Enzo A. Palombo

Full Genome-Based Classification of Rotaviruses Reveals a Common Origin between Human Wa-Like and Porcine Rotavirus Strains and Human DS-1-Like and Bovine Rotavirus Strains

ABSTRACT Group A rotavirus classification is currently based on the molecular properties of the two outer layer proteins, VP7 and VP4, and the middle layer protein, VP6. As reassortment of all the 11 rotavirus gene segments plays a key role in generating rotavirus diversity in nature, a classification system that is based on all the rotavirus gene segments is desirable for determining which genes influence rotavirus host range restriction, replication, and virulence, as well as for studying rotavirus epidemiology and evolution. Toward establishing such a classification system, gene sequences encoding VP1 to VP3, VP6, and NSP1 to NSP5 were determined for human and animal rotavirus strains belonging to different G and P genotypes in addition to those available in databases, and they were used to define phylogenetic relationships among all rotavirus genes. Based on these phylogenetic analyses, appropriate identity cutoff values were determined for each gene. For the VP4 gene, a nucleotide identity cutoff value of 80% completely correlated with the 27 established P genotypes. For the VP7 gene, a nucleotide identity cutoff value of 80% largely coincided with the established G genotypes but identified four additional distinct genotypes comprised of murine or avian rotavirus strains. Phylogenetic analyses of the VP1 to VP3, VP6, and NSP1 to NSP5 genes showed the existence of 4, 5, 6, 11, 14, 5, 7, 11, and 6 genotypes, respectively, based on nucleotide identity cutoff values of 83%, 84%, 81%, 85%, 79%, 85%, 85%, 85%, and 91%, respectively. In accordance with these data, a revised nomenclature of rotavirus strains is proposed. The novel classification system allows the identification of (i) distinct genotypes, which probably followed separate evolutionary paths; (ii) interspecies transmissions and a plethora of reassortment events; and (iii) certain gene constellations that revealed (a) a common origin between human Wa-like rotavirus strains and porcine rotavirus strains and (b) a common origin between human DS-1-like rotavirus strains and bovine rotaviruses. These close evolutionary links between human and animal rotaviruses emphasize the need for close simultaneous monitoring of rotaviruses in animals and humans.

Antibacterial activity of traditional Australian medicinal plants

Fifty-six ethanolic extracts of various parts of 39 plants used in traditional Australian Aboriginal medicine were investigated for their antibacterial activities against four Gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Streptococcus pyogenes) and four Gram-negative (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella typhimurium) bacterial species. In a plate-hole diffusion assay, 12 extracts inhibited the growth of one or more of the bacteria, with five extracts showing broad spectrum antibacterial activity against Gram-positive bacteria. B. cereus was the most susceptible bacterium, with all 12 extracts displaying activity against this organism. Extracts from the leaves of Eremophila species (Myoporaceae) were the most active, with Eremophila duttonii exhibiting the greatest activity (against Gram-positive bacteria). The antibacterial effects of E. duttonii were further investigated by time-course growth assays which showed that significant growth inhibition was observed in cultures incubated in the presence of the extract within 1 h for B. cereus, E. faecalis and S. aureus and 2 h for S. pyogenes.

Traditional medicinal plant extracts and natural products with activity against oral bacteria: potential application in the prevention and treatment of oral diseases

Oral diseases are major health problems with dental caries and periodontal diseases among the most important preventable global infectious diseases. Oral health influences the general quality of life and poor oral health is linked to chronic conditions and systemic diseases. The association between oral diseases and the oral microbiota is well established. Of the more than 750 species of bacteria that inhabit the oral cavity, a number are implicated in oral diseases. The development of dental caries involves acidogenic and aciduric Gram-positive bacteria (mutans streptococci, lactobacilli and actinomycetes). Periodontal diseases have been linked to anaerobic Gram-negative bacteria (Porphyromonas gingivalis, Actinobacillus, Prevotella and Fusobacterium). Given the incidence of oral disease, increased resistance by bacteria to antibiotics, adverse affects of some antibacterial agents currently used in dentistry and financial considerations in developing countries, there is a need for alternative prevention and treatment options that are safe, effective and economical. While several agents are commercially available, these chemicals can alter oral microbiota and have undesirable side-effects such as vomiting, diarrhea and tooth staining. Hence, the search for alternative products continues and natural phytochemicals isolated from plants used as traditional medicines are considered as good alternatives. In this review, plant extracts or phytochemicals that inhibit the growth of oral pathogens, reduce the development of biofilms and dental plaque, influence the adhesion of bacteria to surfaces and reduce the symptoms of oral diseases will be discussed further. Clinical studies that have investigated the safety and efficacy of such plant-derived medicines will also be described.

Are Human P[14] Rotavirus Strains the Result of Interspecies Transmissions from Sheep or Other Ungulates That Belong to the Mammalian Order Artiodactyla?

ABSTRACT A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains—B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)—and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/Río Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.

Extended excretion of rotavirus after severe diarrhoea in young children

BACKGROUND Rotaviruses are the major cause of severe childhood diarrhoea. Knowledge of the natural history of infection, including duration of intestinal virus shedding, is important in the understanding of transmission, sources of infection, and immune responses. METHODS We carried out a study of rotavirus excretion in 37 children admitted to hospital with severe rotavirus diarrhoea. Sequential faecal specimens were collected from each child during 100 days of surveillance, and screened for rotavirus by EIA and by amplification of genome double-stranded RNA by reverse-transcription PCR. IgA coproantibody was estimated by EIA. FINDINGS Duration of rotavirus excretion ranged from 4 to 57 days after onset of diarrhoea. Excretion ceased within 10 days in 16 (43%) children, and within 20 days in 26 (70%) children. Extended excretion was detected for 25-57 days in the remaining 11 (30%) children owing mainly to continued excretion of the primary infecting strain. Extended excretion was significantly associated with antirotavirus IgA coproantibody boosts during 100 days of surveillance (p=0.001, log-rank test), and with recurrence of mild diarrhoea symptoms during convalescence (p=0.006, Fishers exact test). INTERPRETATION Severe rotavirus disease in young children may be followed by extended excretion of rotavirus. The risk of transmission to others may be greater than previously believed. Extended excretion could also explain some cases of the postgastroenteritis syndrome.

Methods used for the detection and subtyping of Listeria monocytogenes

Listeria monocytogenes is an important foodborne pathogen responsible for non-invasive and invasive diseases in the elderly, pregnant women, neonates and immunocompromised populations. This bacterium has many similarities with other non-pathogenic Listeria species which makes its detection from food and environmental samples challenging. Subtyping of L. monocytogenes strains can prove to be crucial in epidemiological investigations, source tracking contamination from food processing plants and determining evolutionary relationships between different strains. In recent years there has been a shift towards the use of molecular subtyping. This has led to the development of new subtyping techniques such as multi-locus variable number tandem repeat analysis (MLVA) and multi-locus sequence based typing (MLST). This review focuses on the available methods for Listeria detection including immuno-based techniques and the more recently developed molecular methods and analytical techniques such as matrix-assisted laser desorption/ionisation time-of-flight based mass spectrometry (MALDI-TOF MS). It also includes a comparison and critical analysis of the available phenotypic and genotypic subtyping techniques that have been investigated for L. monocytogenes.

Investigating the role of perceived stress on bacterial flora activity and salivary cortisol secretion: a possible mechanism underlying susceptibility to illness

This study examined the impact of academic stress on salivary cortisol concentrations and lactic acid bacteria activity. Whole, unstimulated saliva samples and faecal samples were collected from 23 healthy undergraduate students (23.0+/-6.8 years; range 18-44) over two 1-week periods: during the beginning of semester (low-stress baseline condition) and during the first week of exams (high-stress condition). Students also completed a series of questionnaires measuring perceived levels of stress, gastrointestinal symptoms, and nutritional intake. Significant findings indicated that faecal lactic acid bacterial levels were lower during the high-stress condition. Paralleling this, students rated perceived levels of stress as being greater during the exam period compared to the baseline condition. The findings from this study have provided further insight into the link between stress and gastrointestinal flora activity in humans.

Genetic Analysis of Group A Rotaviruses: Evidence for Interspecies Transmission of Rotavirus Genes

Rotaviruses are the major cause of severe gastroenteritis in young children and animals. The rotavirus genome is composed of eleven segments of double-stranded RNA and can undergo genetic reassortment during mixed infections, leading to progeny viruses with novel or atypical phenotypes. There are numerous descriptions of rotavirus strains isolated from human and animals that share genetic and antigenic features of viruses from heterologous species. In many cases, genetic analysis by hybridization has clearly demonstrated the genetic relatedness of gene segments to those from viruses isolated from different species. Together with the observation that some virus strains appear to have been transmitted to a different species as a whole genome constellation, these data suggest that interspecies transmission occurs naturally, albeit at low frequencies. Although interspecies transmission has not been documented directly, there is an increasing number of reports of atypical rotaviruses that are apparently derived from transmission between: humans, cats and dogs; humans and cattle; humans and pigs; pigs and cattle; and pigs and horses. Interspecies evolutionary relationships are supported by phylogenetic analysis of rotavirus genes from different species. The emergence of novel strains derived from interspecies transmission has implications for the design and implementation of successful human rotavirus vaccine strategies.

Molecular Characterisation of Gastrointestinal Microbiota of Children With Autism (With and Without Gastrointestinal Dysfunction) and Their Neurotypical Siblings

Many children with autism spectrum disorders (ASDs) suffer from gastrointestinal problems such as diarrhoea, constipation and abdominal pain. This has stimulated investigations into possible abnormalities of intestinal microbiota in autistic patients. Therefore, we designed this study to identify differences (and/or similarities) in the microbiota of children with autism (without gastrointestinal dysfunction: n = 23; with gastrointestinal dysfunction: n = 28) and their neurotypical siblings (n = 53) who share a similar environment using bacterial tag‐encoded FLX amplicon pyrosequencing. Regardless of the diagnosis and sociodemographic characteristics, overall, Firmicutes (70%), Bacteroidetes (20%) and Proteobacteria (4%) were the most dominant phyla in samples. Results did not indicate clinically meaningful differences between groups. The data do not support the hypothesis that the gastrointestinal microbiota of children with ASD plays a role in the symptomatology of ASD. Other explanations for the gastrointestinal dysfunction in this population should be considered including elevated anxiety and self‐restricted diets. Autism Res 2012, 5: 419–427.

Genetic and Antigenic Characterization of Rotavirus Serotype G9 Strains Isolated in Australia between 1997 and 2001

ABSTRACT Rotavirus serotype G9 is recognized as the most widespread of the emerging serotypes, emerging since 1996 as a frequent cause of severe acute gastroenteritis in children from many countries covering all continents of the world. This study characterized serotype G9 strains collected in three widely separated Australian centers from 1997 to 2001. All G9 strains possessed the VP4 P[8] and VP6 subgroup II genes. The overall prevalence of the G9 strains increased in Australia, from 0.6% of the strains found in 1997 to 29% of the strains found in 2001. The prevalence of G9 relative to all other serotypes varied from year to year and with geographic location. In Melbourne (representing east coast urban centers), G9 made up 11 to 26% of all of the strains found from 1999 to 2001. In Perth (representing west coast urban centers), G9 made up less than 2% of the strains found in 1997 to 2000 but increased to 18.6% of the strains found in 2001. In Alice Springs (representing widely dispersed settlements in northern arid regions), G9 made up 0 to 5% of the strains found from 1997 to 2000 and was the dominant strain in 2001, making up 68.9% of all of the strains found. Three distinct antigenic groups based on reaction with neutralizing monoclonal antibodies (N-MAbs) were identified, including a dominant group (63%) that cross-reacted with the serotype G4 N-MAb. Phylogenetic analysis of the VP7-encoding gene from Australian strains, compared with a worldwide collection of G9 strains, showed that the Australian G9 strains made up a genetic group distinct from other serotype G9 strains identified in the United States and Africa. Future epidemiological studies of the occurrence of G9 strains should combine reverse transcription-PCR and typing with G1 to G4 and G9 N-MAbs to determine the extent of G9 and G4 cross-reactions among rotavirus strains, in order to assess the need to incorporate G9 strains into new candidate vaccines.