Combined antimicrobial action of streptomycin and terpenes against atypical mycobacteria isolated from fish.

The aim of this study was to investigate the antimycobacterial activity of 39 free terpenes and their activity in combination with streptomycin. Antimicrobial activity was first evaluated by screening 39 free terpenes at concentrations from 1.56 to 400 µg/mL. None of these exhibited positive effects against any of the nontuberculous mycobacteria (NTM) strains tested. However, six of the 39 terpenes (isoeugenol, nerol, (+)-α-terpineol, (1R)-(-)-myrtenol, (+)-terpinen-4-ol, and eugenol) were shown to enhance the activity of streptomycin against the NTM strains isolated from diseased ornamental fish.

Selection of Biodegrading Phytosterol Strains.

The phytosterol-biotransforming strains can be selected from Mycobacterium sp. using a high concentration of ß-sitosterol. The selection is made by culturing the strains in a medium enriched with 14 g/L of ß-sitosterol as the unique source of carbon. During 2 months, the bacterial cultures are transferred successively. The extraction of the biotransformation products is made with methanol and ethyl acetate. The qualitative and quantitative analyses are made by means of thin-layer chromatography, gas-liquid chromatography (GLC), and GLC-mass spectrometry. Under these conditions, it is observed that after seven transfers, the strains Mycobacterium sp. MB-3683 and Mycobacterium fortuitum B-11045 increase their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial are androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of ß-sitosterol into steroidal bases.

ß-Sitosterol Bioconversion in Small-Scale Devices: From Microtiter Plates to Microfluidic Reactors.

Small-scale devices are routinely used as low-cost miniaturized bioreactors due to the large number of experiments that can be conducted simultaneously under similar conditions and replicate all functions of bench-scale reactors at dramatically smaller volumes. Microtiter plates, due to the standard footprint, can be integrated with liquid handling systems and associated equipment, expanding considerably their application and use. However, care has to be taken to operate the microtiter plates in optimized mixing and oxygen transfer conditions, preventing medium evaporation in prolonged experiment runs. Recently, to increase data quality, microbioreactors have emerged as an alternative to shaken systems. These systems offer higher degree of control over key process variables and when combined with sensing technology increase dramatically the reliability of translational process data. In this chapter, we describe the production of 4-androstene-3,17-dione (androstenedione (AD)), a key pharmaceutical steroid intermediate, by Mycobacterium sp. NRRL B-3805 via the selective cleavage of the side-chain of ß-sitosterol using 24-well microtiter plates and microfluidic microbioreactors.

Loop pathways are responsible for tuning the accumulation of C19- and C22-sterol intermediates in the mycobacterial phytosterol degradation pathway.

4-Androstene-3,17-dione (4-AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (BA) are the most important and representative C19- and C22-steroidal materials. The optimalization of sterol production with mycobacterial phytosterol conversion has been investigated for decades. One of the major challenges is that current industrial mycobacterial strains accumulate unignorable impurities analogous to desired sterol intermediates, significantly hampering product extractions and refinements. Previously, we identified Mycobacterium neoaurum HGMS2 as an efficient 4-AD-producing strain (Wang et al. in Microb Cell Fact. 19:187, 2020). Recently, we have genetically modified the HGMS2 strain to remove its major impurities including ADD and 9OH-AD (Li et al. in Microb Cell Fact. 20:158, 2021). Unexpectedly, the modified mutants started to significantly accumulate BA compared with the HGMS2 strain. In this work, while we attempted to block BA occurrence during 4-AD accumulation in HGMS2 mutants, we identified a few loop pathways that regulated metabolic flux switching between 4-AD and BA accumulations and found that both the 4-AD and BA pathways shared a 9,10-secosteroidial route. One of the key enzymes in the loop pathways was Hsd4A1, which played an important role in determining 4-AD accumulation. The inactivation of the hsd4A1 gene significantly blocked the 4-AD metabolic pathway so that the phytosterol degradation pathway flowed to the BA metabolic pathway, suggesting that the BA metabolic pathway is a complementary pathway to the 4-AD pathway. Thus, knocking out the hsd4A1 gene essentially made the HGMS2 mutant (HGMS2Δhsd4A1) start to efficiently accumulate BA. After further knocking out the endogenous kstd and ksh genes, an HGMS2Δhsd4A1 mutant, HGMS2Δhsd4A1/Δkstd1, enhanced the phytosterol conversion rate to BA in 1.2-fold compared with the HGMS2Δhsd4A1 mutant in pilot-scale fermentation. The final BA yield increased to 38.3 g/L starting with 80 g/L of phytosterols. Furthermore, we knocked in exogenous active kstd or ksh genes to HGMS2Δhsd4A1/Δ kstd1 to construct DBA- and 9OH-BA-producing strains. The resultant DBA- and 9OH-BA-producing strains, HGMS2Δhsd4A1/kstd2 and HGMS2Δkstd1/Δhsd4A1/kshA1B1, efficiently converted phytosterols to DBA- and 9OH-BA with the rates of 42.5% and 40.3%, respectively, and their final yields reached 34.2 and 37.3 g/L, respectively, starting with 80 g/L phytosterols. Overall, our study not only provides efficient strains for the industrial production of BA, DBA and 9OH-BA but also provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

Humic acid enhanced pyrene degradation by Mycobacterium sp. NJS-1.

The search for nature-based tools to enhance bioremediation is essential for the sustainable restoration of contaminated ecosystems. Humic acid (HA) is an important component of organic matter in soil and water, but its effect on the microbial degradation of organic pollutants remains unclear. In this study, the biodegradation of pyrene by Mycobacterium sp. NJS-1 with and without HA was investigated. Only around 10.5% of pyrene was biodegraded in the pyrene treatment alone, whereas the addition of HA significantly enhanced biodegradation to the point where over 90% of pyrene was biodegraded. The production of 4,5-dihydropyrene-4,5-diol and phenanthrene-3,4-diol indicated the metabolic pathway via attacking of 4,5-positions of pyrene. Interestingly, 1,2-dimethoxypyrene was detected with the addition of HA, suggesting that HA induced a new ring-opening pathway involving the attack on the 1,2-positions of pyrene. The addition of HA first induced protein self-cleavage behavior with a significant increase in phenylalanine, tyrosine, and tryptophan containing large numbers of COO- groups. Furthermore, it altered the intracellular and extracellular ultrastructure of bacterial cells, promoting their growth in size and number as well as reducing the space between them. Overall, HA increased the ring-opening positions of pyrene and facilitated its interaction with bacterial cells, thus improving its biodegradability. Building upon the findings of this study to further research is conducive to the sustainable solution of environmental pollution.

Pathophysiological Responses of Pinna nobilis Individuals Enlightens the Etiology of Mass Mortality Situation in the Mediterranean Populations.

Due to the rapid decrease of Pinna nobilis populations during the previous decades, this bivalve species, endemic in the Mediterranean Sea, is characterized as 'critically endangered'. In addition to human pressures, various pathogen infections have resulted in extended reduction, even population extinction. While Haplosporidium pinnae is characterized as one of the major causative agents, mass mortalities have also been attributed to Mycobacterium sp. and Vibrio spp. Due to limited knowledge concerning the physiological response of infected P. nobilis specimens against various pathogens, this study's aim was to investigate to pathophysiological response of P. nobilis individuals, originating from mortality events in the Thermaikos Gulf and Lesvos and Limnos islands (Greece), and their correlation to different potential pathogens detected in the diseased animals. In isolated tissues, several cellular stress indicators of the heat shock and immune response, apoptosis and autophagy, were examined. Despite the complexity and limitations in the study of P. nobilis mortality events, the present investigation demonstrates the cumulative negative effect of co-infection additionally with H. pinnae in comparison to the non-presence of haplosporidian parasite. In addition, impacts of global climate change affecting physiological performance and immune responses result in more vulnerable populations in infectious diseases, a phenomenon which may intensify in the future.

Coexpression of VHb and MceG genes in Mycobacterium sp. Strain LZ2 enhances androstenone production via immobilized repeated batch fermentation.

Androstenone production is limited by low-efficiency substrate transport and dissolved oxygen levels during fermentation. In this study, the coexpression of the optimized Vitreoscilla hemoglobin (VHb) and sterol transporter ATPase (MceG) genes in Mycobacterium sp. LZ2 (Msp) was investigated to alleviate dissolved oxygen and mass transfer limitations. Results revealed that Msp-vgb/mceG effectively improved the growth, production, and adaptation to dissolved oxygen compared with those of Msp. The increased catalase activity and reduced intracellular ROS levels enhanced cell viability and promoted transcription of genes critical for phytosterol metabolism. Bagasse as an immobilization carrier increased the productivity of Msp-vgb/mceG by 56%. Immobilized repeat batch fermentation reduced the biotransformation period from 60 days to 37 days and improved the productivity from 0.039 g/L/h to 0.069 g/L/h. To the best of our knowledge, this work is the first study on the immobilization of recombinant mycobacteria on bagasse for androstenone production.

Efficient conversion of phytosterols into 4-androstene-3,17-dione and its C1,2-dehydrogenized and 9α-hydroxylated derivatives by engineered Mycobacteria.

4-Androstene-3,17-dione (4-AD), 1,4-androstadiene-3,17-dione (ADD) and 9α-hydroxyl-4-androstene-3,17-dione (9OH-AD), which are important starting compounds for the synthesis of steroidal medicines, can be biosynthetically transformed from phytosterols by Mycobacterium strains. Genomic and metabolic analyses have revealed that currently available 4-AD-producing strains maintain the ability to convert 4-AD to ADD and 9OH-AD via 3-ketosteroid-1,2-dehydrogenase (KstD) and 3-ketosteroid-9α-hydroxylase (Ksh), not only lowering the production yield of 4-AD but also hampering its purification refinement. Additionally, these 4-AD industrial strains are excellent model strains to construct ADD- and 9OH-AD-producing strains. We recently found that Mycobacterium neoaurum HGMS2, a 4-AD-producing strain, harbored fewer kstd and ksh genes through whole-genomic and enzymatic analyses, compared with other strains (Wang et al. in Microbial Cell Fact 19:187, 2020). In this study, we attempted to construct an efficient 4-AD-producing strain by knocking out the kstd and ksh genes from the M. neoaurum HGMS2 strain. Next, we used kstd- and ksh-default HGMS2 mutants as templates to construct ADD- and 9OH-AD-producing strains by knocking in active kstd and ksh genes, respectively. We found that after knocking out its endogenous kstd and ksh genes, one of these knockout mutants, HGMS2Δkstd211 + ΔkshB122, showed a 20% increase in the rate of phytosterol to 4-AD conversion, compared relative to the wild-type strain and an increase in 4-AD yield to 38.3 g/L in pilot-scale fermentation. Furthermore, we obtained the ADD- and 9OH-AD-producing strains, HGMS2kstd2 + Δkstd211+ΔkshB122 and HGMS2kshA51 + Δkstd211+ΔkshA226, by knocking in heterogenous active kstd and ksh genes to selected HGMS2 mutants, respectively. During pilot-scale fermentation, the conversion rates of the ADD- and 9OH-AD-producing mutants transforming phytosterol were 42.5 and 40.3%, respectively, and their yields reached 34.2 and 37.3 g/L, respectively. Overall, our study provides efficient strains for the production of 4-AD, ADD and 9OH-AD for the pharmaceutical industry and provides insights into the metabolic engineering of the HGMS2 strain to produce other important steroidal compounds.

The Implication of Vibrio Bacteria in the Winter Mortalities of the Critically Endangered Pinna nobilis.

Pinna nobilis populations, constituting the largest bivalve mollusk endemic to the Mediterranean, is characterized as critically endangered, threatened by extinction. Among the various factors proposed as etiological agents are the Haplosporidium pinnae and Mycobacterium sp. parasites. Nevertheless, devastation of the fan mussel populations is still far from clear. The current work is undertaken under a broader study aiming to evaluate the health status of Pinna nobilis population in Aegean Sea, after the mass mortalities that occurred in 2019. A significant objective was also (a) the investigation of the etiological agents of small-scale winter mortalities in the remaining populations after the devastating results of Haplosporidium pinnae and Mycobacterium sp. infections, as well as (b) the examination of the susceptibility of the identified bacterial strains in antibiotics for future laboratory experiments. Microbiological assays were used in order to detect the presence of potential bacterial pathogens in moribund animals in combination with molecular tools for their identification. Our results provide evidence that Vibrio bacterial species are directly implicated in the winter mortalities, particularly in cases where the haplosporidian parasite was absent. Additionally, this is the first report of Vibrio mediterranei and V. splendidus hosted by any bivalve on the Greek coastline.

Gut Symbiotic Microbial Communities in the IUCN Critically Endangered Pinna nobilis Suffering from Mass Mortalities, Revealed by 16S rRNA Amplicon NGS.

Mass mortality events due to disease outbreaks have recently affected almost every healthy population of fan mussel, Pinna nobilis in Mediterranean Sea. The devastating mortality of the species has turned the interest of the research towards the causes of these events. After the haplosporidan infestation and the infection by Mycobacterium sp., new emerging pathogens have arisen based on the latest research. In the present study, a metagenomic approach of 16S rRNA next generation sequencing (NGS) was applied in order to assess the bacterial diversity within the digestive gland of diseased individuals as well as to carry out geographical correlations among the biodiversity of microbiome in the endangered species Pinna nobilis. The specimens originated from the mortalities occurred in 2019 in the region of Greece. Together with other bacterial genera, the results confirmed the presence of Vibrio spp., assuming synergistic effects in the mortality events of the species. Alongside with the presence of Vibrio spp., numerous bacterial genera were detected as well, including Aliivibrio spp., Photobacterium spp., Pseudoalteromonas spp., Psychrilyobacter spp. and Mycoplasma spp. Bacteria of the genus Mycoplasma were in high abundance particularly in the sample originated from Limnos island representing the first time recorded in Pinna nobilis. In conclusion, apart from exclusively the Haplosporidan and the Mycobacterium parasites, the presence of potentially pathogenic bacterial taxa detected, such as Vibrio spp., Photobactrium spp. and Alivibrio spp. lead us to assume that mortality events in the endangered Fan mussel, Pinna nobilis, may be attributed to synergistic effects of more pathogens.

Caracterización clínico-epidemiológica de pacientes con tuberculosis en el Departamento de Caaguazú, Paraguay. 2014 a 2017

Resumen Introducción: La tuberculosis (TBC) es una de las diez principales causas de muerte en todo el mundo. Objetivo: Caracterizar clínica y epidemiológicamente los casos de TBC del Departamento de Caaguazú-Paraguay, entre los años 2014 y 2017. Pacientes y Métodos: Se realizó un estudio observacional, retrospectivo, utilizando datos secundarios del Programa Nacional de Control de la Tuberculosis (PNCT). La población: 659 casos de TBC registrados en el PNCT. Las variables: edad, sexo, grupo poblacional, tipo de TBC, co-infección TBC/VIH, y categoría de egreso. Procesamos la base de datos en Excel 2016 © usando Stata 14.0®. Resultado: El 63,3% de los participantes fue del género masculino. La edad promedio fue de 35,8 años. El 39,6% eran indígenas y 14,5% fueron personas privadas de libertad (PPL) conocidos como reclusos. El 89,6% tuvo TBC pulmonar, 2,4% tuvo co-infección TBC/VIH. La tasa de incidencia fue superior a 21,6/100.000 habts en 2014. La incidencia en indígenas fue de 76,5/100.000 habts en 2017. La incidencia en PPL fue de 2.272,1/100.000 habitantes en 2017. Conclusión: La incidencia de TBC en el Departamento de Caaguazú es baja en la población general afectando principalmente a hombres, mientras que en la población indígena y PPL es alta.

Abstract Background: Tuberculosis (TB) is one of the ten leading causes of death worldwide. Aim: To characterize the clinical and epidemiological point of view of TB cases reported in the Department of Caaguazú-Paraguay, from 2014 to 2017. Methods: Observational, descriptive, retrospective study; Population: 659 cases of TB registered in the National Tuberculosis Control Program (NTCP); variables: age, sex, population group, type of TB, TB/HIV coinfection. We procesed database in Excel 2016 © using Stata 14.0®. Results: 63.3% were of male gender, average age: 35.8 years, 39.6% were indigenous and 85.4% were liberty deprived persons known as inmates (LDP), 89.6% had pulmonary TB and 2,4% had TB/HIV coinfection. Incidence rate exceed 21.6/100,000 inhabitants in 2014. Indigenous incidence was 76.5/100,000 inhabitants in 2017, LDP incidence was 2,272.1/ 100,000 inhabitants in 2017. Conclusion: The incidence of TB in the Department of Caaguazú is low, mainly affecting men, while TB incidence in indigenous people and LDP was high.

Whole-genome and enzymatic analyses of an androstenedione-producing Mycobacterium strain with residual phytosterol-degrading pathways.

Mycobacterium neoaurum strains can transform phytosterols to 4-androstene-3,17-dione (4-AD), a key intermediate for the synthesis of advanced steroidal medicines. In this work, we presented the complete genome sequence of the M. neoaurum strain HGMS2, which transforms ß-sitosterol to 4-AD. Through genome annotation, a phytosterol-degrading pathway in HGMS2 was predicted and further shown to form a 9,10-secosteroid intermediate by five groups of enzymes. These five groups of enzymes included three cholesterol oxidases (ChoM; group 1: ChoM1, ChoM2 and Hsd), two monooxygenases (Mon; group 2: Mon164 and Mon197), a set of enzymes for side-chain degradation (group 3), one 3-ketosteroid-1,2-dehydrogenase (KstD; group 4: KstD211) and three 3-ketosteroid-9a-hydroxylases (Ksh; group 5: KshA226, KshA395 and KshB122). A gene cluster encoding Mon164, KstD211, KshA226, KshB122 and fatty acid ß-oxidoreductases constituted one integrated metabolic pathway, while genes encoding other key enzymes were sporadically distributed. All key enzymes except those from group 3 were prepared as recombinant proteins and their activities were evaluated, and the proteins exhibited distinct activities compared with enzymes identified from other bacterial species. Importantly, we found that the KstD211 and KshA395 enzymes in the HGMS2 strain retained weak activities and caused the occurrence of two major impurities, i.e., 1,4-androstene-3,17-dione (ADD) and 9-hydroxyl-4-androstene-3,17-dione (9OH-AD) during ß-sitosterol fermentation. The concurrence of these two 4-AD analogs not only lowered 4-AD production yield but also hampered 4-AD purification. HGMS2 has the least number of genes encoding KstD and Ksh enzymes compared with current industrial strains. Therefore, HGMS2 could be a potent strain by which the 4-AD production yield could be enhanced by disabling the KstD211 and KshA395 enzymes. Our work also provides new insight into the engineering of the HGMS2 strain to produce ADD and 9OH-AD for industrial application.

Multiple Non-Species-Specific Pathogens Possibly Triggered the Mass Mortality in Pinna nobilis.

The fan mussel, Pinna nobilis, represents the largest bivalve endemic to the Mediterranean Sea. Since 2016, dramatic mass mortality of this species has been observed in several areas. The first surveys suggested that Haplosporidium pinnae (currently considered species-specific) was the main etiological agent, but recent studies have indicated that a multifactorial disease may be responsible for this phenomenon. In this study, we performed molecular diagnostic analyses on P. nobilis, P. rudis, and bivalve heterologous host species from the island of Sardinia to shed further light on the pathogens involved in the mass mortality. The results support the occurrence of a multifactorial disease and that Mycobacterium spp. and H. pinnae are not necessarily associated with the illness. Indeed, our analyses revealed that H. pinnae is not species-specific for P. nobilis, as it was present in other bivalves at least three years before the mass mortality began, and species of Mycobacterium were also found in healthy individuals of P. nobilis and P. rudis. We also detected the species Rhodococcus erythropolis, representing the first report in fan mussels of a bacterium other than Mycobacterium spp. and Vibrio spp. These results depict a complicated scenario, further demonstrating how the P. nobilis mass mortality event is far from being fully understood.

First detection of the invasive Haplosporidian and Mycobacteria parasites hosting the endangered bivalve Pinna nobilis in Thermaikos Gulf, North Greece.

Mycobacterium sp. and Haplosporidium pinnae constitute invasive parasite species of bivalves, reported for the first time in the present study in the Aegean Sea and Thermaikos Gulf, respectively. During the last years, the endangered fan mussel (Pinna nobilis) experienced several mortality events in the Mediterranean Sea that caused deaths to 90% or more of their populations and have been attributed to infections by these pathogens. In Greece, two mass mortality events have been recently reported, namely in the Gulf of Kalloni and in Limnos island. In the present study we investigated the presence of both pathogens in P. nobilis from these marine areas as well as from Thermaikos Gulf using both histopathological microscopy and molecular markers. The detected parasite DNA was further quantified in the three populations utilizing a real time qPCR. Histopathological results indicated the presence of a Mycobacterium species alongside with the existence of the Haplosporidian parasite, which was identified in all mortality events in the Mediterranean Sea. The parasite was present in different phases mostly on the digestive gland epithelium. Phylogenetic analysis confirmed the taxonomy of the Haplosporidian parasite as the recently described Haplosporidium pinnae, whereas it failed to identify the Mycobacteria parasite at species level. While Mycobacterium sp. was detected in all examined specimens, H. pinnae was not detected in all diseased fan mussels. Interestingly, monitoring of P. nobilis population from Thermaikos Gulf, an estuary of extremely high importance for bivalve production, revealed the presence of both pathogens in a few specimens in higher quantity but with no symptoms of the disease. Besides, all the specimens from Thermaikos Gulf had inflammatory responses similarly to moribund specimens from mortality events.

Pilot study of specific microbe immobilization cells (SMICs) technology in removing tetramethyl ammonium hydroxide for reuse of low-strength electronics wastewater.

This study describes the specific microbe immobilization cell (SMIC) as an innovative technology for reuse of low-strength electronics wastewater. Pilot tests were performed to evaluate feasibility of this technology for removing slowly biodegradable organics including tetramethyl ammonium hydroxide (TMAH). SMIC pellets were prepared by entrapping concentrated culture of TMAH degrading bacteria inside media through polymerization. The operating conditions including hydraulic retention time, packing ratio of SMIC pellets, and recirculation ratio were optimized. The comparison data with conventional biological activated carbon (BAC) process exhibited superior removals in total organic carbon (TOC) as well as TMAH. SMIC process was applicable to the wastewater stream of up to 10 mg TOC/L. In addition, it was confirmed that sufficient amount of microorganisms were actively survived in SMIC pellets after 150 days of operation. Furthermore, economic analysis results showed that SMIC process was more cost-effective than BAC process.

Structure-based reconstruction of a Mycobacterium hypothetical protein into an active Δ5-3-ketosteroid isomerase.

Protein engineering based on structure homology holds the potential to engineer steroid-transforming enzymes on demand. Based on the genome sequencing analysis of industrial Mycobacterium strain HGMS2 to produce 4-androstene-3,17-dione (4-AD), three hypothetical proteins were predicted as putative Δ5-3-ketosteroid isomerases (KSIs) to catalyze an intramolecular proton transfer involving the transformation of 5-androstene-3,17-dione (5-AD) into 4-AD, which were defined as mKSI228, mKSI291 and mKSI753. Activity assays indicated that mKSI228 and mKSI291 exhibited weak activity, as low as 0.7% and 1.5%, respectively, of a well-studied and highly active KSI from Pseudomonas putida KSI (pKSI), while mKSI753 had no activity similar to Mycobacterium tuberculosis KSI (mtKSI). Although the 3D structures of the putative mKSIs were homologous to pKSI, their amino acid sequences were significantly different from those of pKSI and tKSI. Thus, by use of these two KSIs as homology models, we were able to convert the low-active mKSI291 into a high-active active KSI by site-directed mutagenesis. On the other hand, an X-ray crystallographic structure of mKSI291 identified a water molecule in its active site. This unique water molecule might function as a bridge to connect Ser-OH, Tyr57-OH and C3O of the intermediate form a hydrogen-bonding network that was responsible for its weak activity, compared with that of mtKSI. Our results not only demonstrated the use of a protein engineering approach to understanding KSI catalytic mechanism, but also provided an example for engineering the catalytic active sites and gaining a functional enzyme based on homologous structures.

Concomitant Presence of Aspergillus Species and Mycobacterium Species in the Respiratory Tract of Patients: Underestimated Co-occurrence?

OBJECTIVES: Aspergillus and Mycobacterium are opportunistic pathogens that can cause severe pulmonary diseases. To date, the clinical significance of their concomitant isolation and potential interactions in the lung remains poorly understood. The aim of this study was to assess the prevalence of their concomitant isolation from respiratory samples, and to depict the related clinical and microbiological characteristics. METHODS: A retrospective monocentric study was conducted from January 2011 to December 2017, including all in-patients from whom positive cultures of Aspergillus and Mycobacterium were obtained on respiratory samples within a 3-month period. Clinical, radiological and laboratory data were analyzed. Patients were categorized by a clinical and microbiological committee as "infected" or "colonized" by both pathogens according to current guidelines. RESULTS: Overall, 140 patients had ≥1 respiratory samples positive for Mycobacterium and concomitantly sent for fungal culture, and 708 were positive for Aspergillus, concomitantly sent for mycobacterial culture. Only 50 had at least one positive culture for both Mycobacterium sp. and Aspergillus sp. Men represented 63% of patients, mean age was 61 years. A third of patients were immunocompromised and 92% had underlying lung diseases. Aspergillus was primarily found as a colonizing agent. Proportion of Mycobacterium Avium Complex (p = 0.02) was higher in patients co-carrying Aspergillus spp. CONCLUSION: In this first study focusing on co-isolation of Mycobacteria and Aspergillus in patient's respiratory samples, co-infection remains rare. Further studies are warranted in order to precise the exact relationship between these opportunistic pathogens and the clinical impact of co-isolations.

Enhancing Expression of 3-Ketosteroid-9α-Hydroxylase Oxygenase, an Enzyme with Broad Substrate Range and High Hydroxylation Ability, in Mycobacterium sp. LY-1.

3-Ketosteroid-9α-hydroxylase (KSH) consists of two protein systems, KshA and KshB, and is a key enzyme in microbial degradation pathway of natural sterols. 9α-Hydroxy-4-androstene-3,17-dione (9α-OH-AD) is a valuable steroid pharmaceutical intermediate. The expression of a 3-ketosteroid-9α-hydroxylase oxygenase (KshA1) with a broad substrate range and high hydroxylation ability was enhanced in Mycobacterium sp. LY-1 to improve the yield of 9α-OH-AD. Through whole-genome sequence mining and homologous comparison, the putative genes (kshA1 and kshB) in wild strain LY-1 were firstly identified. Then they were heterogeneously co-expressed in Escherichia coli BL21. The transformation results of recombinant BL21-KshA1/B demonstrated KshA1/B had high hydroxylation ability to AD. Moreover, substrate preference analysis suggested that KshA1LY-1 had a broad substrate range. After enhancing expression of kshA1 and kshB in the strain LY-1, the maximum productivity of 9α-OH-AD in recombinant LY-1-KshA1/B reached 0.064 g/L/h in a 5-L stirred fermenter.

Enhancing the Electricity Generation and Nitrate Removal of Microbial Fuel Cells With a Novel Denitrifying Exoelectrogenic Strain EB-1.

Microbial fuel cells (MFCs) have been tentatively applied for wastewater treatment, but the presence of nitrogen, especially nitrate, induces performance instability by changing the composition of functional biofilms. A novel denitrifying exoelectrogenic strain EB-1, capable of simultaneous denitrification and electricity generation and affiliated with Mycobacterium sp., was isolated from the anodic biofilm of MFCs fed with nitrate containing medium. Polarization curves and cyclic voltammetry showed that strain EB-1 could generate electricity through a direct electron transfer mechanism with a maximum power density of 0.84 ± 0.05 W m-2. Additionally, anodic denitrification, as a concurrent metabolism, was demonstrated with an efficient removal rate of 0.66 ± 0.01 kg N m-3 d-1 at a COD/N ratio of 3.5 ± 0.3. Importantly, voltage output was not negatively influenced by nitrate, indicating that the concurrent process of nitrate removal and electricity generation was a limitation of the electron donor rather than an inhibition of the system. Furthermore, various organic materials were successfully utilized as anode donors for strain EB-1, and demonstrated the exciting performances in terms of simultaneous denitrification and electricity generation. Mycobacterium sp. EB-1 thus expands the diversity of exoelectrogens and contributes to the potential applications of MFC for simultaneous energy recovery and wastewater treatment.

Tuberculosis of the central nervous system in cattle in Paraíba, Brazil / Tuberculose no sistema nervoso central de bovinos na Paraíba

This paper describes six cases of tuberculosis in the central nervous system (CNS) of cattle in the state of Paraíba in northeastern Brazil. We reviewed the autopsy reports of 851 bovine necropsies performed from 2003 to 2016. Seventy-three (8.6%) cattle were diagnosed with tuberculosis and six showed lesions in the CNS. Three cases affected cattle up to two-year-old and other three affected adults. Three cattle presented exclusively nervous signs, two had respiratory signs and weight loss and one did not present any clinical signs. At necropsy, five cattle had thickening of the leptomeninges of the cerebellum, pons, obex, spinal cord and cortex, mainly, in the region near the brain basilar Willis´ circle. Another animal, presented a single focal lesion in the cerebellum. Microscopically we observed moderate to severe granulomatous meningitis and encephalitis. Five cattle presented lesions in the lungs and mediastinal lymph nodes and three of them had disseminated lesions in other organs. In all cattle acid-fast bacilli were observed in the lesions and marked positive for immunohistochemistry with polyclonal antibody anti-Mycobacterium tuberculosis. It is concluded that bovine tuberculosis of central nervous system occurs sporadically in Paraíba, in cattle of different ages, most of them with disseminate lesions in other organs. The location of the lesions suggests that the agent invaded the brain by hematogenous route through the circle of Willis.(AU)

Descrevem-se seis casos de tuberculose no sistema nervoso central (SNC) em bovinos, no semiárido da Paraíba. Foram revisados os laudos de um total de 851 necropsias de bovinos realizadas no período de 2003 a 2016. Destes, 73 (8,6%) foram diagnosticados com tuberculose e seis apresentavam lesões no SNC. Três casos ocorreram em bovinos de até dois anos de idade e três em bovinos adultos. Três bovinos apresentaram exclusivamente sinais nervosos, dois tinham sinais respiratórios e perda de peso e um não apresentava nenhum sinal clínico. Macroscopicamente, em cinco bovinos, havia espessamento das leptomeninges do cerebelo, medula espinhal, ponte, obex, colículos e córtex, principalmente, na região basilar encefálica próxima ao polígono de Willis. Em apenas um bovino houve a presença de tubérculo único no cerebelo. Microscopicamente observou-se moderada a acentuada meningite e encefalite granulomatosa. Cinco bovinos apresentaram lesões pulmonares e nos gânglios mediastínicos e três deles tinham lesões disseminadas em outros órgãos. Em todos os bovinos foram encontrados bacilos ácido-álcool resistentes intralesionais e todos tiveram marcação positiva na técnica de imuno-histoquímica com anticorpo policlonal anti-Mycobacterium tuberculosis. Conclui-se que a tuberculose do sistema nervoso central de bovinos ocorre de forma esporádica na Paraíba, principalmente em bovinos com lesões disseminadas em outros órgãos. Sugere-se que a disseminação do agente ocorre pela via hematógena, possivelmente através do polígono de Willis.(AU)