Methods for Paramecium tetraurelia ciliary membrane protein identification and function.

In this chapter we provide some tools to study the ciliary proteins that make it possible for Paramecium cells to swim by beating their cilia. These proteins include many ion channels, accessory proteins, peripheral proteins, structural proteins, rootlets of cilia, and enzymes. Some of these proteins are also found in the soma membrane, but their distinct and critical functions are in the cilia. Paramecium has 4000 or more cilia per cell, giving it an advantage for biochemical studies over cells that have one primarily cilium per cell. Nonetheless, a challenge for studies of many ciliary proteins in Paramecium is their low abundance. We discuss here several strategies to overcome this challenge and other challenges such as working with very large channel proteins. We also include for completeness other techniques that are critical to the study of swimming behavior, such as genetic crosses, recording of swimming patterns, electrical recordings, expression of very large channel proteins, RNA Interference, among others.

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.

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.

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

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 stx2 positive E. coli O157 of food/animal origin for stx2 gene variants, Shiga toxin production, presence of other virulence genes, stx phage insertion sites, presence of genes associated with functionality of stx phages (Q933 and Q21) and stx2 upstream region. Of the 47 isolates, 46 were positive for both stx2a and stx2d while the remaining isolate was positive for stx2d 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 stx2 promoter. Additionally, all isolates were negative for antiterminator Q933while a majority of isolates were positive for Q21 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.

Microbiological quality assessment of milk at different stages of the dairy value chain in a developing country setting.

The main objective of the study was to assess the microbiological quality of milk at different stages of the dairy value chain from farm to the factory in Bangladesh. A total of 438 raw milk samples (387 from primary producers, 32 from collectors, 15 from chilling plants, 4 from local restaurants) and 95 commercially processed milk samples were collected from northern part of Bangladesh. Almost 72% (n = 280) of samples at producer level and 100% from both collectors (n = 32) and chilling plants (n = 15) were contaminated with coliforms while 57% (n = 220) of samples from producers, 91% (n = 29) of samples from collectors and 100% (n = 15) from chilling plants were contaminated with fecal coliforms. Around 31% (n = 119) of samples from producers were positive for E. coli whereas >60% (n = 20) and 100% (n = 15) samples from collectors and chilling plants, respectively were positive for E. coli. One quarter of samples from collectors were positive for B. cereus and coagulase positive staphylococci and 33% (n = 5) of samples from chilling plants were positive for both of these microorganisms. In case of commercially processed milk, 77% (n = 46) and 37% (n = 22) of pasteurized milk samples had a high aerobic plate count (APC) (104 CFU/ml) and coliform count (>10 CFU/ml), respectively. None of the samples was positive for Shigella spp., Salmonella spp., and Campylobacter spp. Among 158 E. coli positive raw milk samples, 9% (n = 14) contained pathogenic E. coli, and enteroaggregative E. coli (EAEC) and Shiga-toxin producing E. coli (STEC) were found to be the predominant pathotypes. Of the 23 pathogenic E. coli identified from 14 samples based on their gene contents, >95% (n = 22) were resistant to at least one antibiotic and 13% (n = 3) of isolates were resistant to ≥3 classes of antibiotics. Several factors including the time of milking, hygiene practices of the producers, cow breed and amount of milk produced by the cow were found to be significantly associated with high APC of milk samples. In conclusion, both raw and commercially pasteurized milk are highly contaminated with fecal organisms. For intervention, more emphasis should be given at producer's level as microorganisms introduced to milk at this stage get the longest time for survival and multiplication.

Environmental Spread of New Delhi Metallo-ß-Lactamase-1-Producing Multidrug-Resistant Bacteria in Dhaka, Bangladesh.

Resistance to carbapenem antibiotics through the production of New Delhi metallo-ß-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor the possible source of the spread of these organisms in Dhaka, Bangladesh, we conducted a comparative analysis of wastewater samples from hospital-adjacent areas (HAR) and from community areas (COM), as well as public tap water samples, for the occurrence and characteristics of NDM-1-producing bacteria. Of 72 HAR samples tested, 51 (71%) samples were positive for NDM-1-producing bacteria, as evidenced by phenotypic tests and the presence of the blaNDM-1 gene, compared to 5 of 41 (12.1%) samples from COM samples (P < 0.001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among blaNDM-1-positive isolates, followed by Escherichia coli (29%), Acinetobacter spp. (15%), and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes, including blaCTX-M-1 (80%), blaCTX-M-15 (63%), blaTEM (76%), blaSHV (33%), blaCMY-2 (16%), blaOXA-48-like (2%), blaOXA-1 (53%), and blaOXA-47-like (60%) genes. Around 40% of the isolates contained a qnr gene, while 50% had 16S rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30 to 50 MDa carrying blaNDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multidrug-resistant bacteria as a potential public health threat. In view of the existing practice of discharging untreated liquid waste into the environment, hospitals in Dhaka city contribute to the potential dissemination of NDM-1-producing bacteria into the community.IMPORTANCE Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently described carbapenemase, and the blaNDM-1 gene, which encodes NDM-1, is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows a high prevalence of NDM-1-producing organisms in the wastewater samples from hospital-adjacent areas as a potential source for the spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines the characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple-antibiotic-resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

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 ß-lactamase (ESBL). Of the 22 ESBL-producers, 20 were positive for bla CTX-M-15, 7 for bla OXA-1-group (all had bla OXA-47) and 2 for bla CMY-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.

Occurrence and characterization of multidrug-resistant New Delhi metallo-ß-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-ß-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 (n = 1789) and Shigella spp. (n = 90) isolated between 2009 and 2010 from stool samples was resistant or had intermediate susceptibility to imipenem. Among 127 extended-spectrum ß-lactamase-producing strains isolated during 2003-2009, three Klebsiella pneumoniae isolates (2.4 %) were resistant to imipenem and were positive for bla(NDM-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 bla(OXA-1) group, bla(CTX-M-1) group (bla(CTX-M-15)) and bla(SHV) genes, whilst two isolates were positive for 16S rRNA methylase (armA) and qnr (qnrB) genes. One isolate was positive for the bla(CMY) gene and one for the rmtB gene. The bla(NDM-1) gene was located on a conjugative plasmid of ~23-24 MDa. The PFGE patterns of the isolates were different from each other. This study highlights the occurrence of NDM-1-producing organisms in Bangladesh in 2008. The clonal diversity of the isolates and the transferability of bla(NDM-1) plasmids suggest a wider distribution of NDM-1-producing bacteria in Bangladesh.