The unusual occurrence of green algal balls of <i>Chaetomorpha linum</i> on a beach in Sydney, Australia.

In spring 2014, thousands of green algal balls were washed up at Dee Why Beach, Sydney, New South Wales, Australia. Reports of algal balls are uncommon in marine systems, and mass strandings on beaches are even more rare, sparking both public and scientific interest. We identified the algal masses as Chaetomorpha linum by using light microscopy and DNA sequencing. We characterize the size and composition of the balls from Dee Why Beach and compare them to previous records of marine algal balls. We describe the environmental conditions that could explain their appearance, given the ecophysiology of C. linum

Fluorescence analysis detects gp60 subtype diversity in Cryptosporidium infections

Ninety percent of human cryptosporidiosis infections are attributed to two species; the anthroponotic Cryptosporidium hominis and the zoonotic Cryptosporidium parvum. Sequence analysis of the hypervariable gp60 gene, which is used to classify Cryptosporidium to the subtype level, has highlighted extensive intra-species diversity within both C. hominis and C. parvum. The gp60 has also facilitated contamination source tracking and increased understanding of the epidemiology of cryptosporidiosis. Two surface glycoproteins, the gp40 and gp15 are encoded in the gp60 gene; both are exposed to the hosts' immune system and play a pivotal role in the disease initiation process. The extent of genetic diversity observed within the gp60 would support the hypotheses of significant selection pressure placed on the gp40 and gp15. This study used a dual fluorescent terminal-restriction fragment length polymorphism (T-RFLP) analysis to investigate the genetic diversity of Cryptosporidium subtype populations in a single host infection. Terminal-RFLP showed subtype variation within one human Cryptosporidium sample and mouse samples from seven consecutive passages with C. parvum. Furthermore, this was the first study to show that differences in the ratio of subtype populations occur between infections. T-RFLP has provided a novel platform to study infection populations and to begin to investigate the impact of the hosts' immune system on the gp60 gene. © 2011.8 page(s

Screening foodstuffs for class 1 integrons and gene cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.7 page(s

Evaluation of a PCR protocol for sensitive detection of Giardia intestinalis in human faeces

Giardia intestinalis is a protozoan parasite and a human pathogen. It is a leading cause of human diarrheal disease and a significant cause of morbidity worldwide. At the molecular level, G. intestinalis is a species complex, consisting of genetic assemblages (A to G) and subassemblage strains. The genotypes that cause human disease have been characterised to assemblages A and B, and include strains AI, AII, BIII and BIV. PCR amplification of diagnostic loci is used to genotype samples and is required to understand different transmission cycles within communities. A multi-locus approach is required for validation of Giardia genotyping and molecular diagnostic techniques that are efficient across numerous loci have not been established. This study evaluated several published protocols for the 18S small subunit ribosomal RNA (18S rRNA) and glutamate dehydrogenase genes (gdh) genes. Assays were compared using spiked faecal samples and by measuring the concentration of DNA generated following DNA extraction and PCR amplification. An optimal molecular method for G. intestinalis identification was established from direct DNA extraction of faecal material and GC-rich PCR chemistry. The protocol was applied to 50 clinical samples and produced PCR success rates of 90% and 94% at the 18S rRNA and gdh loci. Cyst concentration prior to DNA extraction was not necessary, and the optimal protocol was highly sensitive and an efficient method for testing clinical samples.6 page(s

Wildlife-associated Cryptosporidium fayeri in Human, Australia

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Evolution of class 1 integrons: Mobilization and dispersal via food-borne bacteria

<div><p>Class 1 integrons have played a major role in the global dissemination of antibiotic resistance. Reconstructing the history of class 1 integrons might help us control further spread of antibiotic resistance by understanding how human activities influence microbial evolution. Here we describe a class 1 integron that represents an intermediate stage in the evolutionary history of clinical integrons. It was embedded in a series of nested transposons, carried on an IncP plasmid resident in <i>Enterobacter</i>, isolated from the surface of baby spinach leaves. Based on the structure of this integron, we present a modified hypothesis for integron assembly, where the ancestral clinical class 1 integron was captured from a betaproteobacterial chromosome to form a Tn<i>402</i>-like transposon. This transposon then inserted into a plasmid-borne Tn<i>21</i>-like ancestor while in an environmental setting, possibly a bacterium resident in the phyllosphere. We suggest that the <i>qacE</i> gene cassette, conferring resistance to biocides, together with the mercury resistance operon carried by Tn<i>21</i>, provided a selective advantage when this bacterium made its way into the human commensal flora via food. The integron characterized here was located in Tn<i>6007</i>, which along with Tn<i>6008</i>, forms part of the larger Tn<i>6006</i> transposon, itself inserted into another transposable element to form the Tn<i>21</i>-like transposon, Tn<i>6005</i>. This element has previously been described from the human microbiota, but with a promoter mutation that upregulates integron cassette expression. This element we describe here is from an environmental bacterium, and supports the hypothesis that the ancestral class 1 integron migrated into anthropogenic settings via foodstuffs. Selection pressures brought about by early antimicrobial agents, including mercury, arsenic and disinfectants, promoted its initial fixation, the acquisition of promoter mutations, and subsequent dissemination into various species and pathogens.</p></div

A model for the origin of clinical class 1 integrons.

<p>(A) Diverse chromosomal class 1 integrons that are present in environmental <i>Betaproteobacteria</i> can interact with mobile DNA elements such as transposons and plasmids in the environment. Integrons have access to a vast pool of integron gene cassettes including the <i>qacE</i> cassette that encodes a membrane efflux pump; (B) A single chromosomal integron is captured by a Tn<i>5090</i>-like transposon, to generate Tn<i>402</i>; (C) Now mobilized, the integron is free to move between a range of bacterial species. In particular, the Tn<i>402</i> transposon inserts into the mercury resistance Tn<i>501</i>-like transposon, to generate Tn<i>21</i>. Residence of this complex DNA element on a broad host range plasmid allows the integron to make its way into the human commensal flora via food-borne bacteria; (D) Once resident within the human microbiota, the integron is fixed by selection, driven by mercury and disinfectants, and after introduction of sulfonamide antibiotics, captures the <i>sul1</i> and <i>orf5</i> gene cassettes to delete part of the original <i>qacE</i> cassette; (E) Partial deletions of the <i>tni</i> module, and the collective acquisition of diverse resistance cassettes, lead to the diversity of clinical class 1 integrons that have since disseminated around the globe.</p

Detailed structure of the class 1 integron promoter regions.

<p>The -35 and -10 motifs for the Pc and P<i>intI1</i> promoters are boxed; a point mutation in the -10 motif, which distinguishes the PcW and PcH1 promoters, is highlighted in red; the transcription initiation sites are indicated by arrows; the LexA box is shaded in blue (expression of <i>intI1</i> is regulated by the SOS response). (A) Promoter region within the class 1 integron described in the present study, containing the PcW promoter variant, which is also present in a number of chromosomal class 1 integrons (accession numbers EU316185 and EU327987-EU327991). This strongly suggests PcW is the ancestral promoter in these integrons; (B) Promoter region within the otherwise identical class 1 integron characterized from the human fecal flora by Labbate et al. (23), which contains the PcH1 promoter.</p

Genomic landscape of the class 1 integron reported in the present study.

<p>From left to right, the components are as follows: the direct repeat (DR1) formed by the insertion of Tn<i>6005</i>; the inverted repeat for Tn<i>6005</i> (IRp); Tn<i>21</i>-like transposition genes <i>tnpA</i> and <i>tnpR</i>; the direct repeat (DR2) formed by the insertion of Tn<i>6006</i>; the inverted repeat for Tn<i>6006</i>/<i>6007</i> (IRi); a class 1 integron with <i>intI1</i> and integron-associated recombination site <i>attI1</i>, carrying two gene cassettes, <i>MN039</i> and <i>qacE2</i>, each with a cassette-associated recombination site <i>attC</i>; Tn<i>402</i>-like transposition genes <i>tniR</i>, <i>tniQ</i>, <i>tniB</i>, <i>tniA</i>; the inverted repeat for Tn<i>6007</i> (IRt); genes <i>MN040</i> and <i>MN041</i>; the inverted repeat for Tn<i>6008</i> (IRi); the <i>Tn6008</i> transposon, carrying genes <i>MN042</i>, <i>ahpD</i>, <i>MN043</i>, <i>MN044</i>, <i>MN045</i>, <i>MN046</i>, and <i>tniA</i>; the inverted repeat for Tn<i>6006</i>/<i>6008</i> (IRt); the direct repeat (DR2) formed by the insertion of Tn<i>6006</i>; Tn<i>21</i>-like <i>mer</i> operon consisting of <i>urf-2Y</i>, <i>merE</i>, <i>merD</i>, <i>merA</i>, <i>merC</i>, <i>merP</i>, <i>merT</i> and <i>merR</i>; the inverted repeat for Tn<i>6005</i> (IRm); and the direct repeat (DR1) formed by the insertion of Tn<i>6005</i>. This whole element is embedded in an IncP plasmid whose sequence is lodged as accession number KY126370.</p