Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication

BACKGROUND: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community-acquired gastroenteritis and traveler's diarrhea. Its mechanisms of adaptation to various environmental niches and host defense evasion are largely unknown. During the process of analyzing the L. hongkongensis genome, a complete urease cassette and two adjacent arc gene cassettes were found. We hypothesize that the urease cassette and/or the arc gene cassettes are important for L. hongkongensis to survive in acidic environment and macrophages. In this study, we tested this hypothesis by constructing single, double and triple non-polar deletion mutants of the urease and two arc gene cassettes of L. hongkongensis using the conjugation-mediated gene deletion system and examining their effects in acidic environment in vitro, in macrophages and in a mouse model. RESULTS: HLHK9ureA, HLHK9ureC, HLHK9ureD and HLHK9ureE all exhibited no urease activity. HLHK9arcA1 and HLHK9arcA2 both exhibited arginine deiminase (ADI) activities, but HLHK9arcA1/arcA2 double deletion mutant exhibited no ADI activity. At pH 2 and 3, survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly decreased (p < 0.001) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to wild type L. hongkongensis HLHK9. Survival of HLHK9ureA/arcA1/arcA2 and HLHK9arcA1/arcA2 in macrophages were also markedly decreased (p < 0.001 and p < 0.01 respectively) but that of HLHK9ureA was slightly decreased (p < 0.05), compared to HLHK9, although expression of arcA1, arcA2 and ureA genes were all upregulated. Using a mouse model, HLHK9ureA exhibited similar survival compared to HLHK9 after passing through the murine stomach, but survival of HLHK9arcA1/arcA2 and HLHK9ureA/arcA1/arcA2 were markedly reduced (p < 0.01). CONCLUSIONS: In contrast to other important gastrointestinal tract pathogens, ADI pathway is far more important than urease for acid resistance and intracellular survival in L. hongkongensis. The gene duplication of the arc gene cassettes could be a result of their functional importance in L. hongkongensis.published_or_final_versio

Pseudobacteraemia in a patient with neutropenic fever caused by a novel paenibacillus species: Paenibacillus hongkongensis sp. nov.

Aims: To characterise a strain of Gram negative aerobic straight or slightly curved rods (HKU3) isolated from the blood culture of a 9 year old Chinese boy with neutropenic fever and pseudobacteraemia. Methods: The isolate was phenotypically investigated by standard biochemical methods using conventional biochemical tests, scanning electron microscopy, and transmission electron microscopy. Genotypically, the 16S rRNA gene of the bacterium was amplified by the polymerase chain reaction (PCR) and sequenced. The sequence of the PCR product was compared with known 16S rRNA gene sequences in the Genbank by multiple sequence alignment. The G + C content was determined by thermal denaturation. A phylogenetic tree was constructed by the PileUp method. Results: The cells of the bacterial strain were aerobic, sporulating, Gram negative straight or slight curved rods. The bacterium grew on horse blood agar as non-haemolytic, grey colonies of 1 mm in diameter after 24 hours of incubation at 37°C in ambient air. No enhancement of growth was seen in 5% CO 2. It grew at 50°C as pinpoint colonies after 72 hours of incubation, but did not grow at 65°C or on MacConkey agar. It was non-motile. It produced catalase (weakly positive) and cytochrome oxidase. It reduced nitrate, produced β galactosidase, hydrolysed esculin, and utilised sodium acetate. A scanning electron micrograph of the bacterium showed straight or slightly curved rods. A transmission electron micrograph of the cell wall of the bacterium revealed multiple electron dense layers, including the outer membrane, middle murein layer, and inner cytoplasmic membrane, compatible with its Gram smear appearance. 16S rRNA gene sequencing showed that there were 7.7%, 8.0%, 8.2%, and 8.6% differences between the 16S rRNA gene sequence of the bacterium and those of Paenibacillus macerans, Paenibacillus borealis, Bacillus ehimensis, and Paenibacillus amylolyticus, respectively. The mean (SD) G + C content of the bacterium was 47.6 (2.1) mol%. Phylogenetically, it belongs to the genus paenibacillus (previously called group 3 bacillus). Conclusions: A bacterium that exhibited phenatypic and genotypic characteristics that are very different from closely related members of paenibacillus was the cause of pseudobacteraemia in a patient with neutropenic fever. A new species, Paenibacillus hongkongensis sp. nov. is proposed, for which HKU3 is the type strain.published_or_final_versio

Arginine deiminase pathway is far more important than urease for acid resistance and intracellular survival in Laribacter hongkongensis: a possible result of arc gene cassette duplication

Title in Final Programme: Arginine deiminase pathway is far more important than urease for acid resistance in Laribacter hongkongensis: result of arc gene cassette duplicationPoster Session - Pathogenesis and animal models of bacterial infections: abstract no. P1122INTRODUCTION AND PURPOSE: Laribacter hongkongensis is a Gram-negative, urease-positive bacillus associated with invasive bacteremic infections in liver cirrhosis patients and fish-borne community- acquired gastroenteritis and traveler’s diarrhea (1-2). Its mechanisms of acid resistance are unknown. a complete urease cassette and two adjacent arc gene cassettes (encoding enzymes of ADI pathway) were found in the genome (3). In this study, we investigated the mechanism for resisting acidic environment in vitro, in macrophages and in a mouse model ...published_or_final_versio

Primary infective spondylodiscitis caused by Lactococcus garvieae and a review of human L. garvieae infections

We report the first case of primary infective spondylodiscitis due to Lactococcus garvieae, confirmed by 16S rRNA gene sequencing, in the absence of concomitant endocarditis in a patient with long-standing gastritis on famotidine. He responded to a 6-week course of ampicillin. The gastrointestinal tract is probably the source of infection. © 2011 The Author(s).published_or_final_versionSpringer Open Choice, 21 Feb 201

Comparative evaluation of a point-of-care immunochromatographic test SNAP 4Dx with molecular detection tests for vector-borne canine pathogens in Hong Kong

There are no comprehensive studies on the performance of commonly used point-of-care diagnostic enzyme immunoassay for common arthropod-borne canine pathogens. A comparative evaluation of an immunochromatographic test for these infections with a comprehensive polymerase chain reaction (PCR) test panel was performed on 100 pet dogs and 100 stray dogs without obvious clinical symptoms. Of the 162 positive test results from both immunochromatographic test and PCR, there was 85.2% concordance. The 24 discordant results between serology and PCR occurred in tests involving Ehrlichia canis (14) and Anaplasma platys (10), which may be related to the time of infection. No positive cases of borreliosis or rickettsiosis were detected. One important limitation of the immunochromatographic test was its lack of testing for babesiosis and hepatozoonosis. The former is the most prevalent arthropod-borne canine infection in our cohort (41%). Coinfections were found in 19% stray dogs and 6% of pet dogs with both tests (p<0.01). Seventeen and 8 samples from stray and pet dogs, respectively, were initially positive in the PCR test for Ehrlichia. However, on sequencing of the PCR amplicon, 10 from stray and 2 from pet dogs were found to be Wolbachia sequences instead, with 100% nucleotide identity to the 16S rRNA sequence of Wolbachia endosymbiont of Dirofilaria immitis. The presence of Wolbachia DNAemia (6%) correlated well with the molecular test and immunochromatographic antigen test for D. immitis. © Copyright 2011, Mary Ann Liebert, Inc.published_or_final_versio

Phylogenomic analysis of streptococcus sinensis HKU4T reveals a novel phylogenetic group in the genus streptococcus

Poster Board Number: FRIDAY-022Currently, the genus Streptococcus comprised more than 90 species and was divided into 7 major groups, namely anginosus, mitis, salivarius, mutans, bovis, pyogenic and sanguinis. Streptococcus sinensis has been discovered by our group from patients with infective endocarditis in Hong Kong in 2002. Subsequently, additional cases reported from worldwide suggested that the bacterium is an emerging pathogen of global importance. Phenotypic profile …postprin

First detection of a Vssc allele V1016G conferring a high level of insecticide resistance in Aedes albopictus collected from Europe (Italy) and Asia (Vietnam), 2016. A new emerging threat to controlling arboviral diseases

Introduction Aedes albopictus (Skuse) is an important vector of arboviral diseases, including dengue, chikungunya and Zika virus disease. Monitoring insecticide resistance and mechanisms by which the mosquito develops resistance is crucial to minimise disease transmission. Aim To determine insecticide resistance status and mechanisms in Ae. albopictus from different geographical regions. Methods We sampled 33 populations of Ae. albopictus from Asia, Europe and South America, and tested these for susceptibility to permethrin, a pyrethroid insecticide. In resistant populations, the target site for pyrethroids, a voltage-sensitive sodium channel (Vssc) was genotyped. Three resistant sub-strains, each harbouring a resistance allele homozygously, were established and susceptibilities to three different pyrethroids (with and without a cytochrome P450 inhibitor) were assayed. Results Most populations of Ae. albopictus tested were highly susceptible to permethrin but a few from Italy and Vietnam (4/33), exhibited high-level resistance. Genotyping studies detected a knockdown resistance (kdr) allele V1016G in Vssc for the first time in Ae. albopictus. Two previously reported kdr alleles, F1534C and F1534S, were also detected. The bioassays indicated that the strain homozygous for the V1016G allele showed much greater levels of pyrethroid resistance than other strains harbouring F1534C or F1534S. Conclusion The V1016G allele was detected in bothAsian and Italian Ae. albopictus populations, thus a spread of this allele beyond Italy in Europe cannot be ruled out. This study emphasises the necessity to frequently and regularly monitor the V1016G allele in Ae. albopictus, particularly where this mosquito species is the main vector of arboviruses

Molecular characterization of the arginine deiminase pathways in Laribacter hongkongensis: unique genetic organisation and regulation by transcriptional regulator ArgR and multiple environmental stresses

Conference Theme: Molecular Systems and Community Response

Current status and future directions for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis and traveller's diarrhoea

Purpose of review: Despite extensive investigations, a microbiological cause cannot be found in about half of the patients with infectious disease. Throughout the years, scientists have spent tremendous efforts in looking for microorganisms associated with these 'unexplained infectious disease syndromes'. Recently, a novel bacterium, Laribacter hongkongensis, was discovered and shown to be associated with gastroenteritis and traveller's diarrhoea. This review summarizes the current status, and shares with the readers the authors' experience in the microbiology, classification, epidemiology, clinical disease, laboratory diagnosis, antibiotic resistance and treatment of L. hongkongensis. It also discusses the importance and perspective of describing novel pathogenic bacterial species. Recent findings: L. hongkongensis was shown to be associated with gastroenteritis and traveller's diarrhoea. Consumption of fish was associated with recovery of L. hongkongensis. Freshwater fish was a reservoir of L. hongkongensis. Genotypic typing revealed the possibility of virulent clones of L. hongkongensis. The class C β-lactamase of L. hongkongensis has been cloned and characterized. Summary: In 2001, L. hongkongensis, a novel genus and species, was first discovered in Hong Kong from the blood and empyema pus of a patient with alcoholic cirrhosis. Subsequently, it was isolated from patients in other parts of the world. Recently, this bacterium was found to be associated with community-acquired gastroenteritis and traveller's diarrhoea using cefoperazone MacConkey agar as the selective medium. Further studies, including setting up of animal and tissue culture models and characterization of virulence factors, should be performed. For pathogenic microbes, even one strain of a novel species should be described, so that global concerted efforts can be drawn to look for more cases associated with such a pathogen. © 2005 Lippincott Williams & Wilkins.link_to_subscribed_fulltex