Bca Gene Research Articles

Genomic characterization and resistance features of Streptococcus agalactiae isolated from non-pregnant adults in Shandong, China

BackgroundStreptococcus agalactiae is a recognized pathogen that primarily affects infants and pregnant women. However, its increasingly important role in causing invasive infections among non-pregnant adults has become a significant health concern due to the severity and variety of its clinical impacts. MethodsNonduplicate S. agalactiae clinical strains associated with clinical infections (n = 139) were isolated from non-pregnant adults in Shandong, China. Antibiotic susceptibility testing, whole-genome sequencing and genomic analyses were conducted to characterize the genome and identify resistance features of these strains. ResultsThe strains exhibited universal susceptibility to penicillin, ampicillin, cefotaxime, meropenem, linezolid and vancomycin. Notably, high resistance rates were observed for erythromycin (91.4%), clindamycin (89.2%), levofloxacin (84.2%), tetracycline (54.0%) and, to a lesser extent, chloramphenicol (12.9%). Serotyping revealed seven serotypes and one non-typeable strain. Serotypes Ia, Ib, III and V predominated, representing 95.7% of the strains. Nineteen sequence types were categorized into seven clonal complexes, with CC10 being the most prevalent at 48.9%. The resistance genes mreA (100%), ermB (70.5%) and tetM (46.0%) were commonly detected. All the isolates carried at least one pilus backbone determinant and one alpha-like protein gene, with the PI-1+PI-2a and the bca gene being the most frequent at 84.2% and 54.7%, respectively. ConclusionsWhile S. agalactiae strains in non-pregnant adults retain sensitivity to β-lactam antibiotics, the elevated resistance to erythromycin, clindamycin, levofloxacin and tetracycline is concerning. Given the growing elderly population worldwide, the burden of S. agalactiae infections is significant. Continuous surveillance of serotype distribution and antibiotic resistance patterns is imperative for targeted prevention and therapeutic strategies.

Analysis of Virulence Genes of Staphylococcus aureus, Streptococcus, E. coli Isolated from Bovine Subclinical Mastitis

Background: The major mastitis-producing organisms are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli. Molecular characterization of the major bacterial pathogens for some genes responsible for their virulence should be considered the reduction of risk factors responsible for the presence and spread of contagious pathogens through milk. Methods: The two hundred milk samples were collected from cows and buffaloes of four different tehsils of the Sirohi district of Southern Rajasthan. Out of 200 milk samples, 74 milk samples that were found positive for SCM were cultured for primary isolation of predominant bacterial pathogens. Out of these 74 positive samples, total of 97 isolates were recovered from these milk samples either as a single or mixed infection. To genotypically characterize S. aureus isolates, genes encoding virulence determinants (spa-IgG-binding and Coa), Staphylococ cal enterotoxins (bac and bca) and E. coli Shiga toxins (stx1 and stx2) were investigated. Result: The etiological prevalence of SCM caused by different bacteria was, S. aureus, (27%), Streptococcus spp. (15%) and E. coli (6.5%) respectively either as single and or as mixed infections. All S. aureus isolates were tested by PCR for the presence of the spa gene and coa gene results revealed that 40 isolates (74.0%) carried both spa (IgG-binding) and coa gene. 11 isolates of S. agalactiae (36.6%) carried the bca gene. The bca gene codes for Alpha-C protein, a surface protein that helps the bacteria to enter the host cells. E. coli isolates were also screened for the presence of virulence gene stx1 and stx2 gene. Out of 13 isolates tested, 6 isolates harboured both stx1 and stx2 genes.

Virulence-genes Rib and Bca in Serotypes of Group B Streptococcus (GBS) Isolated from Symptomatic Pregnant Women in East Coast Malaysia

INTRODUCTION: Group B streptococcus (GBS) is a leading cause of maternally-acquired invasive infections in neonates. Nowadays maternal immunization is of utmost demand for prevention of these infections. We undertook capsular serotyping and virulence factor genes identification for local GBS isolates as a pilot study, to identify potential candidates to propagate vaccine development. MATERIALS AND METHODS: This is a descriptive lab -based study to determine GBS serotypes and presence of genes coding virulence factors bca and rib in isolates obtained from symptomatic pregnant women in Hospital Tengku Ampuan Afzan, Kuantan, Pahang, Malaysia. Sixty-two GBS isolates from high vaginal swabs were collected. Latex agglutination test was performed to determine GBS serotypes. Real-time PCR was done to determine the presence of virulence genes. RESULTS: Of the 62 GBS isolates, 77.4% were serologically typeable, and 22.6% were non -typeable. Serotypes Ia and Ib (16.1% each) were the most common capsular types, followed by II, V, and VII (9.7% each), III (8.1%), VI (6.5%), and VIII (1.6 %). Furthermore, 67.7% of the isolates harboured the rib gene while 98.4% possessed the bca gene. CONCLUSION: The five known prevalent serotypes worldwide, do not match the CPS distribution in symptomatic pregnant women in Kuantan. However, the frequency of virulence genes rib and bca is high among our isolates, which if confirmed by further bigger and wider studies makes the proteinaceous vaccine, N-terminal domains of Rib and AlpC a suitable candidate for GBS prevention in this geographical area.

Prevalence, serotypes and virulence genes of Streptococcus agalactiae isolated from pregnant women with 35\u201337\u2009weeks of gestation

BackgroundIn pregnant women Streptococcus agalactiae (GBS) can be transmitted to newborn causing severe infections. It is classified into 10 serotypes (Ia, Ib, II-IX). The severity of neonatal disease is determined by the capsular serotype and virulence factors such as the polysaccharide capsule, encoded by the cps gene, protein C, which includes the Cα surface proteins (bca gene), Rib (rib gene) and Cβ (bac gene); the proteins Lmb (lmb gene), FbsB (fbsB gene), FbsA (fbsA gene), the cyl operon encoding a β-hemolysin (hylB gene), the CAMP factor (cfb gene) and the C5a peptidase (scpB gene). The aim of this work was to determine the degree of GBS colonization in pregnant women, the serotypes distribution and to investigate virulence-associated genes.MethodsWe worked with 3480 samples of vagino-rectal swabs of women with 35–37 weeks of gestation. The identification of the strains was carried out using conventional biochemical tests and group confirmatory serology using a commercial latex particle agglutination kit. Two hundred GBS strains were selected. Their serotype was determined by agglutination tests. The monoplex PCR technique was used to investigate nine virulence-associated genes (cps, bca, rib, bac, lmb, fbsB, fbsA, hylB and scpB).ResultsThe maternal colonization was 9.09%. The serotypes found were: Ia (33.50%), III (19.00%), Ib (15.50%), II (14.00%), V (7.00%) and IX (5.50%). 5.50% of strains were found to be non-serotypeable (NT). The nine virulence genes investigated were detected simultaneously in 36.50% of the strains. The genes that were most frequently detected were scpB (100.00%), fbsA (100.00%), fbsB (100.00%), cylB (95.00%), lmb (94.00%) and bca (87.50%). We found associations between serotype and genes bac (p = 0.003), cylB (p = 0.02), rib (p = 0.01) and lmb (p < 0.001).ConclusionsThe frequency of vaginal-rectal colonization, serotypes distribution and associated virulence genes, varies widely among geographical areas. Therefore, epidemiological surveillance is necessary to provide data to guide decision-making and planning of prevention and control strategies.

Rate of Carriage of Streptococcus agalactiae among Pregnant Women and Role of Some Virulence Genes

Background: Although, GBS is a natural flora of the ano-rectal region, it may colonize vagina and many infants can be infected during the passage through the birth canal. It has emerged as a leading cause of neonatal infections and deaths. Objectives: To estimate the rate of recto-vaginal carriage of GBS among pregnant females, describe its antimicrobial susceptibility profile and to detect some of its virulence genes by multiplex PCR. Methodology: Vaginal, anorectal and neonatal throat swabs which were collected from two-hundred and fifty pregnant women were inoculated in Todd-Hewitt broth for 24 hours then inoculated on blood agar plates. Antimicrobial susceptibility testing for GBS isolates was done and its virulence genes (scpB, bca, rib and HvgA) were identified by PCR. Also, the relation between these virulence genes and antimicrobial susceptibility was studied. Results: Among 250 pregnant females, 36(14.4%) were identified as GBS carriers with exclusive vaginal and anorectal colonization rates of 4% and 10.4% respectively. All isolates were susceptible to penicillin, ampicillin, cefepime, cefotaxime, ceftriaxone, vancomycin and linezolid. On the other hand, 19.4%, 80.6%, 44.4% and 13.9% of GBS isolates were resistant to each of erythromycin and azithromycin, tetracycline, levofloxacin and clindamycin respectively. ScpB, rib, and Hvg-A genes were identified in 100%, 69.4% and 33.3% of GBS isolates respectively. None of them had the bca gene. Conclusion: Screening for GBS colonization of pregnant females is recommended and determination of virulence and different surface proteins would be relevant for better diagnosis and further possible formulation of a vaccine

Group B streptococcal carriage, antimicrobial susceptibility, and virulence related genes among pregnant women in Alexandria, Egypt

Background and aimGroup B Streptococcus (GBS) has emerged as a leading cause of illness and death among neonates. The study was conducted to estimate the prevalence of recto-vaginal carriage of GBS among pregnant women at 35–37weeks, gestation, to describe GBS antimicrobial susceptibility profile and to investigate selected virulence genes by PCR. Subjects and methodsTwo-hundred pregnant women at 35–37weeks of gestation attending antenatal clinic at Al-Shatby University Hospital were enrolled in the study. Both vaginal and rectal swabs were collected from each subject. Swabs were inoculated onto CHROMagar™ StrepB and sheep blood agar plates. All GBS isolates were subjected to antimicrobial susceptibility testing using disc diffusion. Disc approximation test was performed to detect erythromycin resistance phenotype (MLSB). GBS virulence genes scpB, bac, bca, and rib were identified by PCR. ResultsAmong the 200 pregnant women, 53 (26.5%) were identified as GBS carriers. All carriers had vaginal colonization (100%), four (7.5%) had combined recto-vaginal colonization. None of the carriers had rectal colonization alone. All isolates (100%) were susceptible to penicillin, ampicillin, ceftriaxone, cefotaxime, cefepime, vancomycin, and linezolid. On the other hand, 43.4%, 28.3%, 22.6%, and 15% of isolates were resistant to levofloxacin, azithromycin, erythromycin, and clindamycin respectively. Out of 12 erythromycin resistant isolates, six isolates had constitutive while two had inducible MLSB resistance. scpB was identified in 100%, rib in 79.2%, and bac in 35.8% of GBS isolates. None of the isolates possessed the bca gene. ConclusionIntroduction of GBS screening in Egyptian pregnant women is recommended. Penicillin or ampicillin is still the antibiotic of choice for intrapartum prophylaxis.

Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum.

Carbonic anhydrase catalyzes the reversible hydration of carbon dioxide to bicarbonate and maintains the balance of CO2/HCO3- in the intracellular environment, specifically for carboxylation/decarboxylation reactions. In Corynebacterium glutamicum, two putative genes, namely the bca (cg2954) and gca (cg0155) genes, coding for β-type and γ-type carbonic anhydrase, respectively, have been identified. We here analyze the transcriptional organization of these genes. The transcriptional start site (TSS) of the bca gene was shown to be the first nucleotide “A” of its putative translational start codon (ATG) and thus, bca codes for a leaderless transcript. The TSS of the gca gene was identified as an “A” residue located at position -20 relative to the first nucleotide of the annotated translational start codon of the cg0154 gene, which is located immediately upstream of gca. Comparative expression analysis revealed carbon source-dependent regulation of the bca gene, with 1.5- to 2-fold lower promoter activity in cells grown on acetate as compared to glucose as sole carbon source. Based on higher expression of bca in a mutant deficient of the regulator of acetate metabolism RamA as compared to the wild-type of C. glutamicum and based on the binding of His-tagged RamA protein to the bca promoter region, we here present evidence that RamA negatively regulates expression of bca in C. glutamicum. Functional characterization of a gca deletion mutant of C. glutamicum revealed the same growth characteristics of C. glutamicum ∆gca as that of wild-type C. glutamicum and no effect on expression of the bca gene.

Abstract A52: Gene alterations associated with clinical characteristics of bladder cancer

Abstract Next-generation sequencing (NGS) of urologic tumor genomes has identified frequently altered cancer genes. We recently examined the exomes of bladder tumors (BCa) from 54 U.S. patients and 99 Chinese patients to identify novel, altered cancer genes associated with clinical characteristics of disease. Exome capture of DNA from tumor and normal urothelial tissue using probes for ~180,000 protein-coding exons and microRNA loci; DNA fragment libraries were sequenced on a HiSeq 2000 platform (Illumina); and base calls on paired-end, 100 bp reads were generated using the Genome Analyzer Pipeline, v. 1.3 and standard parameters. Gene expression was analyzed using quantitative PCR, telomere length by a standard assay, and KDM6A function using cell-based assays in T24T BCa cells. Copy number variation (CNV), aneuploidy, and CpG methylation were analyzed as recently described. Telomerase (TERT) was the most frequently altered by somatic sequence changes in 37/54 (69%) of U.S. patient tumors. The TERT promoter was altered by frequent germline nucleotide changes in 30/54 (56%) tumors. Most of the TERT germline variants were novel (19/20) and three variants were confirmed in distinct tumors as either somatic or germline, including the most frequent variant, c. –245 T&amp;gt;C (rs2853669). The somatic TERT promoter alterations did not correlate with other somatic BCa gene alterations, but were associated with increased TERT expression and with significantly shorter telomeres in tumors as compared to matched normal tissue. The stromal antigen 2 (STAG2) gene was the most frequently altered novel BCa gene in Chinese patients, by somatic, deleterious sequence changes in 11% of tumors; CNV deletions (5%); and promoter CpG hypermethylation leading to presumed expression silencing (7/30, 23%). STAG2 encodes a component of the cohesion complex involved in sister chromatid cohesion and segregation. STAG2 mutations were associated with an increased number of chromosomal arm copy number changes corresponding to higher tumor cell aneuploidy, and mutations were associated with a significantly reduced patient survival by Kaplan-Meier survival analysis. In total, 32/99 (32%) of tumors displayed SCCS pathway gene alterations. The most frequently altered epigenetic gene was the histone lysine-specific demethylase 6A (KDM6A/UTX) in 24% of U.S. and 30% of Chinese patient tumors. Experimentally, we show KDM6A loss in human BCa T24T cells enhanced in vitro proliferation and cell migration, and in vivo tumor growth in mice. Re-expression reversed these effects, confirming KDM6A loss drives the BCa phenotype. Somatic KDM6A alterations were associated with somatic BRCA1-associated protein-1 (BAP1) alterations observed in 15% of U.S. tumors. Somatic, deleterious sequence changes occurred preferentially in U.S. Caucasian as compared to Chinese BCa patient tumors; are associated with papillary histologic features in a subset of tumors; and contribute to a significant number of tumors (10/14, 71%) that displayed alteration of genes encoding BRCA DNA repair pathway proteins. Genes encoding BRCA pathway proteins, including BAP1, BRCA1, BRCA2, PALB2, and ATM were altered by a combination of somatic and rare germline variants. We identified altered genes associated with clinical characteristics of BCa, including ‘immortalization' (TERT), aneuploidy (STAG2), proliferation (KDM6A), and papillary features in tumors (BAP1). STAG2 alterations are associated with a reduced patient survival, indicating a lethal subtype of disease; and altered X chromosome genes (STAG2 and KDM6A) are single copy in males and likely contribute to the cancer gender bias. Finally, we identified BRCA pathway alterations in BCa that are similar to those in breast, ovarian, prostate and kidney tumors, that have a common DNA repair deficiency that can be exploited by therapy targeting poly (ADP ribose) polymerase. Citation Format: Mike Nickerson, Kate M. Im, Guangwu Guo, Yaoting Gui, Sevilay Turan, James C. Costello, Quan Zhou, Zhiming Cai, Song Wu, M. Scott Lucia, Lee E. Moore, Michael Dean, Dan Theodorescu. Gene alterations associated with clinical characteristics of bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A52.

Pulsed-field gel electrophoresis, virulence determinants and antimicrobial susceptibility profiles of type Ia group B streptococci isolated from humans in Brazil

Group B streptococci (GBS) infections occur worldwide. Although serotyping has been used for epidemiologic purposes, this does not accurately characterize enough members of a genetically heterogeneous bacterial population. The aims of this work were to evaluate the genetic diversity of 45 type Ia GBS strains isolated in Brazil by pulsed-field gel electrophoresis as well as to evaluate antimicrobial susceptibility profiles and identify virulence genes. Twenty-four strains were assigned to cluster A. All strains under study contained the hylB and scpB genes. The bca gene was detected in only 10 strains and none of the streptococci carried the bac gene. Thirty-nine strains were resistant to tetracycline.

Molecular epidemiology of group B streptococci in Ireland: associations between serotype, invasive status and presence of genes encoding putative virulence factors.

Group B streptococcal isolates (n = 159) from the three Dublin maternity hospitals, were serotyped and analysed for the bac, bca, hylB, pepB, and rib genes. The serotype distribution of the isolates was Ia, 19.5%; Ib, 18.9%; II, 10.7%; III, 29.5%; IV, 1.9%; V, 15.1%; non-typeable, 4.4%. There was a statistically significant association between the serotype and invasive status (carriage or infection) of isolates (P < 0.005), but no significant association between serotype and degree of invasiveness was demonstrated. The presence or absence of each analysed gene was not associated with the invasive status of isolates. Statistically significant associations were revealed between bca and hylB (IS1548) (P = 0.0004) and between bac and bca (P=0.014). The bac, bca, hylB (IS1548) and rib genes and the numbers of tandem repeats in the bca gene showed significant associations with serotype. Almost 50% of serotype III isolates possessed at least one of the bac and bca genes and 55-65% of strains of serotypes Ia, Ib and II possessed the rib gene. Most serotype III isolates had IS1548 in their hylB genes. Serotype Ib was the only serotype in which more than half of the strains contained more tandem repeats in the bca gene than the overall mean for the GBS population studied of 7.4 repeats. These findings indicate that some previously reported associations between putative virulence factors and GBS disease require further study and clarification.

A gene homologous to beta-type carbonic anhydrase is essential for the growth of Corynebacterium glutamicum under atmospheric conditions.

Carbonic anhydrase catalyzes the interconversion of CO(2) and bicarbonate. We focused on this enzyme in the amino acid-producing organism Corynebacterium glutamicum in order to assess the availability of bicarbonate for carboxylation reactions essential to growth and for those required for L-lysine overproduction. A whole-genome sequence revealed two genes encoding putative beta-type and gamma-type carbonic anhydrases in C. glutamicum. These genes encode polypeptides containing zinc ligands strictly conserved in each type of carbonic anhydrase and were designated bca and gca, respectively. Internal deletion of the chromosomal bca gene resulted in a phenotype showing severely reduced growth under atmospheric conditions (0.04% CO(2)) on both complete and minimal media. The growth defect of the Delta bca strain was restored under elevated CO(2) conditions (5% CO(2)). Introduction of the red alga Porphyridium purpureum carbonic anhydrase gene ( pca) could compensate for the bca deletion, allowing normal growth under an atmospheric level of CO(2). In contrast, the Delta gca strain behaved identically to the wild-type strain with respect to growth, irrespective of the CO(2) conditions. Attempts to increase the dosage of bca, gca, and pca in the defined L-lysine-producing strain C. glutamicum AHD-2 led to no discernable effects on growth and production. Northern blot analysis indicated that the bca transcript in strain AHD-2 and another L-lysine producer, C. glutamicum B-6, was present at a much higher level than in the wild-type strain, particularly during exponential growth phases. These results indicate that: (1) the bca product is essential to achieving normal growth under ordinary atmospheric conditions, and this effect is most likely due to the bca product's ability to maintain favorable intracellular bicarbonate/CO(2) levels, and (2) the expression of bca is induced during exponential growth phases and also in the case of L-lysine overproduction, both of which are conditions of higher bicarbonate demand.

Distribution and expression of bca, the gene encoding the c alpha protein, by Streptococcus agalactiae.

A total of 52 clinical isolates of group B streptococci (GBS) was tested for expression of the c protein c(alpha) by a fluorescent antibody test (FAT) and by PCR amplification of a 202-bp stretch within the repeat unit of the bca gene encoding the c(alpha) protein. The strains were categorised as follows: c(alpha) FAT positive and PCR positive with amplification products of multiple sizes (category A, n = 12); FAT negative and with PCR products of multiple sizes (category B, n = 11); FAT negative and with a single PCR product of c. 200 bp (category C, n = 5); negative in both tests (category D, n = 24). A single amplification product of minimum size and additional products of larger sizes corresponded to one and more bca repeats, respectively. Five of the 11 category B strains showed expression of low Mr c(alpha) in whole cell-based Western blotting. The results showed that a proportion of the GBS isolates harboured bca gene elements that either were not expressed or they expressed c(alpha) molecular variants which could not be detected by the whole cell-based FAT. This genotype/phenotype discrepancy should be considered in relation to GBS typing, including the selection of antibody reagents and the technical approach to c(alpha) protein detection.

Bca, beta gene, and gene product divergency in reference and prototype strains of streptococcus agalactiae.

Reference and prototype strains of Streptococcus agalactiae (GBS) were originally selected on the basis of phenotypic traits which, however, do not always mirror genotypic traits. A total of 14 reference and prototype GBS strains were examined by PCR designed to detect the bca and beta genes, encoding the c proteins c(alpha) and c(beta), respectively. The cognate proteins were detected by whole-cell-based fluorescent antibody testing and Western blotting. The PCR for beta gene detection and the antibody-based c(beta) protein detection showed concordant results with all of the isolates, whereas 7 of 14 strains which did not express c(alpha) protein at detectable levels contained bca gene elements, consistent with bca gene and gene product divergency in these strains. The results emphasize the importance of genetic characterization of reference and prototype strains of GBS which, in the past, have been selected on the basis of phenotypic traits.

Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis

Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M(r) of 54,200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.