Multiple Functions Of The Striated Rootlet Proteins Of The Paramecium Basal Body

Paramecium ciliary basal bodies align in straight rows from posterior to anterior. Each basal body is connected to three rootlets ((Post Ciliary Rootlet (PCR), Transverse Rootlet (TR) and Striated Rootlet (SR)). The SR, the longest, projects from the basal body toward the anterior past several more anterior basal bodies. The depletion of Meckelin (MKS3) misaligns SRs, disorganizes basal body rows and makes the SRs appear ragged and serpentine. In this study we clarify the composition of the Paramecium ciliary basal body’s SR and demonstrate that the SR plays a critical role in creating the orderly array of basal bodies in rows that run from pole to pole of the cell, likely through the interactions with centrins and other cytoskeletal elements underlying the cell surface. Here in this study we first report the reciprocal relationship between the SR and centrin related infraciliary lattice (ICL) protein that can dictate the cell surface morphology. The SR of Chlamydomonas is the best studied. Using the single SR Chlamydomonas gene SF-assemblin to search in Paramecium DB, we found thirty Paramecium genes in thirteen Paralog Groups. Proteins from 13 paralog groups were confirmed to be in the SR structure using immunofluorescence. LC-MS/MS analyses of density fractions from SRs isolation show all thirty SR members are within the same density fraction. We further categorized all 30 SR genes in five Structural Groups based on their ability to form coiled coil domain and evaluate the function of all five Structural Group using RNA interference (RNAi). Silencing the transcripts of the any of the Structural Group showed misaligned basal body rows and the disordered organization of the SRs with abnormal appearance of SRs all over the cell surface. Silencing of Paralog Group showed normal phenotype except for the two Paralog Group (Paralog Group 1 or Paralog Group 7) which themselves constitute Structural Group individually. Isolated SRs from the control or Paralog Group depleted cells show a characteristic striation pattern that includes characteristic major and minor striations. Isolated SRs from any of the Structural Group depleted cells demonstrate abnormal shapes and striation periodicity. There is a correlation between the SR Structural Group RNAi surface misalignment phenotype and the isolated SR Structural Group RNAi phenotype for shape and periodicity of the SR. Strikingly our study of SR clearly demonstrates the role of SRs in shaping the other cytoskeleton structures of the cell cortex e.g., ICL, epiplasm territory and cortical unit territory. In another follow up study of MKS3 (Picariello et al., 2014), we depleted the transcripts of MKS5 gene in Paramecium tetraurelia. Depletion of MKS5 transcripts in Paramecium causes cilia loss all over the cell surface. Unlike MKS3 depletion, MKS5 depletion does not affect the straight basal body rows and the ordered organization of SRs. Moreover, data presented in this study clearly demonstrates depletion of MKS5 transcripts somehow affect the localization of another transition zone protein, B9D2. It appears when lacking any of the SR Structural Group, the rest fail to interact properly with each other to maintain the SRs structure and directionality toward the anterior. As a result, abnormal SRs appear to lose the interaction with other cytoskeleton structures such as ICL network complex, which eventually results in misaligned basal body rows and altered swimming behavior. From the data presented in this study it is reasonable to postulate ICL1e subfamily and SRs are in a reciprocal relationship to maintain the straight basal body rows and the highly ordered organization of the SRs all over the cell surface

Occurrence and Characterization of Methicillin Resistant Staphylococcus aureus in Processed Raw Foods and Ready-to-Eat Foods in an Urban Setting of a Developing Country

Infections by methicillin-resistant Staphylococcus aureus (MRSA) are gradually increasing in the community. In this study, we investigated a total of 162 food samples including 112 ready-to-eat (RTE) foods and 40 processed raw meat and fish samples collected from retail vendors in Dhaka, Bangladesh and determined the occurrence of toxigenic S. aureus and MRSA. Around 22% of samples were positive for S. aureus, RTE foods being more positive (23%) than the processed raw meat/fish samples (18%). Among 35 S. aureus isolates, 74% were resistant to erythromycin, 49% to ciprofloxacin and around 30% to oxacillin and cefoxitin. Around 37% of isolates were resistant to ≥3 classes of antibiotics and 26% of isolates (n = 9) were identified as MRSA. Majority of the isolates were positive for enterotoxin genes (74%), followed by pvl gene (71%), toxic shock syndrome toxin (tsst) gene (17%) and exfoliative toxin genes (11%). Multi locus sequence typing (MLST) of 9 MRSA isolates identified four different types such as ST80 (n = 3), ST6 (n = 2), ST239 (n = 2) and ST361 (n = 2). spa typing of MRSA isolates revealed seven different types including t1198 (n = 2), t315 (n = 2), t037 (n = 1), t275 (n = 1), t304 (n = 1), t8731 (n = 1) and t10546 (n = 1). To our knowledge, this is the first report entailing baseline data on the occurrence of MRSA in RTE foods in Dhaka highlighting a potential public health risk to street food consumers

Antimicrobial Resistance, Virulence Factors and Genetic Diversity of Escherichia coli Isolates from Household Water Supply in Dhaka, Bangladesh

Background: Unsafe water supplies continue to raise public health concerns, especially in urban areas in low resource countries. To understand the extent of public health risk attributed to supply water in Dhaka city, Bangladesh, Escherichia coli isolated from tap water samples collected from different locations of the city were characterized for their antibiotic resistance, pathogenic properties and genetic diversity. Methodology/Principal Findings: A total of 233 E. coli isolates obtained from 175 tap water samples were analysed for susceptibility to 16 different antibiotics and for the presence of genes associated with virulence and antibiotic resistance. Nearly 36% (n = 84) of the isolates were multi-drug(≥3 classes of antibiotics) resistant (MDR) and 26% (n = 22) of these were positive for extended spectrum β-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for blaCTX-M-15, 7 for blaOXA-1-group(all had blaOXA-47) and 2 for blaCMY-2. Quinolone resistance genes, qnrS and qnrB were detected in 6 and 2 isolates, respectively. Around 7% (n = 16) of the isolates carried virulence gene(s) characteristic of pathogenic E. coli; 11 of these contained lt and/or st and thus belonged to enterotoxigenic E. coli and 5 contained bfp and eae and thus belonged to enteropathogenic E. coli. All MDR isolates carried multiple plasmids (2 to 8) of varying sizes ranging from 1.2 to >120 MDa. Ampicillin and ceftriaxone resistance were co-transferred in conjugative plasmids of 70 to 100 MDa in size, while ampicillin, trimethoprim-sulfamethoxazole and tetracycline resistance were co-transferred in conjugative plasmids of 50 to 90 MDa. Pulsed-field gel electrophoresis analysis revealed diverse genetic fingerprints of pathogenic isolates. Significance: Multi-drug resistant E. coli are wide spread in public water supply in Dhaka city, Bangladesh. Transmission of resistant bacteria and plasmids through supply water pose serious threats to public health in urban areas

SF-Assemblin genes in Paramecium: phylogeny and phenotypes of RNAi silencing on the ciliary-striated rootlets and surface organization

Background: Cilia emanate from basal bodies just underneath the cell membrane. Basal bodies must withstand torque from the ciliary beat and be appropriately spaced for cilia to beat in metachronal waves. Basal body rootlets provide stability for motile cilia. Paramecium has three. Our focus is on the largest one, the striated rootlet (SR). Paramecium basal bodies align in straight rows. Previously we found a potential role for the SR in this alignment. Here we present a phylogeny of the Paramecium homologs of the SF-Assemblin gene of the SR of Chlamydomonas, and the organization of these genes. We describe the phenotypes from RNA interference (RNAi) silencing of genes and gene groups. Methods: Phenotypes of the RNAi depletions were characterized by immunofluorescence (IF), electron microscopy, and mass spectrometry. Results: We found 30 genes for Paramecium SF-Assemblin homologs (SFA) organized into 13 Paralog Groups (further categorized in five Structural Groups). Representatives of Paralog Groups were found in the SRs. Silencing the transcripts of any of the Structural Groups correlates with misaligned rows of basal bodies, SRs, and cortical units. The silencing of Structural Groups was key and gave us the ability to systematically disrupt SR structures and cell surface organization. Conclusions: Silencing of SFA genes and Paralog Groups shows no effects on the SR or the cell surface organization. Silencing of the larger Structural Groups has an enormous impact on rows of basal bodies, SRs and cortical units, and SR striations, and length. Misaligned basal bodies have cilia causing the cells to swim in abnormal paths

Toxigenic properties and stx phage characterization of Escherichia coli O157 isolated from animal sources in a developing country setting

Abstract Background In many Asian countries including Bangladesh E. coli O157 are prevalent in animal reservoirs and in the food chain, but the incidence of human infection due to E. coli O157 is rare. One of the reasons could be inability of the organism from animal origin to produce sufficient amount of Shiga toxin (Stx), which is the main virulence factor associated with the severe sequelae of infection. This study aimed to fill out this knowledge gap by investigating the toxigenic properties and characteristics of stx phage of E. coli O157 isolated from animal sources in Bangladesh. Results We analysed 47 stx 2 positive E. coli O157 of food/animal origin for stx 2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q 933 and Q 21) and stx 2 upstream region. Of the 47 isolates, 46 were positive for both stx 2a and stx 2d while the remaining isolate was positive for stx 2d only. Reverse Passive Latex Agglutination assay (RPLA) showed that 42/47 isolates produced little or no toxin, while 5 isolates produced a high titre of toxin (64 to 128). 39/47 isolates were positive for the Toxin Non-Producing (TNP) specific regions in the stx 2 promoter. Additionally, all isolates were negative for antiterminator Q 933while a majority of isolates were positive for Q 21 gene suggesting the presence of defective stx phage. Of the yehV and wrbA phage insertion sites, yehV was found occupied in 11 isolates while wrbA site was intact in all the isolates. None of the isolates was positive for the virulence gene, cdt but all were positive for hlyA, katP, etpD and eae genes. Isolates that produced high titre Stx (n = 5) produced complete phage particles capable of infecting multiple bacterial hosts. One of these phages was shown to produce stable lysogens in host strains rendering the Stx2 producing ability. Conclusion Despite low frequency in the tested isolates, E. coli O157 isolates in Bangladesh carry inducible stx phages and have the capacity to produce Stx2, indicating a potential risk of E. coli O157 infection in humans

Occurrence and Characterization of Methicillin Resistant Staphylococcus aureus in Processed Raw Foods and Ready-to-Eat Foods in an Urban Setting of a Developing Country

Infections by methicillin-resistant Staphylococcus aureus (MRSA) are gradually increasing in the community. In this study, we investigated a total of 162 food samples including 112 ready-to-eat (RTE) foods and 40 processed raw meat and fish samples collected from retail vendors in Dhaka, Bangladesh and determined the occurrence of toxigenic S. aureus and MRSA. Around 22% of samples were positive for S. aureus, RTE foods being more positive (23%) than the processed raw meat/fish samples (18%). Among 35 S. aureus isolates, 74% were resistant to erythromycin, 49% to ciprofloxacin and around 30% to oxacillin and cefoxitin. Around 37% of isolates were resistant to ≥3 classes of antibiotics and 26% of isolates (n = 9) were identified as MRSA. Majority of the isolates were positive for enterotoxin genes (74%), followed by pvl gene (71%), toxic shock syndrome toxin (tsst) gene (17%) and exfoliative toxin genes (11%). Multi locus sequence typing (MLST) of 9 MRSA isolates identified four different types such as ST80 (n = 3), ST6 (n = 2), ST239 (n = 2) and ST361 (n = 2). spa typing of MRSA isolates revealed seven different types including t1198 (n = 2), t315 (n = 2), t037 (n = 1), t275 (n = 1), t304 (n = 1), t8731 (n = 1) and t10546 (n = 1). To our knowledge, this is the first report entailing baseline data on the occurrence of MRSA in RTE foods in Dhaka highlighting a potential public health risk to street food consumers

Occurrence and characterization of multidrugresistant new delhi metallo-β-lactamase-1- producing bacteria isolated between 2003 and 2010 in Bangladesh

The purpose of this study was to screen for reduced susceptibility against imipenem and the presence of the New Delhi metallo-β-lactamase-1 (NDM-1) gene in a collection of Enterobacteriaceae (Escherichia coli, Shigella spp. and Klebsiella pneumoniae) from different surveillance studies between 2003 and 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh. None of the E. coli (n51789) and Shigella spp. (n590) isolated between 2009 and 2010 from stool samples was resistant or had intermediate susceptibility to imipenem. Among 127 extended-spectrum β-lactamase-producing strains isolated during 2003-2009, three Klebsiella pneumoniae isolates (2.4 %) were resistant to imipenem and were positive for blaNDM-1. All these NDM-1-producing strains were isolated in 2008 and were resistant to all antibiotics tested except for tigecycline and colistin. All three isolates were positive for blaOXA-1 group, blaCTX-M-1 group (blaCTX-M-15) and blaSHV genes, whilst two isolates were positive for 16S rRNA methylase (armA) and qnr (qnrB) genes. One isolate was positive for the blaCMY gene and one for the rmtB gene. The blaNDM-1 gene was located on a conjugative plasmid of