Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis.

The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.

Synthetic engineering and biological containment of bacteriophages.

The serious threats posed by drug-resistant bacterial infections and recent developments in synthetic biology have fueled a growing interest in genetically engineered phages with therapeutic potential. To date, many investigations on engineered phages have been limited to proof of concept or fundamental studies using phages with relatively small genomes or commercially available "phage display kits". Moreover, safeguards supporting efficient translation for practical use have not been implemented. Here, we developed a cell-free phage engineering and rebooting platform. We successfully assembled natural, designer, and chemically synthesized genomes and rebooted functional phages infecting gram-negative bacteria and acid-fast mycobacteria. Furthermore, we demonstrated the creation of biologically contained phages for the treatment of bacterial infections. These synthetic biocontained phages exhibited similar properties to those of a parent phage against lethal sepsis in vivo. This efficient, flexible, and rational approach will serve to accelerate phage biology studies and can be used for many practical applications, including phage therapy.

Jasmonate inhibits plant growth and reduces gibberellin levels via microRNA5998 and transcription factor MYC2.

Jasmonate (JA) and gibberellins (GAs) exert antagonistic effects on plant growth and development in response to environmental and endogenous stimuli. Although the crosstalk between JA and GA has been elucidated, the role of JA in GA biosynthesis remains unclear. Therefore, in this study, we investigated the mechanism underlying JA-mediated regulation of endogenous GA levels in Arabidopsis (Arabidopsis thaliana). Transient and electrophoretic mobility shift assays showed that transcription factor MYC2 regulates GA inactivation genes. Using transgenic plants, we further evaluated the contribution of MYC2 in regulating GA inactivation genes. JA treatment increased DELLA accumulation but did not inhibit DELLA protein degradation. Additionally, JA treatment decreased bioactive GA content, including GA4, significantly decreased the expression of GA biosynthesis genes, including ent-kaurene synthase (AtKS), GA 3ß-hydroxylase (AtGA3ox1), and AtGA3ox2, and increased the expression of GA inactivation genes, including GA 2 oxidase (AtGA2ox4), AtGA2ox7, and AtGA2ox9. Conversely, JA treatment did not significantly affect gene expression in the myc2 myc3 myc4 triple mutant, demonstrating the MYC2-4-dependent effects of JA in GA biosynthesis. Additionally, JA post-transcriptionally regulated AtGA3ox1 expression. We identified microRNA miR5998 as an AtGA3ox1-associated miRNA; its overexpression inhibited plant growth by suppressing AtGA3ox1 expression. Overall, our findings indicate that JA treatment inhibits endogenous GA levels and plant growth by decreasing the expression of GA biosynthesis genes and increasing the expression of GA inactivation genes via miR5998 and MYC2 activities.

DELLA-GAF1 complex is involved in tissue-specific expression and gibberellin feedback regulation of GA20ox1 in Arabidopsis.

KEY MESSAGE: The GAF1 transcription factor is shown to bind to the promoter of the Arabidopsis GA-biosynthetic enzyme GA20ox1 and, in association with DELLA protein, promotes GA20ox1 expression, thereby contributing to its feedback regulation and tissue specificity. Gibberellins (GAs) are phytohormones that promote plant growth and development, including germination, elongation, flowering, and floral development. Homeostasis of endogenous GA levels is controlled by GA feedback regulation. DELLAs are negative regulators of GA signaling that are rapidly degraded in the presence of GAs. DELLAs regulate several target genes, including AtGA20ox2 and AtGA3ox1, encoding the GA-biosynthetic enzymes GA 20-oxidase and GA 3-oxidase, respectively. Previous studies have identified GAI-ASSOCIATED FACTOR 1 (GAF1) as a DELLA interactor, with which DELLAs act as transcriptional coactivators; furthermore, AtGA20ox2, AtGA3ox1, and AtGID1b were identified as target genes of the DELLA-GAF1 complex. Among the five Arabidopsis GA20ox genes, AtGA20ox1 is the most highly expressed gene during vegetative growth; its expression is controlled by GA feedback regulation. Here, we investigated whether AtGA20ox1 is regulated by the DELLA-GAF1 complex. The electrophoretic mobility shift and transactivation assays showed that three GAF1-binding sites exist in the AtGA20ox1 promoter. Using transgenic plants, we further evaluated the contribution of the DELLA-GAF1 complex to GA feedback regulation and tissue-specific expression. Mutations in two GAF1-binding sites obliterated the negative feedback regulation and tissue-specific expression of AtGA20ox1 in transgenic plants. Thus, our results showed that GAF1-binding sites are involved in GA feedback regulation and tissue-specific expression of AtGA20ox1 in Arabidopsis, suggesting that the DELLA-GAF1 complex is involved in both processes.

Isolation and Characterization of a Novel Phage SaGU1 that Infects Staphylococcus aureus Clinical Isolates from Patients with Atopic Dermatitis.

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus.

Engineered Bacteriophages for Practical Applications.

The diversity of advanced genetic engineering techniques that have become available in recent years has enabled a more precise manipulation of genes and genomes. Among these, bacteriophage genomes stand out as an interesting target due to their dependence on a host for replication, which previously complicated their manipulation, and due as well to the many possible fields in which they can be used. In this review, we highlight recent applications for which genetically modified bacteriophages are being employed: as phage therapy in medicine, animal industries and agricultural settings; as a source of new antimicrobials; as biosensors for research, health and environmental purposes; and as genetic engineering tools themselves.

LotA, a Legionella deubiquitinase, has dual catalytic activity and contributes to intracellular growth.

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.

Recovery of mycobacteriophages from archival stocks stored for approximately 50 years in Japan.

Mycobacteriophage archival stocks have been kept for ca. 20-50 years in Japan. In this study, we attempted to recover mycobacteriophages from 50 archival stocks and briefly analyzed the recovered phages. The phages were recovered from 72.2% (13/18) of the lyophilized stocks that had been stored for 47-56 years. Moreover, the analysis of 12 representative recovered phages led to their classification as belonging to the family Siphoviridae, and seven of them were typed by polymerase chain reaction (PCR) targeting the gene that encodes the tape measure protein. Considering these results, lyophilization seems to be suitable for phage archival storage.

Ultraviolet imager on Venus orbiter Akatsuki and its initial results.

The ultraviolet imager (UVI) has been developed for the Akatsuki spacecraft (Venus Climate Orbiter mission). The UVI takes ultraviolet (UV) images of the solar radiation reflected by the Venusian clouds with narrow bandpass filters centered at the 283 and 365 nm wavelengths. There are absorption bands of SO2 and unknown absorbers in these wavelength regions. The UV images provide the spatial distribution of SO2 and the unknown absorber around cloud top altitudes. The images also allow us to understand the cloud top morphologies and haze properties. Nominal sequential images with 2-h intervals are used to understand the dynamics of the Venusian atmosphere by estimating the wind vectors at the cloud top altitude, as well as the mass transportation of UV absorbers. The UVI is equipped with off-axial catadioptric optics, two bandpass filters, a diffuser installed in a filter wheel moving with a step motor, and a high sensitivity charge-coupled device with UV coating. The UVI images have spatial resolutions ranging from 200 m to 86 km at sub-spacecraft points. The UVI has been kept in good condition during the extended interplanetary cruise by carefully designed operations that have maintained its temperature maintenance and avoided solar radiation damage. The images have signal-to-noise ratios of over 100 after onboard desmear processing.

Initiation of a lightning search using the lightning and airglow camera onboard the Venus orbiter Akatsuki.

The existence of lightning discharges in the Venus atmosphere has been controversial for more than 30 years, with many positive and negative reports published. The lightning and airglow camera (LAC) onboard the Venus orbiter, Akatsuki, was designed to observe the light curve of possible flashes at a sufficiently high sampling rate to discriminate lightning from other sources and can thereby perform a more definitive search for optical emissions. Akatsuki arrived at Venus during December 2016, 5 years following its launch. The initial operations of LAC through November 2016 have included a progressive increase in the high voltage applied to the avalanche photodiode detector. LAC began lightning survey observations in December 2016. It was confirmed that the operational high voltage was achieved and that the triggering system functions correctly. LAC lightning search observations are planned to continue for several years.

Venus looks different from day to night across wavelengths: morphology from Akatsuki multispectral images.

Since insertion into orbit on December 7, 2015, the Akatsuki orbiter has returned global images of Venus from its four imaging cameras at eleven discrete wavelengths from ultraviolet (283 and 365 nm) and near infrared (0.9-2.3 µm), to the thermal infrared (8-12 µm) from a near-equatorial orbit. The Venus Express and Pioneer Venus Orbiter missions have also monitored the planet for long periods but from polar or near-polar orbits. The wavelength coverage and views of the planet also differ for all three missions. In reflected light, the images reveal features seen near the cloud tops (~ 70 km altitude), whereas in the near-infrared images of the nightside, features seen are at mid- to lower cloud levels (~ 48-60 km altitude). The dayside cloud cover imaged at the ultraviolet wavelengths shows morphologies similar to what was observed from Mariner 10, Pioneer Venus, Galileo, Venus Express and MESSENGER. The daytime images at 0.9 and 2.02 µm also reveal some interesting features which bear similarity to the ultraviolet images. The nighttime images at 1.74, 2.26 and 2.32 µm and at 8-12 µm reveal features not seen before and show new details of the nightside including narrow wavy ribbons, curved string-like features, long-scale waves, long dark streaks, isolated bright spots, sharp boundaries and even mesoscale vortices. Some features previously seen such as circum-equatorial belts (CEBs) and occasional areal brightenings at ultraviolet (seen in Venus Express observations) of the cloud cover at ultraviolet wavelengths have not been observed thus far. Evidence for the hemispheric vortex organization of the global circulation can be seen at all wavelengths on the day- and nightsides. Akatsuki images reveal new and puzzling morphology of the complex nightside cloud cover. The cloud morphologies provide some clues to the processes occurring in the atmosphere and are thus, a key diagnostic tool when quantitative dynamical analysis is not feasible due to insufficient information.

A silent mutation in mabA confers isoniazid resistance on Mycobacterium tuberculosis.

Drug resistance in Mycobacterium tuberculosis (Mtb) is caused by mutations in restricted regions of the genome. Mutations in katG, the promoter region of the mabA-inhA operon, and inhA are those most frequently responsible for isoniazid (INH) resistance. Several INH-resistant (INH(r) ) Mtb clinical isolates without mutations in these regions have been described, however, indicating that there are as yet undetermined mechanisms of INH resistance. We identified the mabA(g609a) silent mutation in a significant number of INH(r)  Mtb clinical isolates without known INH resistance mutations. A laboratory strain, H37Rv, constructed with mabA(g609a) , was resistant to INH. We show here that the mabA(g609a) mutation resulted in the upregulation of inhA, a gene encoding a target for INH, converting the region adjacent to the mutation into an alternative promoter for inhA. The mabA(g609a) silent mutation results in a novel mechanism of INH resistance, filling in a missing piece of INH resistance in Mtb.

Bacterial community and genome analysis of cytoplasmic incompatibility-inducing Wolbachia in American serpentine leafminer, Liriomyza trifolii.

Liriomyza trifolii, an agricultural pest, is occasionally infected by Wolbachia. A Wolbachia strain present in Liriomyza trifolii is associated with cytoplasmic incompatibility (CI) effects, leading to the death of embryos resulting from incompatible crosses between antibiotic-treated or naturally Wolbachia-free strain females and Wolbachia-infected males. In this study, high-throughput sequencing of hypervariable rRNA genes was employed to characterize the bacterial community in Wolbachia-infected L. trifolii without antibiotic treatment. The analysis revealed that Wolbachia dominates the bacterial community in L. trifolii, with minor presence of Acinetobacter, Pseudomonas, and Limnobacter. To elucidate the genetic basis of the CI phenotype, metagenomic sequencing was also conducted to assemble the genome of the Wolbachia strain. The draft-genome of the Wolbachia strain wLtri was 1.35 Mbp with 34% GC content and contained 1,487 predicted genes. Notably, within the wLtri genome, there are three distinct types of cytoplasmic incompatibility factor (cif) genes: Type I, Type III, and Type V cifA;B. These genes are likely responsible for inducing the strong cytoplasmic incompatibility observed in L. trifolii.

Atypical social cognition processing in bulimia nervosa: an fMRI study of patients thinking of others' mental states.

BACKGROUND: Feeding and eating disorders are severe mental disorders that gravely affect patients' lives. In particular, patients with anorexia nervosa (AN) or bulimia nervosa (BN) appear to have poor social cognition. Many studies have shown the relationship between poor social cognition and brain responses in AN. However, few studies have examined the relationship between social cognition and BN. Therefore, we examined which brain regions impact the ability for social cognition in patients with BN. METHODS: We used task-based functional magnetic resonance imaging (fMRI) to examine brain responses during a social cognition task and the Reading Mind in the Eyes Test (RMET). During the fMRI, 22 women with BN and 22 healthy women (HW) took the RMET. Participants also completed the eating disorder clinical measures Bulimic Investigatory Test, Edinburgh (BITE) and Eating Disorders Examination Questionnaire (EDE-Q), the Patient Health Questionnaire (PHQ-9) measure of depression; and the Generalized Anxiety Disorder (GAD-7) measure of anxiety. RESULTS: No difference was observed in the RMET scores between women with BN and HW. Both groups showed activation in brain regions specific to social cognition. During the task, no differences were shown between the groups in the BOLD signal (p < 0.05, familywise error corrected for multiple comparisons). However, there was a tendency of more robust activation in the right angular gyrus, ventral diencephalon, thalamus proper, temporal pole, and middle temporal gyrus in BN (p < 0.001, uncorrected for multiple comparisons). Moreover, HW showed a positive correlation between RMET scores and the activation of two regions: medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC); however, no significant correlation was observed in women with BN. CONCLUSIONS: While activation in the mPFC and ACC positively correlated to the RMET scores in HW, no correlation was observed in BN patients. Therefore, women with BN might display modulated neural processing when thinking of others' mental states. Further examination is needed to investigate neural processing in BN patients to better understand their social cognition abilities. TRIAL REGISTRATION: UMIN, UMIN000010220. Registered 13 March 2013, https://rctportal.niph.go.jp/s/detail/um?trial_id=UMIN000010220.

Tracking the effects of the COVID-19 pandemic on viral gastroenteritis through wastewater-based retrospective analyses.

The COVID-19 pandemic possibly disrupted the circulation and seasonality of gastroenteritis viruses (e.g., Norovirus (NoV), Sapovirus (SaV), group A rotavirus (ARoV), and Aichivirus (AiV)). Despite the growing application of wastewater-based epidemiology (WBE), there remains a lack of sufficient investigations into the actual impact of the COVID-19 pandemic on the prevalence of gastroenteritis viruses. In this study, we measured NoV GI and GII, SaV, ARoV, and AiV RNA concentrations in 296 influent wastewater samples collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan between October 28, 2018 and January 12, 2023 using the highly sensitive EPISENS™ method. The detection ratios of SaV and ARoV after May 2020 (SaV: 49.8 % (134/269), ARoV: 57.4 % (151/263)) were significantly lower than those before April 2020 (SaV: 93.9 % (31/33), ARoV: 97.0 % (32/33); SaV: p < 3.5×10-7, ARoV: p < 1.5×10-6). Furthermore, despite comparable detection ratios before (88.5 %, 23/26) and during (66.7 %, 80/120) the COVID-19 pandemic (p = 0.032), the concentrations of NoV GII revealed a significant decrease after the onset of the pandemic (p < 1.5×10-7, Cliff's delta = 0.72). NoV GI RNA were sporadically detected (24.7 %, 8/33) before April 2020 and after May 2020 (6.5 %, 17/263), whereas AiV was consistently (100 %, 33/33) detected from wastewater throughout the study period (95.8 %, 252/263). The WBE results demonstrated the significant influence of COVID-19 countermeasures on the circulation of gastroenteritis viruses, with variations observed in the magnitude of their impact across different types of viruses. These epidemiological findings highlight that the hygiene practices implemented to prevent COVID-19 infections may also be effective for controlling the prevalence of gastroenteritis viruses, providing invaluable insights for public health units and the development of effective disease management guidelines.

Wastewater-based prediction of COVID-19 cases using a highly sensitive SARS-CoV-2 RNA detection method combined with mathematical modeling.

Wastewater-based epidemiology (WBE) has the potential to predict COVID-19 cases; however, reliable methods for tracking SARS-CoV-2 RNA concentrations (CRNA) in wastewater are lacking. In the present study, we developed a highly sensitive method (EPISENS-M) employing adsorption-extraction, followed by one-step RT-Preamp and qPCR. The EPISENS-M allowed SARS-CoV-2 RNA detection from wastewater at 50 % detection rate when newly reported COVID-19 cases exceed 0.69/100,000 inhabitants in a sewer catchment. Using the EPISENS-M, a longitudinal WBE study was conducted between 28 May 2020 and 16 June 2022 in Sapporo City, Japan, revealing a strong correlation (Pearson's r = 0.94) between CRNA and the newly COVID-19 cases reported by intensive clinical surveillance. Based on this dataset, a mathematical model was developed based on viral shedding dynamics to estimate the newly reported cases using CRNA data and recent clinical data prior to sampling day. This developed model succeeded in predicting the cumulative number of newly reported cases after 5 days of sampling day within a factor of √2 and 2 with a precision of 36 % (16/44) and 64 % (28/44), respectively. By applying this model framework, another estimation mode was developed without the recent clinical data, which successfully predicted the number of COVID-19 cases for the succeeding 5 days within a factor of √2 and 2 with a precision of 39 % (17/44) and 66 % (29/44), respectively. These results demonstrated that the EPISENS-M method combined with the mathematical model can be a powerful tool for predicting COVID-19 cases, especially in the absence of intensive clinical surveillance.

Impact of the COVID-19 pandemic on the prevalence of influenza A and respiratory syncytial viruses elucidated by wastewater-based epidemiology.

Since the COVID-19 pandemic, a decrease in the prevalence of Influenza A virus (IAV) and respiratory syncytial virus (RSV) has been suggested by clinical surveillance. However, there may be potential biases in obtaining an accurate overview of infectious diseases in a community. To elucidate the impact of the COVID-19 on the prevalence of IAV and RSV, we quantified IAV and RSV RNA in wastewater collected from three wastewater treatment plants (WWTPs) in Sapporo, Japan, between October 2018 and January 2023, using highly sensitive EPISENS™ method. From October 2018 to April 2020, the IAV M gene concentrations were positively correlated with the confirmed cases in the corresponding area (Spearman's r = 0.61). Subtype-specific HA genes of IAV were also detected, and their concentrations showed trends that were consistent with clinically reported cases. RSV A and B serotypes were also detected in wastewater, and their concentrations were positively correlated with the confirmed clinical cases (Spearman's r = 0.36-0.52). The detection ratios of IAV and RSV in wastewater decreased from 66.7 % (22/33) and 42.4 % (14/33) to 4.56 % (12/263) and 32.7 % (86/263), respectively in the city after the COVID-19 prevalence. The present study demonstrates the potential usefulness of wastewater-based epidemiology combined with the preservation of wastewater (wastewater banking) as a tool for better management of respiratory viral diseases.

Identification of SARS-CoV-2 variants in wastewater using targeted amplicon sequencing during a low COVID-19 prevalence period in Japan.

Wastewater-based epidemiology is expected to be able to identify SARS-CoV-2 variants at an early stage via next-generation sequencing. In the present study, we developed a highly sensitive amplicon sequencing method targeting the spike gene of SARS-CoV-2, which allows for sequencing viral genomes from wastewater containing a low amount of virus. Primers were designed to amplify a relatively long region (599 bp) around the receptor-binding domain in the SARS-CoV-2 spike gene, which could distinguish initial major variants of concern. To validate the methodology, we retrospectively analyzed wastewater samples collected from a septic tank installed in a COVID-19 quarantine facility between October and December 2020. The relative abundance of D614G mutant in SARS-CoV-2 genomes in the facility wastewater increased from 47.5 % to 83.1 % during the study period. The N501Y mutant, which is the characteristic mutation of the Alpha-like strain, was detected from wastewater collected on December 24, 2020, which agreed with the fact that a patient infected with the Alpha-like strain was quarantined in the facility on this date. We then analyzed archived municipal wastewater samples collected between November 2020 and January 2021 that contained low SARS-CoV-2 concentrations ranging from 0.23 to 0.43 copies/qPCR reaction (corresponding to 3.30 to 4.15 log10 copies/L). The targeted amplicon sequencing revealed that the Alpha-like variant with D614G and N501Y mutations was present in municipal wastewater collected on December 4, 2020 and later, suggesting that the variant had already spread in the community before its first clinical confirmation in Japan on December 25, 2020. These results demonstrate that targeted amplicon sequencing of wastewater samples is a powerful surveillance tool applicable to low COVID-19 prevalence periods and may contribute to the early detection of emerging variants.

Downregulation of katG expression is associated with isoniazid resistance in Mycobacterium tuberculosis.

Isoniazid (INH) is a key agent in the treatment of tuberculosis. In Mycobacterium tuberculosis, INH is converted to its active form by KatG, a catalase-peroxidase, and attacks InhA, which is essential for the synthesis of mycolic acids. We sequenced furA-katG and fabG1-inhA in 108 INH-resistant (INH(r) ) and 51 INH-susceptible (INH(s) ) isolates, and found three mutations in the furA-katG intergenic region (Int(g-7a) , Int(a-10c) and Int(g-12a) ) in four of 108 INH(r) isolates (4%), and the furA(c41t) mutation with an amino acid substitution in 18 INH(r) isolates (17%). These mutations were not found in any of 51 INH(s) isolates tested. We reconstructed these mutations in isogenic strains to determine whether they conferred INH resistance. We found that the Int(g-7a) , Int(a-10c) and Int(g-12a) single mutations in the furA-katG intergenic region decreased katG expression and conferred INH resistance. In contrast, the furA(c41t) mutation was not sufficient to confer INH resistance. These results suggested that downregulation of katG is a mechanism of INH resistance in M. tuberculosis and that mutations in the furA-katG intergenic region play a role in this resistance mechanism.