- Volume 2, Issue 7A, 2020

Introduction: Gram-negative bacteria are a major cause of urinary tract infections (UTIs) and particularly Klebsiella pneumoniae (K. pneumoniae), which is a causative agent of 60-70% of community-acquired infections, about 30% of nosocomial UTIs and 20% of recurrent infections.

Materials and methods: Nine urine samples were collected from patients from various clinical departments in King Abdulaziz University Hospital from 2/3/2019 to 2/4/2019. The microbial contents in the urine samples was analysed by urine culture and VITEK analyses. Here, we compared K. pneumoniae proteins profiles to find possible proteins which could shed a light on host-pathogen interactions. The colonies were suspended in a lysing buffer, which then were sonicated, and the proteins contents were separated using 1D SDS-PAGE, analyzed using liquid chromatography-mass spectrometry LC/MS. Proteins showing different expressions in samples were identified by TripleTOF 5600 mass spectrometer.

Results: All of the Klebsiella pneumoniae isolates were ESBL+ and KPC+ as shown on ChromAgar plates. There was no available data on resistance, ESBL or KPC from VITEK2. Hence, ESBL+ or KPC+ data were only obtained from ChromAgar. Additionally, proteomics analysis revealed the following, the total number of different proteins that are expressed from all of the isolates is 2958 proteins. Where in sample U-102, 328 different proteins expressed, 300 different proteins expressed from isolate U-871, 350 different proteins expressed from isolate U-713, in isolate U-755, 378 different proteins were expressed, 207 different proteins expressed from isolate U-754, 305 different proteins expressed from sample U-134, 290 different proteins expressed from isolate U-968, 600 different proteins expressed from isolate U-104, and 200 different proteins expressed from isolate U-659.

Conclusion: The different proteins between the UTI patients indicated specific host pathogen interaction, each isolate expressed different proteins than the other isolate could be reasoned by host pathogen interaction. Host pathogen interaction are influenced by numbers of factor: age, gender, immunity, underling health conditions, antibiotic treatment, acute UTI or recurrent infection all of these factor could have an impact on the type of proteins that are expressed during infection. Even though the isolates are Klebsiella pneumoniae from young females with UTI, they expressed different proteins, these proteins could explain evolutionary development of pathogens and survival in urinary niche, as pathogens need to express certain type of proteins to enable them to live and survival in urinary niche.

Absorbent hygiene wastes like nappies and incontinence pads are ubiquitous in municipal and healthcare waste streams around the world as they are convenient products used in child-care and adult incontinence management. Absorbent Hygiene Product (AHP) manufacturing is resource-intensive as the products are required to be of the highest value as they are in almost-constant contact with sensitive body parts. The potential for recovering such valuable resources such as cellulose-based fibres and super-absorbent polymers for reuse in non-food sectors like the construction and wastewater industries has been considered in this study. Appropriate decontamination via chemical methods have been examined using AHPs contaminated with human-associated bacteria.

Findings suggest that for simulated AHP wastes inoculated with 108–109 CFU g-1 of human-associated bacteria like Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes, a 1:1 ratio of 0.5% calcium hypochlorite/AHP waste is adequate to inactivate the bacteria particularly when combined with an inorganic salt for at least 60 min. Specifically, 4 to 5 log10 reductions were observed. Following such disinfection, material storage and temperatures above 25ºC minimise incidences of microbial regrowth. The disinfection protocol was not found to adversely affect the AHP quality. Overall, such findings suggest that AHP recycling is a potential alternative to current AHP waste disposal practices like incineration (with or without energy recovery) and landfilling.

Weak and unstable soils can limit the building of new infrastructure. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate, which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase their mechanical properties. A commonly used bacterium is Sporosarcina pasteurii. A range of factors influences MICCP which presents challenges with process optimisation. Some studies have made use of computational models to predict biocementation at a larger scale, however aspects of models are based on assumption of conditions instead of experimental data.

An aim of this project is to investigate urease activity in S. pasteurii by comparing different growth media, growth stages, pH and temperatures. Ureolysis kinetics of S. pasteurii will be investigated at different urea and calcium chloride concentrations in liquid media. Finally, the biocementation of S. pasteurii in sand syringe setups will also be investigated to compare the effects of changing influencing factors such as growth stage and cell concentration of S. pasteurii, sand particle size, cementation media concentration, duration between cementation media applications and overall number of cementation treatments. Experimental work will be particularly focused towards gaps in the experimental data used in computational models, to help improve these models and bring MICCP biocementation closer to commercial use.

Klebsiella spp. are associated with 3 to 7% of nosocomial infections and can be responsible for a range of conditions including pneumonia, bloodstream infections, meningitis, and necrotizing enterocolitis in infants. The role of Klebsiella pneumoniae in causing disease is well-characterised but, to date, the closely related species Klebsiella oxytoca has not received the same attention, despite often encoding extended-spectrum beta-lactamases and carbapenemases in clinical settings. K. oxytoca is the causative agent of Clostridiodes difficile-negative antibiotic-associated haemorrhagic colitis, a rare condition seen in some individuals receiving antibiotics. Whole-genome sequence analyses have shown K. oxytoca to be a complex comprising at least six species (K. oxytoca, K. michiganensis, K. grimontii, K. huaxiensis, ‘K. pasteurii’, ‘K. spallanzanii’). Our study aims to better characterise the K. oxytoca complex using a polyphasic approach. Preliminary investigations into the genomes of three K. michiganensis clinical isolates revealed the presence of a plasmid-borne ccdABlocus. ccdAB is a toxin-antitoxin (TA) system known to maintain plasmids in other pathogenic enterobacteria. We aim to functionally validate this TA system by cloning and conducting toxicity assays on the CcdB toxin, and cloning and assessing the ability of CcdA to function as an antidote. We also aim to sequence and generate Illumina/Oxford Nanopore hybrid genome assemblies of a larger collection of K. oxytoca complex clinical isolates and investigate their plasmids and TA systems in the same manner.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is a pathogen of humans and cattle, in which it is responsible for carriage or invasive disease and subclinical mastitis, respectively. From the 1950s to 1970s, thanks to successful mastitis control programs, the prevalence of GBS fell in the Swedish cattle population, but it re-emerged in the late 1990s. GBS was thought to consist of host-specific subpopulations but recent studies have shown that human and cattle subpopulations overlap, with different accessory genome elements providing survival advantages in each host species. We hypothesized that cattle-adapted GBS was eradicated and replaced by new GBS strains of human origin.

Our aim was to explore the differences in GBS cattle population over six decades (pre-post non-detection), with a focus on the possible role of MGE in the evolution of these strains. Historical (n = 44, 1953 to 1978) and contemporary (n = 76, 1997 to 2012) GBS isolates from bovine milk samples were sequenced and analysed for WGS-MLST. Phylogenetic network analysis revealed the presence of six major clades: two of these were detected only up to 1970, two were only detected after 2004, and two were detected in both periods. Historical isolates were all tetracycline sensitive, whereas 51% of recent isolates harboured tet(M), which is considered a marker of human adaptation. Our data support the elimination of a bovine specific clade (CC61/67) and the emergence of new clades (CC1, CC103/314) that are likely of human of origin.

Staphylococcus aureus is routinely found in sputum samples obtained from people with Cystic Fibrosis (CF). However, its role in the progression of the disease is unclear. This is important, as antibiotic clearance of S. aureus in CF yields unclear clinical results and there is debate around the utility of anti-Staphylococcal antibiotic treatment. We used an ex vivo porcine lung model (EVPL) to compare the growth and virulence of S. aureus isolates from acute CF exacerbations, with isolates from the same donors when they were stable.

There was no significant difference in mean bacterial load between donors, strains or clinical state. However, when we compared the variance in bacterial load of each pair of exacerbation/stable isolates across experimental replicates of the lung model, we found that stable samples grew more consistently in the EVPL compared to those taken from the same donor during an exacerbation.

Virulence factor assay results were mixed, with results implying greater virulence in either stable or acute samples after passage through the EVPL. We could not detect the AIP quorum sensing signal, which control expression of numerous acute virulence factors, using a reporter assay. We hypothesise that S. aureus might down-regulate Agr expression in the model, consistent with a role as a silent persister, rather than as a pathogenic agent. Further work using the EVPL model will determine how well this reflects the clinical reality in CF.

Staphylococcus aureus uses several strategies to evade the host immune reaction including expression of the genes on the immune evasion cluster (IEC), which target the innate immune response, and tarP, which alters the structure of the bacterial wall teichoic acids to avoid binding of host antibodies. IEC is carried on an ΦSa3 prophage, while the tarP gene is found located on diverse prophages. Livestock-associated methicillin-resistant S. aureus of clonal complex 398 (LA-MRSA CC398) typically lacks the IEC, but a number of Danish isolates have been found to carry the elements regardless.

In this study, whole-genome sequences of 96 isolates from humans and 45 isolates from pigs from the North Denmark Region were used to establish a maximum-likelihood phylogeny from core-genome SNPs and to investigate the prevalence of IEC and tarP. Furthermore, epidemiological and national surveillance data were used to investigate household transmission and the prevalence of IEC in LA-MRSA CC398 isolates from the general population. The study documents several independent acquisitions of IEC on distinct ΦSa3 prophages in humans and an almost 3-fold higher human-to-human transmission rate of LA-MRSA CC398 in households with strains carrying IEC than in households with strains lacking it. No such effect was found for tarP, which is widespread among LA-MRSA CC398 isolates from both humans and pigs. Moreover, IEC also seems to promote spread of LA-MRSA CC398 in the general population. Thus, LA-MRSA CC398 is capable of re-adapting to the human host by acquisition of human-specific immune evasion factors encoded on mobile genetic elements.

The evolutionarily ubiquitous multidrug and toxin efflux (MATE) proteins mediate anticancer and antibiotic resistance, while transporting toxins, ions and flavonoids in plants. MATEs of the model amoeba Dictyostelium discoideum have not been studied although sequences of its pair group with the two Homo sapiensMATEs. Ddmate1 and 2 are both transcribed, Ddmate2 more so, with peaks in vegetative and slug life-cycle stages. Ddmate1 was upregulated in response to a toxin, ethidium bromide, at the lowest concentration tested. Removing MATE function by inhibitor or mutation increased intracellular levels of various compounds, confirming these as efflux transporters. Plasma membrane localisation was revealed using a GFP-MATE1 reporter-line. MATE1 and MATE2 phenotypes indicated roles beyond detoxification: on Klebsiella lawns these mutants produced significantly smaller plaques than WT, and their axenic growth rates were also lower. The transporters’ impact on use of Dictyostelium for novel drug research was tested using flavonoids. LCMS and fluorescence-imaging revealed differential flavonoid uptake. Flavanones such as naringenin did not cross into cells, whereas flavonols localised to mitochondria and cytoplasm. Ddmate1 transcription was upregulated, however, in response to naringenin, which is known to reduce levels of kidney-disease protein PKD2 in both Dictyostelium and animal cells. Increased flavonol intracellular concentrations confirmed that efflux not import was impeded in MATE1 and MATE2, and kaempferol therefore further reduced MATE1-cells’ growth. These D. discoideum MATEs may usefully model the HsMATEs, aid understanding of flavonoids’ effects, and should be considered when using this model eukaryote to screen drugs.

The global uncontrolled rise of antimicrobial resistance (AMR) is a major societal threat and it is well documented that AMR is already negatively affecting healthcare and intensive livestock farming systems. Nonetheless, capture fisheries are still essential to the global food supply providing over 50% of the world’s aquatic organism production. To facilitate improved management, it is therefore imperative that we better understand reservoirs of AMR and how gene transmission occurs in the aquatic environment. In order to discern which AMR bacteria and genes are present in the marine environment, sediment and seawater samples were collected and Atlantic Mackerel were captured at the beginning of summer and autumn in the Solent Strait (Portsmouth, U.K). In addition, commercially available Atlantic mackerel were purchased at a local fish market. Using culture-dependent techniques we obtained more than 700 bacterial and 20 fungal isolates from skin, intestinal lining, and intestinal content. Ten different cefotaxime-resistant Pseudomonas spp. were isolated from the seawater and market fish skin samples, and one cefotaxime-resistant Rhanella sp. was isolated from wild fish digesta. Results from ongoing whole-genome shotgun metagenomics analysis will be discussed, as well as the connection between AMR bacteria and AMR gene presence within the marine coastal environment and the local fish markets, which are the last link between capture fisheries and consumers.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of nosocomial infections, and has been categorised by the World Health Organisation as a “Priority 1: Critical” target for research and development of novel antibiotics owing to its intrinsic multi-resistance and ability to acquire novel resistance mechanisms.

One strategy for discovering novel antibiotics is the identification and characterisation of metabolites with antimicrobial activity. Members of the bacterial phylum Actinobacteria are historic source of these metabolites, in particular the genus Streptomyces. However, other genera have not received this same level of interest despite sharing the capacity to biosynthesise a diverse array of metabolites. One such genus is Micromonospora, responsible for production of the broad-spectrum aminoglycoside antibiotic gentamicin (M. purpurea).

Here we present three Micromonospora species isolated from the Atacama Desert, Chile; that possess anti-pseudomonad bioactivity inducible by culture on International Streptomyces Project (ISP) Media. In addition, preliminary data indicates that this activity can be affected by the addition of P. aeruginosa conditioned media. In parallel, short-read Illumina sequencing was used to assemble draft genomes for these strains, enabling antiSMASH analysis of putative biosynthetic gene clusters. In addition to this, estimated Average Nucleotide Identity (ANI) as calculated by the autoMLST server indicates that these strains may all be novel Micromonospora species.

The results of this work serve to highlight the biosynthetic capacity of an understudied genus of bacteria, as well as the value of examining underexplored environments and habitats in the search for novel bioactive molecules.

Japanese encephalitis virus (JEV) is the leading cause of viral encephalitis worldwide. Annually it causes between 13,000 and 20,000 deaths mainly in South-East Asia. Vaccination programmes have drastically reduced the number of JEV cases however outbreaks do still occur. Recently G1 has overtaken G3 as the dominant genotype in most endemic countries and as all currently licensed vaccines are based on G3 it is necessary to evaluate the potential of emerging genotypes to break through current control measures.

This project seeks to generate a virus-like-particle (VLP) neutralisation assay in order to investigate the potential for emergent or divergent genotypes of JEV to infect vaccinated individuals. Using this assay, we will evaluate the ability of vaccinee sera to neutralise emergent genotypes as well as to introduce a variety of SNPs that have been shown to enhance virulence and investigate whether any combination of these are able to invalidate vaccine protection.

There is also concern that recombination between wild-type and live attenuated vaccine strains may occur in the vaccine setting which could result in rescue of the virulent potential of the attenuated virus. Evidence of this is being sought in silico using publicly available data and existing techniques.

These data will provide a clearer picture of the nature of JEV in endemic areas and whether the current strategy of vaccination is sufficient to prevent naturally acquired infection in the long term or if other strategies must now be considered.

To human health worldwide. Existing treatments are becoming inefficacious and therefore there is an urgent need for the development of treatments with alternative modes of action. The use of gallium as an antimicrobial agent has been of interest due to its unconventional mode of action involving the inhibition of iron acquisition and metabolism. The structural similarity and inability to reduce from a trivalent to divalent form under normal physiological conditions allows gallium to act as an iron mimetic and inhibit many iron-dependent biological pathways, respectively.

The antimicrobial potential of gallium maltolate (GaM), Ga(III) coordination complex of maltol, was investigated on the opportunistic pathogen Pseudomonas aeruginosa. In vitro and in vivo analyses using Galleria mellonella (greater wax moth) larvae demonstrated the potent bacteriostatic and non-toxic effect of the complex. Subsequent analysis of GaM treated P. aeruginosa via label-free quantitative proteomics provided an insight into the intrinsic mechanisms of action of GaM. Increased expression of iron-storage protein Bacterioferritin B, the HemO component of iron-sulfur clusters and several stress response proteins (Chaperone Proteins ClpB, HtpG and DnaJ) indicate cell stress in response to inhibited iron uptake. Decreased expression of LasA Protease and LasB Elastase quorum-sensing proteins and flagellar motility proteins FlgM and FlgG further demonstrate the growth inhibitory effect of GaM. These findings provide a basis for a better understanding of the mode of action of GaM, a requirement for the improvement of synthesis and efficacy of the treatment.

Static microcosms are a well-established system used to study the adaptive radiation of Pseudomonas fluorescens SBW25 and the adaptive biofilm-forming mutants known as the Wrinkly Spreaders (WS). We have developed this system to investigate selection within multi-species communities using a soil-wash inoculum dominated by biofilm-competent pseudomonads. Here we present community and isolate-level analyses of one serial-transfer experiment in which replicate populations were selected for over ten transfers and 60 days. Although no significant trends in improving community biofilm characteristics or total microcosm productivity were observed, a significant shift in biofilm-formation and microcosm growth by individual isolates recovered from the initial soil-wash inoculum and final transfers indicated that these communities were subject to selection for growth in these microcosms. Surprisingly, the fitness of the archetypal WS was poor when competing against community samples, and having compared the cell densities in the low-O2 region of liquid column below the biofilm, we suggest that part of the community’s fitness advantage comes from the ability to colonise this under-utilised niche as well as to compete at the A-L interface. Samples from the community biofilms and the low-O2 region were able to re-colonize both niches and many final transfer isolates grew throughout the liquid column as well as forming A-L interface biofilms. This suggests that there is a trade-off between fast growth under highly competitive conditions at the A-L interface and slower growth with less competition in the low-O2 region, with some isolates taking a bet-hedging approach a colonizing both niches in our microcosm system.

Air pollution is the single largest environmental health risk worldwide. Particulate matter (PM) air pollution is released as a result of fossil fuel combustion and vehicle motion, breaking and tyre wear. It has been shown that exposure to PM can cause increased levels of respiratory disease, including the exacerbation of COPD, which is frequently associated with bacterial infection. Despite this, the effects of air pollution exposure on COPD associated respiratory bacteria, includingHaemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are largely unknown. Our recent publication was the first to document that as well as damaging the host, PM has a direct impact on bacteria that can cause respiratory infections. We showed that exposure to black carbon (BC), an important component of PM, results in alterations in biofilm structure in both Streptococcus pneumoniae and Staphylococcus aureus, and increases dissemination of colonising S. pneumoniaein in vivo models.

Following on from this work, we aim to determine how BC impacts the growth, behaviour and virulence of bacteria associated with the COPD exacerbation, including non-typeable Haemophilus influenzaeand Moraxella catarrhalis. Current data show that BC exposure is decreasing the biofilm forming ability of NTHistrains 162 and 375. M. catarrhalis strain M61 biofilm formation is also decreased in the presence of BC, while its growth rate is increased. In addition, pre-exposing NTHi375 cells to BC, prior to infection of A549 cells, increases their ability to adhere to human epithelial cells. This suggests that the frequency of bacterial infection induced COPD exacerbation may be altered in patients from highly polluted areas.

The excessive use of antibiotics in agriculture is routinely described as a major contributor to bacterial resistance. Globally, antibiotics are widely used as growth supplements in livestock. This has led to concerns regarding human-use antibiotics in food and food-producing animals. Lately, organic acids (OAs) such as propionic acid (PA) and formic acid (FA) have been increasingly used as alternative antimicrobials or preservatives instead of antibiotics. These are particularly efficient at removing salmonella.

Recently, we have shown that exposure of a Crohn’s Disease associated bacterial pathotype, Adherent-invasive Escherichia coli (AIEC), to PA significantly alters its phenotype resulting in increased adhesion and invasion of epithelial cells and increased persistence through biofilm formation. AIEC are both evolutionarily and phylogenetically related to avian pathogenic Escherichia coli (APEC), however the virulence mechanisms of APEC in poultry remain unclear. The widespread use of OAs as growth supplements and antimicrobials in the poultry industry is a rising concern due to the ability of OAs to alter the bacterial pathotype. In this study, we examined the effect of FA on the phenotype of APEC. We observed that following FA-exposure, APEC showed an increased ability to adhere to and invade human intestinal epithelial cells and form better biofilms both aerobically and anaerobically. Worryingly, these isolates also showed an increased resistance to several antibiotics. These results suggest that the increasing use of alternative antimicrobial such as FA in the poultry industry may lead to APEC strains that are increasingly virulent towards human cells with a potential for increased horizontal transmission

Halogenated compounds constitute one of the largest groups of environmental pollutants and are a worldwide issue accumulating readily across even remote environments. Contamination by halogenated flame retardants such as polybrominated diphenyl ether (PBDE) is widespread, including marine and freshwater sediments, soil and human tissue. This has been hypothesised to lead to multiple health issues following exposure such as endocrine disruption and immune system alterations. Despite a recent reduction in use, PBDE containing products will continue to leach into the environment for years. Marine sponges are a natural reservoir of brominated compounds and dehalogenation activities of the associated microbiota have been previously observed. It has been postulated that mechanisms for microbial mediated degradation of halogenated compounds also exist within environmental samples commonly contaminated by PBDEs, for example widespread areas of soil sediment. In order to detect and investigate the dehalogenation capabilities of environmental microorganisms, sediment samples from recycling plants have been used to create microcosms with various PBDE congeners (PBDE 47, 99, 153, 209) added, to establish bacterial communities in the laboratory associated with potential PBDE degradation. Once dehalogenation is detected characterisation of debrominating communities from environmental samples will be performed using microbial community 16S rRNA community profiling and QRTPCR. This will provide valuable insight to bacterial communities associated with haloaromatic degradation and further useful information for potential bioremediation of PBDE contaminated soils and sediments.

The evolution of tubercle bacilli correlates closely with changes in cell envelope surface lipid composition (Donoghue et al. Diversity 2017, 9:46; Jankute et al. Scientific Reports 2017, 7:1315). Smooth, hydrophilic “Mycobacterium canettii” is the first recognisable member of the Mycobacterium tuberculosis complex, but it has reduced pathogenicity and poor aerosol transmission. In contrast, rough M. tuberculosis is very hydrophobic and readily spread in aerosols. Starting from hydrophilic surface lipids in environmental Mycobacterium kansasii, intermediate “M. canettii” adds hydrophobic lipids but retains overall cell hydrophilicity. Eliminating hydrophilic lipooligosaccharides (LOSs) and phenolic glycolipids (PGLs) from “M. canettii” leads to M. tuberculosis with a refined selection of hydrophobic lipids, namely phthiocerol dimycocerosates (PDIMs), pentaacyl trehaloses (PATs) and sulfoglycolipids (SGLs). The relative hydrophobicity of M. tuberculosis is double that of representatives of M. kansasii and “M. canettii”.

The above changes have implications both for the onset of tuberculosis and pinpointing evolutionary hosts. Tuberculosis has not been found in Homo sapiens during the Late Pleistocene, but megafauna are the most likely hosts; characteristic bone lesions have been validated by TB DNA amplification and lipid biomarkers in bison metacarpals up to 17,000 years old. Late Pleistocene enhanced TB hydrophobicity and aerosolisation may have produced megafaunal pandemics, with extinction of bison, mastodons and contemporary taxa. The oldest H. sapiens tuberculosis is from the “Fertile Crescent” back to 9-11ka BP at the start of the Holocene. Naïve humans arriving “Out of Africa” may have encountered newly virulent tubercle bacilli of megafaunal origin, recently refined through a distinct “bottleneck”.

Modernisation has thrown humanity and other forms of life on our planet into ditch of problems. Poverty, climate change, injustice, environmental degradation are few of the shared global problems. The United Nations SDGs are set as blueprint to achieve a better and more sustainable future for all. The SDGs are well structured to address the global challenges we face including poverty, inequalities, hunger, climate change, environmental degradation, peace and justice. The SDGs have been driven mainly by international donors and ‘professional’ international development organisations. The world is left with 10 years to achieve these ambitious goals and targets. Various reviews indicated that little has been achieve on overall, and the SDGs will not be reality if new strategy is not in place to bring inclusion. Microbiology, the scientific discipline of microbes, their effects and practical uses has insightful influence on our day to day living. We present how microbiology and microbiologists could increase the scorecard and accelerate these global goals. Microbiology contribution to peace, justice, gender equality, decent work and economic growth will be also highlighted among others. The pledge of Leave No One Behind will fast track progress and microbiology is better position to make this work.

Cordycepin is an anticancer metabolite produces by a zombie fungus species of Cordyceps militaris. They are capable to infect and hijack insect’s nervous neuron system. Hypoxic environment commonly must be faced by the pathogenic fungus during infection either this zombie fungus. They activate oxygen sensing mode, heme, siderophore, and sterol biosynthesis to overcome it. Underlined our previous study that liquid surfaced culture of C. militaris NBRC103752 produced a higher amount of cordycepin than submerged culture, suggesting that hypoxic conditions might induce it. However, when and how the mechanism of cordycepin production started in liquid surfaced culture is not understood, yet. In our present study, the combination of transcriptomics and gas chromatography-mass spectrometry were carried out during the production phases of cordycepin (5d, 12d, and 19d of incubation periods) and the mechanism of cordycepin production was figured out. The expression of genes in the fermentation pathway and the oxidative phosphorylation pathway were significantly upregulated and down regulated, respectively. Expression of four genes in the heme biosynthesis, including 5-aminolevulinic acid synthase (CCM_01504), delta-aminolevulinic acid dehydratase (CCM_00935), coproporphyrinogen III oxidase (CCM_07483) and cytochrome c oxidase15 (CCM_05057) were upregulated at the beginning of the exponential phase (12d). Further, the activation of Zn(2)-C6 transcription factor that regulates the iron acquisition and ergosterol biosynthesis significantly upregulated and a metabolite reporter adenosine was detected only at 12d. The results in the present study show the correlation between hypoxia and the accumulation of heme before cordycepin biosynthesis.