Novel Effective Bacillus cereus Group Species "Bacillus clarus" Is Represented by Antibiotic-Producing Strain ATCC 21929 Isolated from Soil.

Gram-positive, spore-forming members of the Bacillus cereus group species complex are widespread in natural environments and display various degrees of pathogenicity. Recently, B. cereus group strain Bacillus mycoides Flugge ATCC 21929 was found to represent a novel lineage within the species complex, sharing a relatively low degree of genomic similarity with all B. cereus group genomes (average nucleotide identity [ANI] < 88). ATCC 21929 has been previously associated with the production of a patented antibiotic, antibiotic 60-6 (i.e., cerexin A); however, the virulence potential and growth characteristics of this lineage have never been assessed. Here, we provide an extensive genomic and phenotypic characterization of ATCC 21929, and we assess its pathogenic potential in vitro. ATCC 21929 most closely resembles Bacillus paramycoides NH24A2T (ANI and in silico DNA-DNA hybridization values of 86.70 and 34.10%, respectively). Phenotypically, ATCC 21929 does not possess cytochrome c oxidase activity and is able to grow at a range of temperatures between 15 and 43°C and a range of pH between 6 and 9. At 32°C, ATCC 21929 shows weak production of diarrheal enterotoxin hemolysin BL (Hbl) but no production of nonhemolytic enterotoxin (Nhe); at 37°C, neither Hbl nor Nhe is produced. Additionally, at 37°C, ATCC 21929 does not exhibit cytotoxic effects toward HeLa cells. With regard to fatty acid composition, ATCC 21929 has iso-C17:0 present in highest abundance. Based on the characterization provided here, ATCC 21929T (= PS00077AT = PS00077BT = PSU-0922T = BHPT) represents a novel effective B. cereus group species, which we propose as effective species "Bacillus clarus"IMPORTANCE The B. cereus group comprises numerous closely related lineages with various degrees of pathogenic potential and industrial relevance. Species-level taxonomic classification of B. cereus group strains is important for risk evaluation and communication but remains challenging. Biochemical and phenotypic assays are often used to assign B. cereus group strains to species but are insufficient for accurate taxonomic classification on a genomic scale. Here, we show that antibiotic-producing ATCC 21929 represents a novel lineage within the B. cereus group that, by all metrics used to delineate prokaryotic species, exemplifies a novel effective species. Furthermore, we show that ATCC 21929 is incapable of producing enterotoxins Hbl and Nhe or exhibiting cytotoxic effects on HeLa cells at human body temperature in vitro These results provide greater insight into the genomic and phenotypic diversity of the B. cereus group and may be leveraged to inform future public health and food safety efforts.

Unique inducible filamentous motility identified in pathogenic Bacillus cereus group species.

Active migration across semi-solid surfaces is important for bacterial success by facilitating colonization of unoccupied niches and is often associated with altered virulence and antibiotic resistance profiles. We isolated an atmospheric contaminant, subsequently identified as a new strain of Bacillus mobilis, which showed a unique, robust, rapid, and inducible filamentous surface motility. This flagella-independent migration was characterized by formation of elongated cells at the expanding edge and was induced when cells were inoculated onto lawns of metabolically inactive Campylobacter jejuni cells, autoclaved bacterial biomass, adsorbed milk, and adsorbed blood atop hard agar plates. Phosphatidylcholine (PC), bacterial membrane components, and sterile human fecal extracts were also sufficient to induce filamentous expansion. Screening of eight other Bacillus spp. showed that filamentous motility was conserved amongst B. cereus group species to varying degrees. RNA-Seq of elongated expanding cells collected from adsorbed milk and PC lawns versus control rod-shaped cells revealed dysregulation of genes involved in metabolism and membrane transport, sporulation, quorum sensing, antibiotic synthesis, and virulence (e.g., hblA/B/C/D and plcR). These findings characterize the robustness and ecological significance of filamentous surface motility in B. cereus group species and lay the foundation for understanding the biological role it may play during environment and host colonization.

Characterization of Emetic and Diarrheal Bacillus cereus Strains From a 2016 Foodborne Outbreak Using Whole-Genome Sequencing: Addressing the Microbiological, Epidemiological, and Bioinformatic Challenges.

The Bacillus cereus group comprises multiple species capable of causing emetic or diarrheal foodborne illness. Despite being responsible for tens of thousands of illnesses each year in the U.S. alone, whole-genome sequencing (WGS) is not yet routinely employed to characterize B. cereus group isolates from foodborne outbreaks. Here, we describe the first WGS-based characterization of isolates linked to an outbreak caused by members of the B. cereus group. In conjunction with a 2016 outbreak traced to a supplier of refried beans served by a fast food restaurant chain in upstate New York, a total of 33 B. cereus group isolates were obtained from human cases (n = 7) and food samples (n = 26). Emetic (n = 30) and diarrheal (n = 3) isolates were most closely related to B. paranthracis (group III) and B. cereus sensu stricto (group IV), respectively. WGS indicated that the 30 emetic isolates (24 and 6 from food and humans, respectively) were closely related and formed a well-supported clade distinct from publicly available emetic group III genomes with an identical sequence type (ST 26). The 30 emetic group III isolates from this outbreak differed from each other by a mean of 8.3 to 11.9 core single nucleotide polymorphisms (SNPs), while differing from publicly available emetic group III ST 26 B. cereus group genomes by a mean of 301.7-528.0 core SNPs, depending on the SNP calling methodology used. Using a WST-1 cell proliferation assay, the strains isolated from this outbreak had only mild detrimental effects on HeLa cell metabolic activity compared to reference diarrheal strain B. cereus ATCC 14579. We hypothesize that the outbreak was a single source outbreak caused by emetic group III B. cereus belonging to the B. paranthracis species, although food samples were not tested for presence of the emetic toxin cereulide. In addition to showcasing how WGS can be used to characterize B. cereus group strains linked to a foodborne outbreak, we also discuss potential microbiological and epidemiological challenges presented by B. cereus group outbreaks, and we offer recommendations for analyzing WGS data from the isolates associated with them.

Telomere length as a potential biomarker of coronary artery disease.

Coronary artery disease (CAD) is a multifactorial disease whose prevalence remains unabated especially in developing countries. Both lifestyle factors and genetic predisposition contribute to this disorder. Though notable achievements have been made in the medical, interventional and surgical management of CAD, the need for its prevention is more important. Among other modalities, this calls for defining evidence-based new biomarkers, which on their own or in combination with other known biomarkers may predict the risk of CAD to enable institution of appropriate preventive strategies. In the present communication, we have discussed the usefulness of shortening of telomeres as a potential biomarker of CAD. Clinical research evidence in favour of telomere shortening in CAD is well documented in different ethnic populations of the world. Establishing a well-standardized and accurate method of evaluating telomere length is essential before its routine use in preventive cardiology.

Study on influence of age, gender and genetic variants on lactose intolerance and its impact on milk intake in adult Asian Indians.

BACKGROUND: Lactase non-persistence (LNP) has been associated with the CC genotype of -13910C > T and GG genotype of -22018G > A polymorphisms present upstream of the lactase gene. Lactose intolerance (LI) is caused when gastrointestinal symptoms develop in individuals with low lactase activity. OBJECTIVE: To analyse association of LNP genotype and LI symptoms with milk intake and determine whether factors such as age, gender and genotype affect LI status. SUBJECTS AND METHODS: Genetic analysis and lactose tolerance test (LTT) were performed on 205 healthy Indian adults. The pattern of milk consumption was recorded using a dietary questionnaire. RESULTS: LI was strongly associated with -13910CC genotype (OR = 10.28, 95% CI = 2.32-45.55, p = 0.002). Females were found to be at a higher risk of developing LI (OR = 2.47, 95% CI = 1.33-4.59, p = 0.004). The association of the ≥50 years age group with LI was marginally significant (OR = 1.86, 95% CI = 0.995-3.47, p = 0.05). Frequency and quantity of milk intake were lower in subjects belonging to the LNP genotype and LI groups (p < 0.05). CONCLUSIONS: Subject study suggests that gender and genotype may be associated with development of LI. Association of age with LI was marginal. The data also indicate that LNP genotype and LI may play a role in influencing milk intake in individuals.

Association of paraoxonase1 gene Q192R polymorphism and apolipoprotein B in Asian Indian women with coronary artery disease risk.

BACKGROUND/AIMS: Human serum paraoxonase1 (PON1), a high-density lipoprotein-associated enzyme, prevents oxidative modification of low-density lipoprotein and, thus, arrests the development of atheroma formation, whose major consequence is the development of coronary artery disease (CAD). A single-nucleotide polymorphism (SNP) Q192R in the coding region at pon1 locus is a determinant of PON1 activity. The relationship between PON1 activity and vascular disease may be influenced by the relationship of PON1 activity or PON1 SNP genotype to lipid and apolipoprotein (Apo) levels. The aim of the study is to ascertain the prevalence of PON1 Q192R polymorphism in male and female subjects with and without CAD along with its influence on ApoA-I and ApoB levels in Asian Indians. METHODS: Determination of genotypes was carried out in 249 diagnosed CAD cases and in 243 age-, gender-matched asymptomatic controls by polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma Apo-levels were estimated by immunoturbidimetric assay. RESULTS: The genotype frequencies did not differ markedly between the overall CAD and control groups and in male and female subjects, suggesting a lack of any genotype-CAD correlation. ApoB levels were found to be higher in female patients carrying RR when compared with QQ genotypes (p=0.03) with no effect on controls. This may be attributed to the postmenopausal state of the women. CONCLUSION: PON1 Q192R can be used as the DNA marker test to evaluate the risk of CAD in postmenopausal Indian women with high ApoB.

Full-gene-sequencing analysis of N-acetyltransferase-2 in an adult Indian population.

AIMS: Drug-metabolizing enzymes play a major role in determining the outcome of drug therapy. N-acetyltransferase-2 (NAT2) is one of the main enzymes involved in metabolism of isoniazid used in treatment of tuberculosis (TB). Several variations in the NAT2 gene give rise to multiple haplotypes that phenotypically code for different acetylator status. The objective was to generate a more unambiguous picture of the NAT2 scenario in India as compared to that obtained from polymerase chain reaction-restriction fragment length polymorphism methods. METHODS: Full-gene-sequencing analysis of NAT2 was carried out in 181 healthy Indian subjects from different regional groups. RESULTS: A total of 33 diplotypes were recorded from six known single-nucleotide polymorphisms. The overall frequency of the slow acetylator haplotypes detected in this study was 65%, followed by 26% and 9% intermediate and rapid acetylators, respectively. Of the slow acetylator alleles, the NAT2*5B/*6A occurred in 25% of the study subjects. CONCLUSIONS: The study indicates that the frequency of slow acetylator alleles is high in the adult Indian population. Since the prevalence of TB is high in this population, pharmacogenetic testing for NAT2 alleles may be advisable before start of therapy with isoniazid to prevent drug toxicity.

A study on genetic test of lactase persistence in relation to milk consumption in regional groups of India.

BACKGROUND: Lactase nonpersistence (LNP) is characterized by the decrease in lactase expression in the small intestine. Studies have shown that -13910 C>T and -22018 G>A single-nucleotide polymorphisms (SNPs) located upstream of the lactase gene are associated with an LNP/lactase persistence (LP) trait. OBJECTIVE: The current study evaluated the LP allelic frequency in 227 healthy Indian subjects consisting of North Indians, Maharashtrians, Gujaratis, Parsis, and South Indians, and for the first time assessed its relation with milk consumption pattern in Indian subjects. METHODS: The two SNPs were genotyped using the polymerase chain reaction and restriction fragment length polymorphism methods. The milk consumption pattern for the studied subjects was noted by questionnaire. RESULTS: The two SNPs were present in a strong linkage disequilibrium. LP prevalence varied in these Indian regional groups. The LP frequency was highest for North Indians and lowest for Parsis (p=0.03 CC vs. CT+TT, p=0.008 GG vs. GA+AA). South Indians had a lower LP frequency compared to North Indians (p=0.07 for each SNP). The milk consumption pattern varied in these Indian subgroups, with the Gujaratis exhibiting the highest milk intake and Parsis the lowest (p=0.04). CONCLUSION: Our study indicates that the milk intake in Indians might be influenced by their dietary habits in addition to their ancestral history. An overall correlation, however, between milk consumption and LP genotypes was not observed.

Genotype frequencies of drug-metabolizing enzymes responsible for purine and pyrimidine antagonists in a healthy Asian-Indian population.

Purine and pyrimidine antimetabolites are used to treat leukemias, autoimmune diseases, and solid tumors. Detection of slow metabolizers before administration of the drugs is necessary to prevent any subsequent drug toxicity. With this aim, we determined the frequencies of normal and slow alleles in our population. Polymorphisms in genes encoding cytidine deaminase (CDA), dihydropyrimidine dehydrogenase (DPYD), and thiopurine-S-methyltransferase (TPMT) were documented in 225 healthy volunteers. The polymorphisms typed included CDA*3, DPYD*2A, TPMT*2A, TPMT*3B, and TPMT*3C. Methods used for genotyping included standard PCR-RFLP and allele-specific PCR reactions. The frequencies were 0.44 % for DPYD*2A, 0.67 % for TPMT*3B, and 0.89 % for TPMT*3C. The CDA*3 and TPMT*2A alleles were not detected. Although these polymorphisms have been demonstrated to be associated with drug toxicity in other populations, they appear to be very rare in the adult Indian population.

Gene polymorphisms and low dietary intake of micronutrients in coronary artery disease.

BACKGROUND/AIMS: Coronary artery disease (CAD) is a complex disorder involving genetic and non-genetic factors. Food is an important component of the latter. We examined if DNA polymorphisms in genes encoding enzymes of one-carbon metabolism coupled with low consumption of micronutrients such as folate, vitamins B(6) and B(12) might increase the risk of CAD. METHODS: A case-control study consisting of 252 CAD patients and 252 controls were included. Three single nucleotide polymorphisms (SNP), 2 insertion/deletion and 1 repeat polymorphism were typed. The micronutrient intake was estimated from a standard 24-hour dietary recall coupled to a food frequency questionnaire. RESULTS: The results suggest an association of 'early-onset CAD' with betaine homocysteine S-methyl transferase (BHMT) 742G→A SNP (odds ratio = 1.52; 95% confidence interval, 0.96-2.41; p = 0.04). No association was observed for all age of onset, but more patients than controls whose micronutrient intake was in the lowest quintile also carried the minor allele (50% patients vs. 37% controls; p = 0.042). Furthermore, dietary intake of folate micronutrients below the recommended daily allowance was observed in a larger percent of patients than controls with the minor BHMT allele (51% patients vs. 44% controls; p = 0.021). CONCLUSIONS: In the presence of the minor BHMT allele, a decreased consumption of folate micronutrients might increase the risk of CAD.

Genetic studies on the APOA1-C3-A5 gene cluster in Asian Indians with premature coronary artery disease.

BACKGROUND: The APOA1-C3-A5 gene cluster plays an important role in the regulation of lipids. Asian Indians have an increased tendency for abnormal lipid levels and high risk of Coronary Artery Disease (CAD). Therefore, the present study aimed to elucidate the relationship of four single nucleotide polymorphisms (SNPs) in the Apo11q cluster, namely the -75G>A, +83C>T SNPs in the APOA1 gene, the Sac1 SNP in the APOC3 gene and the S19W variant in the APOA5 gene to plasma lipids and CAD in 190 affected sibling pairs (ASPs) belonging to Asian Indian families with a strong CAD history. METHODS & RESULTS: Genotyping and lipid assays were carried out using standard protocols. Plasma lipids showed a strong heritability (h2 48% - 70%; P < 0.0001). A subset of 77 ASPs with positive sign of Logarithm of Odds (LOD) score showed significant linkage to CAD trait by multi-point analysis (LOD score 7.42, P < 0.001) and to Sac1 (LOD score 4.49) and -75G>A (LOD score 2.77) SNPs by single-point analysis (P < 0.001). There was significant proportion of mean allele sharing (pi) for the Sac1 (pi 0.59), -75G>A (pi 0.56) and +83C>T (pi 0.52) (P < 0.001) SNPs, respectively. QTL analysis showed suggestive evidence of linkage of the Sac1 SNP to Total Cholesterol (TC), High Density Lipoprotein-cholesterol (HDL-C) and Apolipoprotein B (ApoB) with LOD scores of 1.42, 1.72 and 1.19, respectively (P < 0.01). The Sac1 and -75G>A SNPs along with hypertension showed maximized correlations with TC, TG and Apo B by association analysis. CONCLUSION: The APOC3-Sac1 SNP is an important genetic variant that is associated with CAD through its interaction with plasma lipids and other standard risk factors among Asian Indians.

Prevalence and component analysis of metabolic syndrome: an Indian atherosclerosis research study perspective.

Asian Indians have a high predisposition to metabolic syndrome (MS) and coronary artery disease (CAD). The present study aimed to estimate MS prevalence in 531 Asian Indian families comprising of 2318 individuals. Anthropometrics and lipid profile were assessed. MS prevalence was estimated using standard Adult Treatment Panel III (ATP-III) and World Health Organisation (WHO) criteria and modified definitions which included lowered cut-offs for waist circumference (WC) (> or =90 cm for men and > or =80 cm for women], body mass index (BMI) (> or =23 kg/m2) and impaired fasting glucose (IFG) levels. ATP-III criteria identified a significantly higher proportion of people with MS (N = 933; 40.3%) compared with WHO (N = 708; 30.6%; p < 0.0001) while modified ATP-III showed maximum gain in percent prevalence among the revised criteria (17.3%; p = 0.0056). The IDF criteria identified similar proportion of subjects with MS (N = 809; 34.9%) as the revised WHO criteria (N = 792; 34.2%). The number of MS subjects was highest in the 50-59 years age group. MS was diagnosed a decade earlier in unaffected subjects compared with those with CAD/diabetes using the modified MS criteria. WC correlated significantly with BMI and waist-hip ratio (WHR) (p = 0.000). Among MS components, high density lipoprotein cholesterol and BMI contributed significantly in males (71.4% and 85.9%) and females (86.8% and 88.8%), respectively. The higher percentage contribution of WC among males and WHR among females indicates the influence of gynecoid/android pelvis on WHR measures. In conclusion, the revision of definition criteria for MS with lowered cut-offs for WC and BMI is critical for the accurate assessment of MS among Asian Indians.

Adult nontwin sib concordance rates for type 2 diabetes, hypertension and metabolic syndrome among Asian Indians: the Indian Atherosclerosis Research Study.

Diabetes (DM), hypertension (HTN), and metabolic syndrome (MS) are established cardiovascular risk factors with a complex etiology. The aim of the present study was to estimate the rates of concordance for the above coronary risk factors between siblings in Asian Indian families with premature coronary artery disease (CAD). Spouse concordance rates were used to evaluate the relative contribution of shared genes and lifestyle towards these traits. A total of 508 families comprising of 1250 sib-pairs and 463 corresponding spouse-pairs were analyzed. Concordance rates were manually determined. Plasma lipids were estimated by standard enzymatic assay. The concordance rates among sib-pairs for DM, HTN, and MS was 11% (N = 136), 14% (N = 174), and 23% (N = 287), while the corresponding concordance for spouse-pairs was 2.8% (N = 13), 6.3% (N = 29), and 28.1% (N = 130), respectively. Employing Chi-square test, sib-pairs showed significantly higher concordance for diabetes (p < or = 0.0001) and hypertension (p < 0.0001) while spouse-pairs had higher concordance for metabolic syndrome (p = 0.033) in our study. These findings suggest a probable dominant genetic component in the causation of DM and HTN and a predominantly nongenetic component for metabolic syndrome among Asian Indians.