Paolo Martelli

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Rapid Identification of Burkholderia pseudomallei: Importance of Expanding Databases with Pathogens Endemic to Different Localities

Burkholderia pseudomallei is the causative agent of melioidosis, a serious disease endemic in southeast Asia. Accurate identification of B. pseudomallei is important, since treatment of melioidosis requires prolonged antibiotics to prevent relapse ([9][1]). Although B. pseudomallei differs greatly

Novel Pan-Genomic Analysis Approach in Target Selection for Multiplex PCR Identification and Detection of Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia cepacia Complex Species: a Proof-of-Concept Study

ABSTRACT Burkholderia pseudomallei, Burkholderia thailandensis, and the Burkholderia cepacia complex differ greatly in pathogenicity and epidemiology. Yet, they are occasionally misidentified by biochemical profiling, and even 16S rRNA gene sequencing may not offer adequate discrimination between certain species groups. Using the 23 B. pseudomallei, four B. thailandensis, and 16 B. cepacia complex genome sequences available, we identified gene targets specific to each of them (a Tat domain protein, a 70-kDa protein, and a 12-kDa protein for B. pseudomallei, B. thailandensis, and the B. cepacia complex, respectively), with an in-house developed algorithm. Using these targets, we designed a robust multiplex PCR assay useful for their identification and detection from soil and simulated sputum samples. For all 43 B. pseudomallei, seven B. thailandensis, and 20 B. cepacia complex (B. multivorans, n = 6; B. cenocepacia, n = 3; B. cepacia, n = 4; B. arboris, n = 2; B. contaminans, B. anthina, and B. pyrrocinia, n = 1 each; other unnamed members, n = 2) isolates, the assay produced specific products of predicted size without false positives or negatives. Of the 60 soil samples screened, 19 (31.6%) and 29 (48.3%) were positive for B. pseudomallei and the B. cepacia complex, respectively, and in four (6.7%) soil samples, the organisms were codetected. DNA sequencing confirmed that all PCR products originated from their targeted loci. This novel pan-genomic analysis approach in target selection is simple, computationally efficient, and potentially applicable to any species that harbors species-specific genes. A multiplex PCR assay for rapid and accurate identification and detection of B. pseudomallei, B. thailandensis, and the B. cepacia complex was developed and verified.

Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus

ABSTRACT While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 103 to 1 × 105 copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks ) to the number of nonsynonymous substitutions per nonsynonymous site (Ka ) in multiple regions of the genome, especially the S gene (Ka /Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

Complete Genome Sequence of a Novel Picobirnavirus, Otarine Picobirnavirus, Discovered in California Sea Lions

ABSTRACT We discovered a novel otarine picobirnavirus in fecal samples of California sea lions. Its genome contains a large segment with two open reading frames (ORFs), ORF1 encoding a putative protein of 163 amino acids with unknown function and ORF2 encoding capsid protein, and a small segment with one ORF encoding RNA-dependent RNA polymerase.

Burkholderia pseudomallei in soil samples from an oceanarium in Hong Kong detected using a sensitive PCR assay

Melioidosis, caused by Burkholderia pseudomallei, is an emerging infectious disease with an expanding geographical distribution. Although assessment of the environmental load of B. pseudomallei is important for risk assessment in humans or animals in endemic areas, traditional methods of bacterial culture for isolation have low sensitivities and are labor-intensive. Using a specific polymerase chain reaction (PCR) assay targeting a Tat domain protein in comparison with a bacterial culture method, we examined the prevalence of B. pseudomallei in soil samples from an oceanarium in Hong Kong where captive marine mammals and birds have contracted melioidosis. Among 1420 soil samples collected from various sites in the oceanarium over a 15-month period, B. pseudomallei was detected in nine (0.6%) soil samples using bacterial culture, whereas it was detected in 96 (6.8%) soil samples using the specific PCR assay confirmed by sequencing. The PCR-positive samples were detected during various months, with higher detection rates observed during summer months. Positive PCR detection was significantly correlated with ambient temperature (P<0.0001) and relative humidity (P=0.011) but not with daily rainfall (P=0.241) or a recent typhoon (P=0.787). PCR-positive samples were obtained from all sampling locations, with the highest detection rate in the valley. Our results suggest that B. pseudomallei is prevalent and endemic in the oceanarium. The present PCR assay is more sensitive than the bacterial culture method, and it may be used to help better assess the transmission of melioidosis and to design infection control measures for captive animals in this unique and understudied environment.

Metabolomic profiling of Burkholderia pseudomallei using UHPLC-ESI-Q-TOF-MS reveals specific biomarkers including 4-methyl-5-thiazoleethanol and unique thiamine degradation pathway

BackgroundBurkholderia pseudomallei is an emerging pathogen that causes melioidosis, a serious and potentially fatal disease which requires prolonged antibiotics to prevent relapse. However, diagnosis of melioidosis can be difficult, especially in culture-negative cases. While metabolomics represents an uprising tool for studying infectious diseases, there were no reports on its applications to B. pseudomallei. To search for potential specific biomarkers, we compared the metabolomics profiles of culture supernatants of B. pseudomallei (15 strains), B. thailandensis (3 strains), B. cepacia complex (14 strains), P. aeruginosa (4 strains) and E. coli (3 strains), using ultra-high performance liquid chromatography-electrospray ionization-quadruple time-of-flight mass spectrometry (UHPLC-ESI-Q-TOF-MS). Multi- and univariate analyses were used to identify specific metabolites in B. pseudomallei.ResultsPrincipal component and partial-least squares discrimination analysis readily distinguished the metabolomes between B. pseudomallei and other bacterial species. Using multi-variate and univariate analysis, eight metabolites with significantly higher levels in B. pseudomallei were identified. Three of the eight metabolites were identified by MS/MS, while five metabolites were unidentified against database matching, suggesting that they may be potentially novel compounds. One metabolite, m/z 144.048, was identified as 4-methyl-5-thiazoleethanol, a degradation product of thiamine (vitamin B1), with molecular formula C6H9NOS by database searches and confirmed by MS/MS using commercially available authentic chemical standard. Two metabolites, m/z 512.282 and m/z 542.2921, were identified as tetrapeptides, Ile-His-Lys-Asp with molecular formula C22H37N7O7 and Pro-Arg-Arg-Asn with molecular formula C21H39N11O6, respectively. To investigate the high levels of 4-methyl-5-thiazoleethanol in B. pseudomallei, we compared the thiamine degradation pathways encoded in genomes of B. pseudomallei and B. thailandensis. While both B. pseudomallei and B. thailandensis possess thiaminase I which catalyzes degradation of thiamine to 4-methyl-5-thiazoleethanol, thiM, which encodes hydroxyethylthiazole kinase responsible for degradation of 4-methyl-5-thiazoleethanol, is present and expressed in B. thailandensis as detected by PCR/RT-PCR, but absent or not expressed in all B. pseudomallei strains. This suggests that the high 4-methyl-5-thiazoleethanol level in B. pseudomallei is likely due to the absence of hydroxyethylthiazole kinase and hence reduced downstream degradation.ConclusionEight novel biomarkers, including 4-methyl-5-thiazoleethanol and two tetrapeptides, were identified in the culture supernatant of B. pseudomallei.

High Diversity of Genogroup I Picobirnaviruses in Mammals

In a molecular epidemiology study using 791 fecal samples collected from different terrestrial and marine mammals in Hong Kong, genogroup I picobirnaviruses (PBVs) were positive by RT-PCR targeting the partial RdRp gene in specimens from five cattle, six monkeys, 17 horses, nine pigs, one rabbit, one dog, and 12 California sea lions, with 11, 9, 23, 17, 1, 1, and 15 sequence types in the positive specimens from the corresponding animals, respectively. Phylogenetic analysis showed that the PBV sequences from each kind of animal were widely distributed in the whole tree with high diversity, sharing 47.4–89.0% nucleotide identities with other genogroup I PBV strains based on the partial RdRp gene. Nine complete segment 1 (viral loads 1.7 × 104 to 5.9 × 106/ml) and 15 segment 2 (viral loads 4.1 × 103 to 1.3 × 106/ml) of otarine PBVs from fecal samples serially collected from California sea lions were sequenced. In the two phylogenetic trees constructed using ORF2 and ORF3 of segment 1, the nine segment 1 sequences were clustered into four distinct clades (C1–C4). In the tree constructed using RdRp gene of segment 2, the 15 segment 2 sequences were clustered into nine distinct clades (R1–R9). In four sea lions, PBVs were detected in two different years, with the same segment 1 clade (C3) present in two consecutive years from one sea lion and different clades present in different years from three sea lions. A high diversity of PBVs was observed in a variety of terrestrial and marine mammals. Multiple sequence types with significant differences, representing multiple strains of PBV, were present in the majority of PBV-positive samples from different kinds of animals.

Fatal Systemic Necrotizing Infections Associated with a Novel Paramyxovirus, Anaconda Paramyxovirus, in Green Anaconda Juveniles

ABSTRACT Beginning in July 2011, 31 green anaconda (Eunectes murinus) juveniles from an oceanarium in Hong Kong died over a 12-month period. Necropsy revealed at least two of the following features in 23 necropsies: dermatitis, severe pan-nephritis, and/or severe systemic multiorgan necrotizing inflammation. Histopathological examination revealed severe necrotizing inflammation in various organs, most prominently the kidneys. Electron microscopic examination of primary tissues revealed intralesional accumulations of viral nucleocapsids with diameters of 10 to 14 nm, typical of paramyxoviruses. Reverse transcription (RT)-PCR results were positive for paramyxovirus (viral loads of 2.33 × 104 to 1.05 × 108 copies/mg tissue) in specimens from anaconda juveniles that died but negative in specimens from the two anaconda juveniles and anaconda mother that survived. None of the other snakes in the park was moribund, and RT-PCR results for surveillance samples collected from other snakes were negative. The virus was isolated from BHK21 cells, causing cytopathic effects with syncytial formation. The virus could also replicate in 25 of 27 cell lines of various origins, in line with its capability for infecting various organs. Electron microscopy with cell culture material revealed enveloped virus with the typical “herringbone” appearance of helical nucleocapsids in paramyxoviruses. Complete genome sequencing of five isolates confirmed that the infections originated from the same clone. Comparative genomic and phylogenetic analyses and mRNA editing experiments revealed a novel paramyxovirus in the genus Ferlavirus, named anaconda paramyxovirus, with a typical Ferlavirus genomic organization of 3′-N-U-P/V/I-M-F-HN-L-5′. Epidemiological and genomic analyses suggested that the anaconda juveniles acquired the virus perinatally from the anaconda mother rather than from other reptiles in the park, with subsequent interanaconda juvenile transmission.

Anaconda paramyxovirus infection in an adult green anaconda after prolonged incubation: Pathological characterization and whole genome sequence analysis

From July 2011 to June 2012, 31 out of 33 green anaconda juveniles from an oceanarium in Hong Kong died over a 12-month period. These anacondas were progeny of a female anaconda purchased from Japan and added to the collection in May 2011. The juvenile anacondas were born in July 2011. A novel paramyxovirus, named anaconda paramyxovirus (AnaPV), was isolated from these affected juvenile anacondas. In July 2015, one of the remaining two anacondas, that survived the cluster of fatal infections, died at the age of four. Pathologically, both the death of the four-year-old anaconda and the previous deaths of the anaconda juveniles involved multiple, similar organs. However, the organ that was primarily affected in the juvenile anacondas that died in 2011 was the kidney, whereas the most remarkable lesions in the four-year-old anaconda involved the lungs. Granulomas previously observed in the juvenile anacondas with AnaPV infections were not obvious in the four-year-old anaconda. RT-PCR for the L gene of AnaPV was positive for the lungs, kidneys, ovary, spleen, liver, tracheal content and gall bladder of the four-year-old anaconda, with a median viral load of 1.32×106AnaPVRNAcopies/mg. Complete genome sequencing revealed that there were only 12-14 nucleotide changes in the AnaPV genome of the four-year old anaconda compared to those of the AnaPV found in anaconda juveniles in 2011/2012. Among these nucleotide changes, only four were non-synonymous mutations, with one in the N gene, one in the M gene and two in the HN gene. Both epidemiological and molecular data supported that the four-year-old green anaconda probably acquired the AnaPV from its mother or its siblings that died 3-4years ago, and its death is a result of an unprecedented extended incubation period or latency of AnaPV followed by a subsequent manifestation of clinical disease and death.

Two novel noroviruses and a novel norovirus genogroup in California sea lions

In this study, two novel noroviruses (NoVs) were discovered from faecal samples from California sea lions from an oceanarium in Hong Kong, and named California sea lion NoV 1 (Csl/NoV1) and California sea lion NoV 2 (Csl/NoV2). Whole-genome sequencing showed that the genome organization and amino acid motifs of both Csl/NoV1 and Csl/NoV2 were typical of those of other NoVs in their open reading frames (ORFs). Csl/NoV1 possessed only 52.6-52.8 % amino acid identity in VP1 to the closest matches in genogroup GII. Therefore, Csl/NoV1 should constitute a novel genogroup of NoV. Shifting of the phylogenetic position of Csl/NoV1 in the RdRp, VP1 and VP2 trees was observed, which may have been due to recombination events and/or biased mutations. Csl/NoV2 possessed 55.4-56.2 % amino acid identity in VP1 to its closest relatives in genogroup GVI, which means that it represents a new genotype in genogroup GVI. Further studies will reveal what diseases these NoVs can cause in marine mammals.