent genes i.e. hbla, bceT and entFM was screened by PCR. Results: B. cereus and enterotoxigenic B. cereus were found to be in 36.7 and 29.41 per cent of fish samples, respectively. All the diarrhoeal enterotoxin producing isolates showed the presence of hbla gene, but hbla gene was not present in any of the non-enterotoxigenic isolates tested in this study. Interpretation & conclusions: Our findings indicated that hbla gene specific PCR can be employed for differentiation of enterotoxigenic B. cereus isolates from non-enterotoxigenic isolates.

Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples / Detección de determinantes de resistencia a tetraciclina y oxitetraciclina en cepas de Bacillus cereus aisladas de muestras de miel

The aim of this study was to investigate the presence of tetracycline and oxytetracycline resistance determinants in Bacillus cereus strains isolated from honey samples. Of a total of 77 isolates analyzed, 30 (39%) exhibited resistance to tetracyclines according to the results of a disk diffusion method. Resistant strains (n=30) were screened by PCR for the presence of the resistant determinants tetK, tetL, tetM, tetO, tetW, otrA and otrB and their MIC values for tetracycline, oxytetracycline and minocycline were assessed. According to the PCR results, 23 isolates (77%) presented at least one tetracycline or oxytetracycline resistance determinant. The tetK genotype was present in 10 isolates while the tetL, tetM, and otrA genotypes were present in 3, 2, and 5 isolates, respectively. In addition, 2 isolates of the tetK plus tetM genotype, 1 of the tetK plus tetL genotype, and 1 of the tetK plus otrA genotype were found. All isolates were tetW, tetO and otrB negatives. On the other hand, 7 isolates (23%) showed a tetracycline-resistant and/or minocyclineresistant phenotype (MIC) but did not carry any of the tet or otr determinants investigated in this study. This research has shown that B. cereus isolates from honey samples contain a variety of tetracycline and oxytetracycline resistance genes, including the tetK and tetL determinants which encode for efflux proteins, and tetM and otrA, which encode for ribosomal protection proteins. These findings indicate that strains isolated from honeys could represent a reservoir for tetracycline resistance genes. To our knowledge, this is the first report of tetracycline-resistant and oxytetracyclineresistant B. cereus strains carrying the tetK determinant, and also the first report of oxytetracycline-resistant and tetracycline- resistant Bacillus species carrying the otrA determinant.

El objetivo del presente estudio ha sido investigar la presencia de diversos determinantes de resistencia a tetraciclina y oxitetraciclina en las poblaciones de Bacillus cereus presentes en la miel. De un total de 77 aislamientos evaluados, 30 (39%) resultaron resistentes a tetraciclina y/o minociclina de acuerdo con los resultados de las pruebas de difusión en disco. Dentro del grupo que presentó un fenotipo resistente, se investigó la presencia de los determinantes tetK, tetL, tetM, tetO, tetW, otrA y otrB por PCR y se determinaron los valores de CIM para tetraciclina, oxitetraciclina y minociclina. De acuerdo con los resultados obtenidos por PCR, 23 aislamientos (77%) presentaron al menos un determinante de resistencia a tetraciclina o a oxitetraciclina; el genotipo tetK se encontró en 10 de esos aislamientos, mientras que los genotipos tetL, tetM y otrA se hallaron en 3, 2 y 5 aislamientos, respectivamente. Ningún aislamiento presentó los genotipos tetW, tetO ni otrB. Adicionalmente, se encontraron los genotipos tetK plus tetM (2 aislamientos); tetK plus tetL (1 aislamiento) y tetK plus otrA (1 aislamiento). Por otra parte, 7 cepas (23%) resultaron resistentes a tetraciclina, oxitetraciclina y/o minociclina por CIM, pero no presentaban ninguno de los determinantes tet u otr estudiados. Estos resultados indican la existencia de un alto porcentaje de cepas de B. cereus aisladas de miel con genes de resistencia a tetraciclina y oxitetraciclina, incluyendo los determinantes tetK, tetL, tetM y otrA. Este estudio constituye el primer registro de la presencia del determinante tetK de resistencia a tetraciclina en B. cereus, como así también la presencia del determinante otrA dentro del género Bacillus.

Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting

The bacterial strain Bacillus cereus is closely related to Bacillus thuringiensis, although any genetic relationship between the two strains is still in debate. Using rep-PCR genomic fingerprinting, we established the genetic relationships between Brazilian sympatric populations of B. cereus and B. thuringiensis simultaneously collected from two geographically separate sites. We observed the formation of both B. thuringiensis and B. cereus clusters, as well as strains of B. cereus that are more closely related to B. thuringiensis than to other B. cereus strains. In addition, lower genetic variability was observed among B. thuringiensis clusters compared to B. cereus clusters, indicating that either the two species should be categorized as separate or that B. thuringiensis may represent a clone from a B. cereus background.

Influence of cellular differentiation on ultraviolet induced DNA damage and its repair mechanisms in B. cereus.

Susceptibility to UV irradiation of B. cereus BIS-59 spores undergoing germination at various stages-dormant spores to vegetative cell stage and their ability to recover from radiation damage were studied. For a given dose of radiation, the number of spore photoproducts (SPP) formed in the DNA of dormant spores was about 5-times greater than that of thymine dimers (TT) formed in the DNA of vegetative cells. At intermediate stages of the germination cycle, there was a rapid decline in the UV radiation-induced SPP formed in DNA with a concomitant increase in the UV radiation-induced TT formed in DNA. Bacterial spores undergoing germination (up to 3 hr) in the low nutrient medium (0.3% yeast extract) displayed much higher resistance to UV radiation than those germinating in the rich nutrient medium, even though there was no discernible difference under the two incubation conditions in respect of the extent of germination and the time at which the outgrowth stage appeared (3 hr). This was due to the formation TT in the DNA of spores germinating in the low nutrient as compared to that of spores germinating in the rich-nutrient medium. In UV-irradiated dormant spores, SPP formed in the spore DNA did not disappear even after prolonged incubation in the non-germinating medium. However, when the UV-irradiated dormant spores were germinated in low or rich nutrient medium, a significant proportion of SPP in DNA was eliminated. The dormant spores incubated in either of the germinating media for 15 min and then UV-irradiated were capable of eliminating SPP (presumably by monomerization) even by incubation in a non-germinating medium and in the complete absence of protein synthesis (buffer holding recovery), thereby implying that spore-repair enzymes were activated in response to initial's germination. The acquisition of photo-reactivation ability appeared in spores subjected to germination only in the rich-nutrient medium at the outgrowth stage and required de novo synthesis of the required enzymes.