Access Microbiology
Optimising synthetic cystic fibrosis sputum media for growth of non-typeable Haemophilus influenzae
Non-typeable Haempophilus influenzae (NTHi) is an early pathogen isolated from the lungs of children with cystic fibrosis (CF). However its role in the progression of CF lung infection is poorly understood. Additionally whether it forms biofilms in the lungs of people with CF is an open question. The development of synthetic cystic fibrosis sputum media has given key insights into the microbiology of later CF pathogens Pseudomonas aeruginosa and Staphylococcus aureus through replicating the chemical composition of CF sputum. However growth of NTHi in these media has not previously been reported. We show that NTHi grows poorly in three commonly-used variants of synthetic cystic fibrosis sputum media commonly used to induce in vivo-like growth of P. aeruginosa and S. aureus (SCFM1 SCFM2 and SCFM3). The addition of NAD and hemin to SCFM1 and SCFM2 promoted the planktonic growth and biofilm formation of both laboratory and clinical NTHi isolates and we were able to develop a modified variant of SCFM2 that allows culture of NTHis. We show that NTHi is unable to be identified in an established ex-vivo model of CF infection which uses SCFM and porcine bronchiolar tissue. This may in part be due to the presence of endogenous bacteria on the pig lung tissue which outcompete NTHi but the lack of selective agar to isolate NTHi from endogenous bacteria and the fact that NTHi is an exclusively human pathogen make it hard to conclude that this is the case. Through spiking modified SCFM2 with filter sterilized lung homogenate biofilm growth of clinical NTHi isolates was enhanced. Our results highlight that there are crucial components present in the lung tissue which NTHi require for growth that are not present in any published variant of SCFM from the Palmer et al. 2007 lineage. Our results may inform future modifications to SCFM recipes to truly mimic the environment of CF lung sputum and thus to facilitate study of a wide range of CF pathogens.
Diagnostic stewardship – Optimisation of superficial wound swab cultures can reduce the environmental impact of the microbiology laboratory
Introduction
Optimisation of diagnostic testing is essential for the sustainable delivery of laboratory services. To date little consideration has been given to the potential benefits of diagnostic stewardship to laboratories looking to reduce their environmental footprint.
Hypothesis
Implementing a pre-analytical diagnostic stewardship intervention for the testing of superficial wound swabs would result in a measurable reduction in the environmental footprint of the microbiology laboratory.
Aim
To assess the consequential impact of a diagnostic stewardship intervention on test volume carbon footprint and quantity of non-recyclable plastic waste generated.
Methodology
Superficial wound swabs received in the absence of clinical details suggestive of active skin and soft tissue infection were rejected by the laboratory. The Carbon footprint of testing was estimated using Publicly available specification (PAS) 2050:2011 methodology in a cradle-grave attributional life-cycle assessment within a defined system boundary. The mass of laboratory plastic waste was calculated through the accurate weighing of associated laboratory consumables.
Results
The intervention resulted in a reduction of 35.77 kgCO2e and 9.06kg of unrecyclable plastic waste over an 8-day period without measurable patient harm.
Conclusion
This study demonstrates in relation to specific testing pathways that the optimisation of microbiology laboratory diagnostic testing can result in a reduction in greenhouse gas emissions and non-recyclable plastic waste generation without negatively impacting patient care.
RSYD-BASIC: a bioinformatics pipeline for Routine Sequence analYsis and Data processing of BActerial iSolates for clInical miCrobiology
Background
Whole genome sequencing of bacterial isolates is increasingly becoming routine in clinical microbiology; however subsequent analysis often needs to be started by a bioinformatician even for comprehensive pipelines. To increase the robustness of our workflow and free up bioinformatician work hours for development and advanced analysis we aimed to produce a robust customizable bioinformatic pipeline for bacterial genome assembly and routine analysis results that could be initiated by non-bioinformaticians.
Results
We introduce the RSYD-BASIC pipeline for bacterial isolate sequence analysis and provide a demonstration of its functionality with two datasets composed of publicly available sequences in which comparable results are obtained in most cases. In some instances the pipeline provided additional information corresponding to in vitro results where these could be obtained. In routine use at our department the pipeline has already yielded clinically relevant results allowing us to type a variety of bacterial pathogens isolated in our clinical laboratory. We also demonstrate how RSYD-BASIC results aided in disproving a potential outbreak.
Conclusion
With the RSYD-BASIC pipeline we present a configurable reads-to-results analysis pipeline operated by non-expert users that greatly eases investigation of potential outbreaks by expert end-users. Results obtained with publicly available sequences show comparable performance to the original methods while underlining the importance of standardized methods.
Rapid Detection of Aspergillus spp. and Quantitative Simultaneous Analysis of Aflatoxins (B1, B2, G1 and G2) in Kenyan Hybrid Maize Cultivars Using FT-IR and LC-ESI-MS/MS Spectro-analysis Techniques
The present study combined Fourier-Transform Infrared Spectroscopy (FT-IR) analysis with high resolution liquid chromatography tandem mass spectroscopy equipped with an electrospray ionization source (LC-ESI-MS/MS) in the screening and quantification of four naturally occurring aflatoxins (AFs) AFB1 AFB2 AFG1 and AFG2 in milled hybrid maize cultivars (n = 171) from selected counties in the Rift-Valley region of Kenya. Samples were extracted in a single-step process using acetonitrile/water 80:20 v/v in 0.5% formic acid without further clean-up steps. Aflatoxins were analyzed in both positive and negative ionization modes using the multi-reaction monitoring (MRM) acquisition process that allowed confirmation and quantification of the aflatoxins in a single run. The MRM mode was utilized for quantitative analysis while qualitative analysis was done using the enhanced product ion mode. Aflatoxin presence was detected in the following ranges and frequencies: AFB1 (0-9.36μg/kg) AFB2 (0-6.62μg/kg) AFG1 (0-7.72μg/kg) and AFG2 (0-9.18μg/kg). Additional mycotoxins recovered included the following; sterigmatocystin (0-8.3μg/kg) and deoxynivalenol (0-7.3μg/kg). In overall low amounts of aflatoxins were recovered from the milled maize samples with an additional low total aflatoxin content above the stipulated regulatory limit of 10μg/kg. Given that slow continued exposure to aflatoxins (especially in low amounts) has been proven to be a leading cause of various forms of cancer urgent intervention measures and national surveillance programs ought to be introduced to protect consumers from gradual aflatoxin poisoning emanating from the consumption of contaminated Kenyan maize.
Whole-genome sequencing of the Streptomyces coelicolor bldA39 mutant (J1700) of reveals hundreds of previously unknown mutations.
We report the genome sequence of the bldA39 (J1700) mutant Streptomyces coelicolor a historically important strain that is deficient in sporulation and antimicrobial production. The S. coelicolor J1700 strain was used extensively from the 1980s onwards to underpin important discoveries in development and antibiotic production in Streptomyces. The bldA gene encodes a leucyl transfer RNA (tRNA) required for the translation of the rare TTA codon found in ~2% of genes in Streptomyces. The whole genome of S. coelicolor J1700 was obtained via Illumina sequencing and mapped to the of S. coelicolor M145 reference genome. Analysis of the genome sequence compared to S. coelicolor M145 identified the known bldA39 mutation (T>C) and revealed more than 300 further mutations likely associated with the S. coelicolor J1501 genetic background the strain was created in including the nature of the hisA1 and uraA1 alleles used extensively in genetic mapping experiments and several mutations in natural product biosynthetic gene clusters. This work highlights the importance of whole genome sequencing of historically important strains.
Genome sequences of pathogenic and non-pathogenic Pantoea ananatis strains in maize (Zea mays L.)
We performed genome sequencing and comparative analysis of Pantoea ananatis strains isolated from corn leaves expressing typical bacterial leaf streak (BLS) and maize white spot (MWS) symptoms to confirm bacterial identity and to understand the relationship among these strains and P. ananatis strains isolated from different plant hosts in Brazil. In pathogenicity tests strains 4.2 and 13.3 isolated from symptomatic BLS leaves were non-pathogenic on corn. In contrast strain B13 isolated from MWS diseased leaf tissue caused symptoms typical of MWS. Our comparative analysis revealed that all three strains are very genetically similar. The G+C (%) content of the strains 4.2 and 13.3 was 53.5% while B13 was 53.7%. Average nucleotide identity (ANI) analysis showed that strains B13 and 13.3 B13 and 4.2 and 4.2 and 13.3 shared ANIs of 99.17% 99.15% and 99.99% respectively. Strains 13.3 B13 and 4.2 shared ~99% ANI with P. ananatis type strain LMG 2665. To our knowledge these are the first genome sequences of P. ananatis strains isolated from corn in Brazil.
Draft genome sequence of Flavobacterium aquidurense strain, isolated from untreated wastewater
Here we report the draft 5.8 Mb genome sequence of a Flavobacterium aquidurense isolate from untreated wastewater in Liverpool UK. The reported isolate has the potential to produce both flexirubin and β-carotene and contains an additional biosynthetic gene cluster for a putative novel beta-lactone. The genome also contains a gene for a putative beta-lactamase blaJOHN-1 analogue and putative novel insertion sequences of the IS3 family.
Antibacterial activity of Cinnamomum verum and Thymus vulgaris essential oils on multidrug-resistant zoonotic bacteria isolated from dogs in southern Benin
Objective Antibiotic resistance is a major public health problem. The search for new therapeutic alternatives is becoming urgent. Essential oils are a promising alternative. This study aimed to evaluate the antibacterial activities of essential oils from selected plants on multidrug-resistant zoonotic strains isolated from dogs. Methods Essential oils from dried Thymus vulgaris leaves Cinnamomum verum bark and Cuminum cyminum seeds were extracted and tested on five multidrug-resistant Escherichia coli and four S. aureus isolated from dogs in southern Benin. Results The study showed that Thymus vulgaris essential oil was bacteriostatic with a Minimum Inhibitory Concentration (MIC) equal to 2.5 μl/ml and a Minimum Bactericidal Concentration (MBC) of 17 μl/ml for E. coli strains and 11.25 μl/ml for S. aureus strains. Regarding Cinnamomum verum essential oil its bacteriostatic power was characterized by an MIC of 1.25 μl/ml for the isolates tested and an average MBC of 11.50 μl/ml for E. coli and 12.19 μl/ml for S. aureus. On the other hand Cuminum cyminum essential oil was ineffective on the strains investigated. Additionally T. vulgaris essential oil contained predominantly thymol (36.57%) p-cymene (30.51%) and carvacrol (7.62%) while C. verum essential oil contained cinnamaldehyde (88.76%). Conclusion This study reveals the antibacterial activity of T. vulgaris dry leaf and C. verum bark essential oils on multi-resistant E. coli and S. aureus isolated from dogs. These two essential oils may be alternative candidates for combating antibiotic-resistant E. coli and S. aureus infections.
“Investigating Colistin Resistance in Carbapenem Resistant Acinetobacter baumanii Isolates during a Neonatal Intensive Care Unit outbreak "
Purpose: Colistin resistant Acinetobacter baumannii poses a growing challenge in neonatal intensive care units (NICUs) due to the emergence of plasmid-mediated Mobile Colistin Resistance (mcr) genes that can rapidly spread among neonates. This pioneering study conducted a comprehensive analysis of mcr-1 mcr-2 mcr-3 mcr-4 and mcr-5 genes in A. baumannii strains isolated from NICU. Notably this investigation marks the first of its kind in this setting.
In addition to genotypic analyses the study incorporated phenotypic assays to identify the most effective method for detecting colistin resistance in these A. baumannii strains.
Methods: In the genotypic investigation DNA was extracted from strains of colistin-resistant A. baumannii collected from the NICU of a local hospital of Islamabad Pakistan. The specific set of primers for each mcr gene was used to detect their presence. Various phenotypic methods such as disc diffusion broth macro dilution Minimum Inhibitory Concentration (MIC) disc elusion and colistin agar methods were performed using predetermined calculations for phenotypic studies.
Results:The study confirms the distribution of the mcr-1 gene among the eight strains which contributes 62% of the total number whereas mcr-2 3 4 and 5 genes were not detected in all strains. Based on phenotypic analysis A. baumannii had shown resistance against colistin at < 2µg/mL. In contrast MIC varies from strain to strain.
Conclusions: The horizontal transfer of the mcr-1 gene is responsible for developing resistance against colistin among A. baumannii in neonates. All phenotypic methods adopted in this study generated the same results so the method selection depends upon individual comfort. However we propose that the colistin agar method offers multiple advantages over other methods as it is more economical easy to perform multiple samples can be assessed simultaneously and fewer calculations are involved for colistin resistance determination and finding out clinical breakpoints.
Exploring the genomic landscape of Chlamydiifrater species: Novel features include multiple truncated major outer membrane proteins (MOMP), unique genes, and chlamydial plasticity zone orthologs
Recently discovered obligate intracellular bacteria belonging to the genus Chlamydiifrater (Cf.) with the species of Cf. phoenicopteri and Cf. volucris were studied to explore the composition of their genomes and their relatedness to Chlamydia the other genus of the family Chlamydiaceae.
We investigated four isolates of Cf. volucris two of them newly sequenced and one of Cf. phoenicopteri alongside 12 representatives of the Chlamydia species. Our study uncovers previously unrecognized genomic structures within Cf. using a hybrid sequencing approach and advanced annotation pipelines providing insights into species-specific adaptations and evolutionary dynamics. The integration of long-read sequencing data comprehensive re-annotation strategies and pan-genomics enabled localization of the unique plasticity zone and identification of novel gene clusters in Chlamydiifrater strains which improves our understanding of chlamydial genome architecture and plasticity in the family Chlamydiaceae. Our analysis revealed that 761 CDS (approx. 80 %) are shared among members of both genera. We further identified 158 unique genes of Cf. species but their annotation remains challenging because of the absence of functionally annotated orthologs in public databases. A full-length ompA gene encoding the major outer membrane porin (MOMP) was seen in all Cf. strains. We also describe the localization and structure of multiple truncated CDS of ompA family members representing one of this study's most interesting findings.
While genome analysis of Chlamydiifrater spp. confirmed numerous common features shared with representatives of the genus Chlamydia many unique genomic elements were identified that underpin the distinct phenotype and separate genetic position of these new microorganisms.
Phytochemical screening and in vitro antibacterial evaluation of Plectranthus amboinicus (Lour) spreng against selected four pathogenic bacteria
Background: Traditional medicine is still utilized around the globe particularly in developing countries such as Ethiopia where the majority of people maintain their health through the use of plant medicines. The primary challenges that needed to be addressed were the requirement for scientific backing for adequate documentation and drug development. The current study intended to screen phytochemical and antibacterial activities of Plectranthus amboinicus crude extracts against selected pathogenic bacteria.
Methods:The study were designed in Complete Randomized Design based on laboratory bioassay with three replications involves a 2x2x3x4x3 (2 sources i.e. leaf and stem; 2 solvent i.e. Ethanol and aqueous water; 3 concentration levels i.e. 100mg/ml 120 mg/ml and 150mg/ml; 4 bacterial species; and 3 replications). Qualitative analysis or susceptibility was carried out using standard protocol and phytochemical and antimicrobial activities were determined by disc diffusion and broth dilution method.
Result: According of study indicated the presence of six phytochemical compounds among the tested compounds while Saponin and Terpenoid were absent in ethanol extract and present in water extract. Phlobotanin possess negative at all in both ethanol and water extract. Based on observed susceptibility test the highest inhibition zone 14.6mm was recorded against S. Pyogenes at a concentration of 150 mg/ml while 9.1 mm inhibition zone was observed against E. coli in concentration of 100 mg/ml. Minimum inhibitory concentration values in the range 25 mg/mL to 100 mg /ml gram positive and gram negative minimum bactericidal concentration values ranged 50 mg/ml to 100 mg/ml respectively recorded.
Conclusion: Plectranthus amboincus plant showed presence of secondary metabolites possesses bio-active compounds against selected human pathogenic bacterial species thereby justifying the application of their crude extracts as medicinal plants. Further studies are required against more bacteria and fungus pathogens.
Comparison of six COVID-19 serology assays for detection of antibodies from patients infected with ancestral and a spectrum of SARS-CoV-2 variants
Serology assays against spike receptor binding domain and nucleocapsid proteins of the severe acute respiratory syndrome coronavirus 2 are essential for serosurveillance. We performed a comparison of four medium-high throughput commercial assays (Abbott Laboratories Ortho Clinical Diagnostics Meso Scale Diagnostics (MSD)) one point-of-care test (ZEKMED) and a laboratory developed plaque reduction neutralization test using a reference panel and clinical specimens. Overall the assays showed high positive percent agreement of ≥85% and negative percent agreement of ≥90% with the MSD spike IgG assay having the best performance (100% in both). Notably assays were able to detect antibodies from individuals infected with different variants - Alpha Beta Gamma Delta and Omicron. The limit of detection (LOD) of the assays ranged from 9.9 - 62.0 BAU/mL with Abbott anti-RBD having the lowest LOD. COVID-19 serology assays will continue to be useful in determining seroprevalence from infection and vaccination.
Interaction with refuse piles is associated with co-occurrence of core gut microbiota in workers of the ant Aphaenogaster picea
Comparing the diversity of gut microbiota between and within social insect colonies can illustrate interactions between bacterial community composition and host behavior. In many eusocial insect species different workers exhibit different task behaviors. Evidence of compositional differences between core microbiota in different worker types could suggest a microbial association with division of labor among workers. Here we present the core microbiota of Aphaenogaster picea ant workers with different task behaviors. The genus Aphaenogaster is abundant worldwide yet the associated microbiota of this group is unstudied. Bacterial communities from A. picea gut samples in this study consist of 19 phyla dominated by Proteobacteria Cyanobacteria and Firmicutes. Analysis of 16S rRNA gene sequences reveals distinct similarity clustering of A. picea gut bacterial communities in workers that have more interactions with the refuse piles. Though gut bacterial communities of nurse and foraging ants are similar in overall composition and structure the worker groups differ in relative abundances of dominant taxa. Gut bacterial communities from ants that have more interactions with refuse piles are dominated by ASVs associated with Entomoplasmataceae. Interaction with fecal matter via refuse piles seems to have the greatest impact on microbial taxa distribution and this effect appears to be independent of worker type. This is the first report surveying the gut microbiome community composition of Aphaenogaster ants.
Comparison of the virulence of community and hospital isolated Acinetobacter baumannii in HeLa cell line and insect model, Galleria mellonella
Acinetobacter baumannii is an important nosocomial pathogen causing high infections and morbidity among affected individuals and most studies focus on nosocomial strains. However A. baumannii can also be isolated from community healthy individuals. This study compared the pathogenicity of hospital and community A. baumannii isolates using Galleria mellonella and human cell cultures. The insect model G. mellonella and in vitro HeLa cell line were used with ten A. baumannii isolates (six community and four hospital isolates from Segamat Malaysia). G. mellonella killing assays and HeLa cell adherence invasion and cytotoxicity assays were performed to investigate the virulence and invasion potential of the isolates. Out of the ten isolates investigated three community and two hospital isolates were found to be highly virulent in G. mellonella infection model killing 100 % larvae within 96 hours. These strains were also found to be invasive and have significant cytotoxicity in HeLa cells. Our study revealed that community and hospital isolated A. baumannii could be equally virulent judged by both model systems. Undoubtedly besides hospital settings the presence of highly virulent A. baumannii in community reservoirs poses a significant public health risk and requires additional investigation.
The impact of taxonomic family and supplemental zinc on minimum inhibitory concentration against carbapenem antibiotics among bacteria expressing IMP metallo-beta-lactamase
Antibiotic-resistant infections cause an estimated 2.8 million illnesses and 35900 deaths annually in the US. Carbapenems are a class of antibiotics that are generally reserved to treat life-threatening invasive infections including sepsis. Accurate diagnosis of carbapenem-resistant infections is critical for early and appropriate treatment. blaIMP encodes bacterial production of the IMP metallo-beta-lactamase (MBL) which can confer resistance to all the beta-lactams including carbapenems. Zinc is an essential co-factor in the IMP MBL enzymatic hydrolysis of carbapenems. Tests for the presence of IMP carbapenemase such as the Carba NP include zinc-sulfate (ZnSO4) although broth dilution methods for determining minimum inhibitory concentration (MIC) for carbapenems do not. We hypothesized that ZnSO4 availability would improve the accuracy of carbapenem MIC determination for Enterobacterales expressing blaIMP. Thus the objective of this study was to determine if supplemental ZnSO4 affects the carbapenem MICs of Enterobacterales and other bacteria expressing blaIMP. Isolates utilized for this study were originally recovered from environmental samples collected at farms wastewater treatment plants and from surface water. They were selected based on phenotypic non-susceptibility to carbapenems and genetic confirmation of bacterial carriage of blaIMP. Cation-adjusted Mueller-Hinton broth suspensions of each isolate standardized to a 0.5 MacFarland standard were tested with and without ZnSO4 at 0.1 mmol/L concentration to determine MICs using standard extended-spectrum beta-lactamase micro-broth dilution MIC panels. Although we observed that Morganellaceae imipenem MICs were higher (P < 0.001) than those from other Enterobacterales harboring blaIMP the inclusion of supplemental ZnSO4 did not influence carbapenem MIC. Additional research will be required to identify important factors that may influence the expression of carbapenemase including IMP and the accurate determination of clinical MICs which is critical to appropriate therapeutic decision-making.
Investigating the presence of hypervirulent carbapenemase-producing Klebsiella pneumoniae, through three phenotypic tests, in the Maltese islands
Hypervirulence and multi-drug resistance are two separate qualities of Klebsiella pneumoniae that have posed a public health threat in separate strains for decades now. The convergence of these two phenotypes into singular strains of Klebsiella pneumoniae have created a super bug causing both severe infections in the young and healthy and having limited antibiotic treatment available against them. These strains were first discovered in Asia in 2013 but are now emerging all around the globe. In this study hypervirulence was assessed in 493 known carabapenemase-producing Klebsiella pneumoniae strains isolated and cryopreserved between 2019 and 2021 from various specimen types from health institutions around the Maltese islands. Three phenotypic tests were used namely the string test for hypermucoviscosity lateral flow assays for the identification of K1 and K2 capsular serotypes and quantification of siderophore production. Since the latter test was the one that was known to be the most significant for the detection of hypervirulence and none of the strains surpassed the set limit of a concentration of 30µg/ml none of the 493 assayed strains were deemed hypervirulent. Hypermucoviscous K1 and K2 capsular serotype belonging strains were found though so much so that a statistically significant relationship was found between hypermucoviscosity and capsular serotype. Other statistical tests carried out exploring the relationship between hypermucoviscosity and siderophore concentration and hypermucoviscosity and type of carabapenemase gene were not found to be statistically significant. According to this research hypervirulent carabapenemase-producing Klebsiella pneumoniae strains have not yet arrived in the Maltese archipelago.
Fungal positivity seen in tertiary care hospital during COVID-19 pandemic
Coronavirus disease 2019 (COVID-19) pandemic has been prevailing for more than a year associated with increased number of opportunistic invasive fungal infections in patients who have been critically ill or immunocompromised. In this retrospective study details of various clinical specimens received from suspected patients of fungal infections were studied. The fungal cultures were seen to be positive in 64% (51/79) COVID-19 positive patients and 43% (163/381) COVID-19 negative patients during the year 2021 in the second wave of COVID-19. Among COVID-19 infected patients the fungal isolations mostly observed were Candida spp. (63%) followed by Aspergillus spp. (15% ) and Mucor spp. (6%). The maximum samples (17% - COVID-19 positive and 83% - COVID-19 negative patients) which had shown positive results in COVID-19 infected patients were urine followed by serum (for aspergillus galactomannan). The candida isolation was seen in 27% (21/79) urine and 15%(12/79) respiratory samples (BAL tracheal aspirate sputum) in COVID-19 positive patients. Rhizopus arrhizus and Rhizopus homothallicus isolations were seen in nasal samples and tissue samples of 6% COVID-19 positive patients. There was an overall increase in fungal co-isolations during the COVID-19 pandemic (64% in COVID-19 positive and 43% in COVID-19 negative patients) which is a matter of great concern. The correlation of clinical symptomatology and laboratory isolation is important for the diagnosis and effective management of these patients.
Anaerobic HgII reduction is driven by cellular HgII-thiol interactions
Redox reactions play a critical role in determining the availability of mercury species HgII and Hg0 to anaerobic microbes responsible for methylating inorganic mercury into toxic monomethylmercury. Some anaerobes also contribute to Hg cycling in methylation hotspots by reducing HgII to its gaseous elemental form Hg0. However their contributions remain poorly quantified due to limited mechanistic insights and the absence of genetic targets. In this study we investigated the mechanisms of anaerobic HgII reduction in the versatile anoxygenic photoheterotroph and fermenter Heliomicrobium modesticaldum Ice1. Given HgII strong electrophilic affinity for thiol groups we hypothesized that cellular thiols are key interaction sites mediating HgII reduction. Exposure of H. modesticaldum to the thiol-alkylating agent N-ethylmaleimide (NEM) which irreversibly binds thiols resulted in a concentration-dependent inhibition of Hg0 production during both photoheterotrophy and fermentation. Hg partitioning assays with Escherichia coli cells revealed no significant differences in Hg-cell partitioning in the presence or absence of NEM suggesting that HgII reduction is dependent on intracellular thiol interactions. These findings highlight the importance of thiol-mediated pathways in Heliobacterial HgII reduction. Although the exact cellular components remain unidentified we discuss potential thiol-containing coupling sites that warrant further investigation.