Development of a sensitive competitive enzyme-linked immunosorbent assay for serodiagnosis of Burkholderia mallei, a Tier 1 select agent.

Glanders is a highly contagious and potentially serious disease caused by Burkholderia mallei, a Tier 1 select agent. In this study, we raised a monoclonal antibody (mAb) against the lipopolysaccharide (LPS) of B. mallei and developed a competitive enzyme-linked immunosorbent assay (cELISA) for B. mallei infection. Using the titrated optimal conditions of B. mallei-LPS (2 ng) for microtiter plate coating, sample serum dilution at 1:20 and 3.5 ng/µL anti-LPS mAb B5, the cutoff value of the cELISA was determined using serum samples from 136 glanders-free seronegative horses in Hong Kong. All calculated percentage inhibition (PI) values from these seronegative samples were below 39.6% inhibition (1.5 standard deviations above mean PI) and was used as the cutoff value. The diagnostic sensitivity of the developed LPS-based cELISA was first evaluated using sera from donkeys and mice inoculated with B. mallei. An increasing trend of PI values above the defined cELISA cutoff observed in the donkey and mouse sera suggested positive detection of anti-LPS antibodies. The sensitivity and specificity of the LPS-based cELISA was further evaluated using 31 serologically positive horse sera from glanders outbreaks in Bahrain and Kuwait, of which 30 were tested positive by the cELISA; and 21 seronegative horse sera and 20 seronegative donkey sera from Dubai, of which all were tested negative by the cELISA. A cELISA with high sensitivity (97.2%) and specificity (100%) for the detection of B. mallei antibodies in different animals was developed.

High Prevalence and Mechanism Associated With Extended Spectrum Beta-Lactamase-Positive Phenotype in Laribacter hongkongensis.

In this study, we reported the prevalence and mechanism associated with the extended-spectrum beta-lactamase (ESBL)-positive phenotype in Laribacter hongkongensis isolated from patients and fish. Using the inhibition zone enhancement test, 20 (95.2%) of the 21 patient strains and 8 (57.1%) of the 14 fish strains were tested ESBL-positive. However, ESBL genes, including SHV, TEM, CTX-M, GES, and PER, were not detected in all of these 28 L. hongkongensis isolates. No ESBL gene could be detected in either the complete genome of L. hongkongensis HLHK9 or the draft genome of PW3643. PCR and DNA sequencing revealed that all the 35 L. hongkongensis isolates (showing both ESBL-positive and ESBL-negative phenotypes) were positive for the ampC gene. When the AmpC deletion mutant, HLHK9ΔampC, was subject to the zone enhancement test, the difference of zone size between ceftazidime/clavulanate and ceftazidime was less than 5 mm. When boronic acid was added to the antibiotic disks, none of the 28 "ESBL-positive" isolates showed a ≥ 5 mm enhancement of inhibition zone size diameter between ceftazidime/clavulanate and ceftazidime and between cefotaxime/clavulanate and cefotaxime. A high prevalence (80%) of ESBL-positive phenotype is present in L. hongkongensis. Overall, our results suggested that the ESBL-positive phenotype in L. hongkongensis results from the expression of the intrinsic AmpC beta-lactamase. Confirmatory tests should be performed before issuing laboratory reports for L. hongkongensis isolates that are tested ESBL-positive by disk diffusion clavulanate inhibition test.

Tsukamurella asaccharolytica sp. nov., Tsukamurella conjunctivitidis sp. nov. and Tsukamurella sputi sp. nov., isolated from patients with bacteraemia, conjunctivitis and respiratory infection in Hong Kong.

Three bacterial strains, HKU70T, HKU71T and HKU72T, were isolated from the conjunctival swab, blood and sputum samples of three patients with conjunctivitis, bacteraemia and respiratory infection, respectively, in Hong Kong. The three strains were aerobic, Gram-stain positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from currently recognized Tsukamurella species. 16S rRNA, secA, rpoB and groEL gene sequence analyses revealed that the three strains shared 99.6-99.9, 94.5-96.8, 95.7-97.8 and 97.7-98.9 % nucleotide identities with their corresponding closest Tsukamurella species respectively. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella (26.2±2.4 to 36.8±1.2 % DNA-DNA relatedness), in line with results of in silico genome-to-genome comparison (32.2-40.9 % Genome-to-Genome Distance Calculator and 86.3-88.9 % average nucleotide identity values]. Fatty acids, mycolic acids, cell-wall sugars and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content determined based on the genome sequence of strains HKU70T, HKU71T and HKU72T were 69.9, 70.2 and 70.5 mol%, respectively. Taken together, our results supported the proposition and description of three new species, i.e. Tsukamurella sputi HKU70T (=JCM 33387T=DSM 109106T) sp. nov., Tsukamurella asaccharolytica HKU71T (=JCM 33388T=DSM 109107T) sp. nov. and Tsukamurella conjunctivitidis HKU72T (=JCM 33389T=DSM 109108T) sp. nov.

Geochemical and mineralogical constraints in iron ore tailings limit soil formation for direct phytostabilization.

The present study aimed to characterize key physico-chemical and mineralogical attributes of magnetite iron (Fe) ore tailings to identify potential constraints limiting in situ soil formation and direct phytostabilization. Tailings of different age, together with undisturbed local native soils, were sampled from a magnetite mine in Western Australia. Tailings were extremely alkaline (pH > 9.0), with a lack of water stable aggregate and organic matter, and contained abundant primary minerals including mica (e.g., biotite), with low specific surface area (N2-BET around 1.2 m2 g-1). These conditions remained relatively unchanged after four years' aging under field conditions. Chemical extraction and spectroscopic analysis [e.g., X-ray diffraction (XRD) and synchrotron-based Fe K edge X-ray absorption fine structure spectroscopy (XAFS) analysis] revealed that the aging process decreased biotite-like minerals, but increased hematite and magnetite in the tailings. However, the aged tailings lacked goethite, a compound abundant in natural soils. Examination using backscattered-scanning electron microscope - energy dispersive X-ray spectrometry (BSE-SEM-EDS) revealed that aged tailings contained discrete sharp edged Fe-bearing minerals that did not physically integrate with other minerals (e.g., Si/Al bearing minerals). In contrast, Fe minerals in native soils appeared randomly distributed and closely amassed with Si/Al rich phyllosilicates, with highly eroded edges. The lack of labile organic matter and the persistence of alkaline-saline conditions may have significantly hindered the bioweathering of Fe-minerals and the biogenic formation of secondary Fe-minerals in tailings. However, there is signature that a native pioneer plant, Maireana brevifolia can facilitate the bioweathering of Fe-bearing minerals in tailings. We propose that eco-engineering inputs like organic carbon accumulation, together with the introduction of functional microbes and pioneer plants, should be adopted to accelerate bioweathering of Fe-bearing minerals as a priority for initiating in situ soil formation in the Fe ore tailings.

Differential Microbial Communities of Omnivorous and Herbivorous Cattle in Southern China.

In Hong Kong, cattle were traditionally raised by farmers as draft animals to plough rice fields. Due to urbanization in the 20th century, they were gradually abandoned and became wild cattle straying in suburban Hong Kong. Recently, these cattle were observed to have become omnivorous by eating leftover barbeque food waste in country parks. Microbiome analysis was performed on fecal samples of the omnivorous cattle using deep sequencing and the resulting microbiome was compared with that of traditional herbivorous cattle in Southern China. A more diverse gut microbiome was observed in the omnivorous cattle, suggesting that microbiota diversity increases as diet variation increases. At the genus level, the relative abundance of Anaeroplasma, Anaerovorax, Bacillus, Coprobacillus and Solibacillus significantly increased and those of Anaerofustis, Butyricimonas, Campylobacter, Coprococcus, Dehalobacterium, Phascolarctobacterium, rc4.4, RFN20, Succinivibrio and Turicibacter significantly decreased in the omnivorous group. The increase in microbial community levels of Bacillus and Anaerovorax likely attributes to the inclusion of meat in the diet; while the decrease in relative abundance of Coprococcus, Butyricimonas, Succinivibrio, Campylobacter and Phascolarctobacterium may reflect the reduction in grass intake. Furthermore, an increased consumption of resistant starch likely resulted in the increase in abundance of Anaeroplasma. In conclusion, a significant change in the gut microbial community was observed in the omnivorous cattle, suggesting that diet may be one of the factors that may signal an adaptation response by the cattle to maintain feed efficiency as a consequence of the change in environment.

Tsukamurella ocularis sp. nov. and Tsukamurella hominis sp. nov., isolated from patients with conjunctivitis in Hong Kong.

Three bacterial strains, HKU63T, HKU64 and HKU65T, were isolated from the conjunctival swabs of three patients with conjunctivitis in Hong Kong. The three strains were aerobic, Gram-stain-positive, catalase-positive, non-sporulating and non-motile bacilli and exhibited unique biochemical profiles distinguishable from closely related Tsukamurella species. 16S rRNA gene sequence analysis revealed that the three strains shared identical sequences with each other, being most closely related to Tsukamurella tyrosinosolvens and Tsukamurella pulmonis, sharing 99.9 % sequence identity. Sequence analysis of three additional housekeeping genes, groEL, secA and rpoB, revealed 100 % nucleotide sequence identity between HKU63T and HKU64, 94.2-97.0 % nucleotide sequence identities between HKU63T/HKU64 and HKU65T and the three strains shared 82.9-98.9 % sequence identities with other currently recognized Tsukamurella species. DNA-DNA hybridization confirmed that they were distinct from other known species of the genus Tsukamurella(23.0±4.2 to 50.7±3.7 % DNA-DNA relatedness), of which HKU63T and HKU64 represented the same species (≥95.2±4.8 % DNA-DNA relatedness) while HKU65T represented another species. Fatty acid, mycolic acid, cell-wall sugar and peptidoglycan analyses showed that they were typical of members of Tsukamurella. The G+C content of strains HKU63T, HKU64 and HKU65T were 71.3±1.9, 71.3±2.0 and 71.2±2.3 mol% (mean±sd; n=3), respectively. A novel species, Tsukamurella ocularis sp. nov. is proposed to accommodate strains HKU63T and HKU64, with HKU63T (=JCM 31969T=DSM 105034T) designated as the type strain whilst another novel species, Tsukamurella hominis sp. nov., is proposed to accommodate the third strain, HKU65T, which is designated as the type strain (=JCM 31971T=DSM 105036T).

Hepatitis E Virus Genotypes and Evolution: Emergence of Camel Hepatitis E Variants.

Hepatitis E virus (HEV) is a major cause of viral hepatitis globally. Zoonotic HEV is an important cause of chronic hepatitis in immunocompromised patients. The rapid identification of novel HEV variants and accumulating sequence information has prompted significant changes in taxonomy of the family Hepeviridae. This family includes two genera: Orthohepevirus, which infects terrestrial vertebrates, and Piscihepevirus, which infects fish. Within Orthohepevirus, there are four species, A-D, with widely differing host range. Orthohepevirus A contains the HEV variants infecting humans and its significance continues to expand with new clinical information. We now recognize eight genotypes within Orthohepevirus A: HEV1 and HEV2, restricted to humans; HEV3, which circulates among humans, swine, rabbits, deer and mongooses; HEV4, which circulates between humans and swine; HEV5 and HEV6, which are found in wild boars; and HEV7 and HEV8, which were recently identified in dromedary and Bactrian camels, respectively. HEV7 is an example of a novel genotype that was found to have significance to human health shortly after discovery. In this review, we summarize recent developments in HEV molecular taxonomy, epidemiology and evolution and describe the discovery of novel camel HEV genotypes as an illustrative example of the changes in this field.

Wastewater-Enhanced Microbial Corrosion of Concrete Sewers.

Microbial corrosion of concrete in sewers is known to be caused by hydrogen sulfide, although the role of wastewater in regulating the corrosion processes is poorly understood. Flooding and splashing of wastewater in sewers periodically inoculates the concrete surface in sewer pipes. No study has systematically investigated the impacts of wastewater inoculation on the corrosion of concrete in sewers. This study investigated the development of the microbial community, sulfide uptake activity, and the change of the concrete properties for coupons subjected to periodic wastewater inoculation. The concrete coupons were exposed to different levels of hydrogen sulfide under well-controlled conditions in laboratory-scale corrosion chambers simulating real sewers. It was evident that the periodic inoculation induced higher corrosion losses of the concrete in comparison to noninoculated coupons. Instantaneous measurements such as surface pH did not reflect the cumulative corrosion losses caused by long-term microbial activity. Analysis of the long-term profiles of the sulfide uptake rate using a Gompertz model supported the enhanced corrosion activity and greater corrosion loss. The enhanced corrosion rate was due to the higher sulfide uptake rates induced by wastewater inoculation, although the increasing trend of sulfide uptake rates was slower with wastewater. Increased diversity in the corrosion-layer microbial communities was detected when the corrosion rates were higher. This coincided with the environmental conditions of increased levels of gaseous H2S and the concrete type.