Quorum sensing mediated response of Achromobacter denitrificans SP1 towards prodigiosin production under phthalate stress.

Quorum sensing is a density-dependent chemical process between bacteria, which may be intergenus or intragenus. N-acyl homoserine lactones (HSLs) are a type of small signaling molecules associated with Gram-negative bacteria for monitoring their own population density. The present study unveils the mechanism of HSLs in Achromobacter denitrificans SP1 while transforming di(2-ethylhexyl) phthalate (DEHP) into prodigiosin in a simple basal salt medium. The primary detection of HSLs was done by the colorimetric method. Fourier-transform infrared spectroscopy and liquid chromatography-mass spectrometry-quadrupole time-of-flight confirmed and identified the HSLs. The maximum production of HSLs was observed between 24 and 72 h of incubation, which is noted to be a peak time of DEHP degradation. A total of 57.2% of DEHP was degraded within 30 h and complete degradation was observed within 72 h of incubation. Regulation in the synthesis of various acyl-HSL molecules, viz. 3OC6-HSL in the initial stage of DEHP stress, 3OC8-HSL, and C10-HSL during the time of degradation and 3OC12-HSL on completion of degradation was noticed. The role of HSLs on the production of prodigiosin was confirmed using vanillin as an HSL inhibitor. Through the selective activation of HSL molecules, A. denitrificans SP1 sustain the changing stressful conditions. Supplementation of acyl-HSL signal molecules may boost up the efficacy of A. denitrificans SP1 in both DEHP degradation and prodigiosin production which offers great potential towards the management of DEHP containing plastic wastes.

Achromobacter xylosoxidans Cellular Pathology Is Correlated with Activation of a Type III Secretion System.

Achromobacter xylosoxidans is increasingly recognized as a colonizer of cystic fibrosis (CF) patients, but the role that A. xylosoxidans plays in pathology remains unknown. This knowledge gap is largely due to the lack of model systems available to study the toxic potential of this bacterium. Recently, a phospholipase A2 (PLA2) encoded by a majority of A. xylosoxidans genomes, termed AxoU, was identified. Here, we show that AxoU is a type III secretion system (T3SS) substrate that induces cytotoxicity to mammalian cells. A tissue culture model was developed showing that a subset of A. xylosoxidans isolates from CF patients induce cytotoxicity in macrophages, suggestive of a pathogenic or inflammatory role in the CF lung. In a toxic strain, cytotoxicity is correlated with transcriptional activation of axoU and T3SS genes, demonstrating that this model can be used as a tool to identify and track expression of virulence determinants produced by this poorly understood bacterium.

Isolation and identification of salt-tolerant plant-growth-promoting rhizobacteria and their application for rice cultivation under salt stress.

Growth and productivity of rice are negatively affected by soil salinity. However, some salt-tolerant rhizosphere-inhabiting bacteria can improve salt resistance of plants, thereby augmenting plant growth and production. Here, we isolated a total of 53 plant-growth-promoting rhizobacteria (PGPR) from saline and non-saline areas in Bangladesh where electrical conductivity was measured as >7.45 and <1.80 dS/m, respectively. Bacteria isolated from saline areas were able to grow in a salt concentration of up to 2.60 mol/L, contrary to the isolates collected from non-saline areas that did not survive beyond 854 mmol/L. Among the salt-tolerant isolates, Bacillus aryabhattai, Achromobacter denitrificans, and Ochrobactrum intermedium, identified by comparing respective sequences of 16S rRNA using the NCBI GenBank, exhibited a higher amount of atmospheric nitrogen fixation, phosphate solubilization, and indoleacetic acid production at 200 mmol/L salt stress. Salt-tolerant isolates exhibited greater resistance to heavy metals and antibiotics, which could be due to the production of an exopolysaccharide layer outside the cell surface. Oryza sativa L. fertilized with B. aryabhattai MS3 and grown under 200 mmol/L salt stress was found to be favoured by enhanced expression of a set of at least four salt-responsive plant genes: BZ8, SOS1, GIG, and NHX1. Fertilization of rice with osmoprotectant-producing PGPR, therefore, could be a climate-change-preparedness strategy for coastal agriculture.

Adhesion of Stenotrophomonas maltophilia, Delftia acidovorans, and Achromobacter xylosoxidans to Contact Lenses.

PURPOSE: Contact lens cases become contaminated with microbes during use. We wished to compare the adhesion of uncommon bacterial contaminants isolated from lens cases to contact lenses with and without organic soil. METHODS: Strains of Delftia acidovorans (001), Stenotrophomonas maltophilia (002 and 006), and Achromobacter xylosoxidans (001) isolated from contact lens cases (test strains) and Pseudomonas aeruginosa (Paer1) isolated from eyes at the time of infiltrative response (control strain) were used. Bacteria were grown and resuspended in phosphate-buffered saline (PBS) or 10% organic soil (heat-killed Saccharomyces cerevisiae resuspended in complement inactivated bovine serum). Two silicone hydrogel (senofilcon A and comfilcon A) and one hydrogel lens (etafilcon A) lens materials were used. Bacteria (1.0×10 and 1.0×10 colony-forming units/mL; CFU/mL) adhered to lenses for 24 hr and the numbers of bacteria adherent to each lens type (with and without organic soil) were estimated by culture. RESULTS: All the four test strains adhered in significantly greater numbers to contact lenses after incubation in inoculum prepared with organic soil compared with PBS-D. acidovorans 001 (0.7 log10 CFU; P<0.05), S. maltophilia 002 (1.7 log10 CFU; P<0.05), S. maltophilia 006 (0.9 log10 CFU; P<0.05), and A. xylosoxidans 001 (0.4 log10 CFU; P<0.05). However, the presence of organic soil did not increase adhesion of P. aeruginosa Paer1 (-0.1 log10 CFU; P>0.05). Achromobacter xylosoxidans 001 (P<0.01), D. acidovorans 001 (P<0.01), and S. maltophilia 002 (P<0.01) significantly differed in their adhesion to the three contact lens materials. CONCLUSION: Bacteria that are commonly found in contact lens cases adhered to contact lenses in relatively high numbers in the presence of organic soil. This might indicate that a similar phenomenon occurs in the presence of tears. This may facilitate their transfer from the lens to the cornea and the production of corneal infiltrates.

Leukotriene B4 is essential for lung host defence and alpha-defensin-1 production during Achromobacter xylosoxidans infection.

Leukotriene B4 (LTB4) is essential for host immune defence. It increases neutrophil recruitment, phagocytosis and pathogen clearance, and decreases oedema and inflammasome activation. The host response and the role of LTB4 during Achromobacter xylosoxidans infection remain unexplored. Wild-type (129sv) and LTB4 deficient (Alox5 -/-) mice were intratracheally infected with A. xylosoxidans. Wild-type 129sv infected mice survived beyond the 8th day post-infection, exhibited increased levels of LTB4 in the lung on the 1st day, while levels of PGE2 increased on the 7th day post-infection. Infected Alox5 -/- mice showed impaired bacterial clearance, increased lung inflammation, and succumbed to the infection by the 7th day. We found that exogenous LTB4 does not affect the phagocytosis of A. xylosoxidans by alveolar macrophages in vitro. However, treatment of infected animals with LTB4 protected from mortality, by reducing the bacterial load and inflammation via BLT1 signalling, the high affinity receptor for LTB4. Of importance, we uncovered that LTB4 induces gene and protein expression of α-defensin-1 during the infection. This molecule is essential for bacterial clearance and exhibits potent antimicrobial activity by disrupting A. xylosoxidans cell wall. Taken together, our data demonstrate a major role for LTB4 on the control of A. xylosoxidans infection.

Biofilm formation of Achromobacter xylosoxidans on contact lens.

Achromobacter spp. may contaminate lenses, lens cases, and contact lens solutions and cause ocular infections. The aim of this study was to investigate the possibility of isolated strain of Achromobacter xylosoxidans to form biofilm on the surface of soft contact lenses (CL), to quantify the production of the formed biofilm, and compare it with the reference strains (Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae). Bacterial strain isolated from one contact lens case was identified as A. xylosoxidans using Vitek2 Automated System. Biofilm forming capacity of isolated strain of A. xylosoxidans and reference strains of P. aeruginosa, S. aureus, and H. influenzae on soft CL were analyzed by commonly used microtitre plate method. Our results showed that isolated strain of A. xylosoxidans was capable to form biofilm on the surface of soft contact lens. A. xylosoxidans was strong biofilm producer while all examined reference strains were moderate biofilm producers. A. xylosoxidans appears to be superior biofilm producer on soft CL compared to reference strains.

Pseudobacteremia outbreak of biofilm-forming Achromobacter xylosoxidans - environmental transmission.

BACKGROUND: Achromobacter xylosoxidans (AX) is known for intrinsic resistance to disinfectants. Our laboratory routine surveillance system detected an unexpected rise in AX bloodstream infections in a 2200-bed hospital. An epidemiological investigation was conducted to find the source and disrupt further transmission. METHODS: Outbreak cases were defined as patients with at least one positive blood culture positive for AX from May 2014 to May 2015. Medical records were reviewed, affected wards, as well as the microbiology laboratory were audited. Additionally, microbiologic culture and biofilm staining for suspected antiseptic reusable tissue dispensers were performed, and isolated AX strains were typed using RAPD PCR and PFGE. RESULTS: During the outbreak period, AX were isolated from blood cultures from 26 patients. The retrospective cohort study did not reveal common risk factors. The clinical features of the case patients suggested a pseudobacteremia. The reusable tissue dispensers containing Incidin® Plus solution product were found to be contaminated with biofilm-forming AX. Typing of the isolates revealed that blood culture isolates were identical with the strains found in the dispensers. CONCLUSIONS: After changing the usage of the product to single-use and educating staff, the outbreak was terminated. Contamination of dispensers occurred due to insufficient reprocessing, since biofilm disrupting steps were not included in the process.

Impact of Delftia tsuruhatensis and Achromobacter xylosoxidans on Escherichia coli dual-species biofilms treated with antibiotic agents.

Recently it was demonstrated that for urinary tract infections species with a lower or unproven pathogenic potential, such as Delftia tsuruhatensis and Achromobacter xylosoxidans, might interact with conventional pathogenic agents such as Escherichia coli. Here, single- and dual-species biofilms of these microorganisms were characterized in terms of microbial composition over time, the average fitness of E. coli, the spatial organization and the biofilm antimicrobial profile. The results revealed a positive impact of these species on the fitness of E. coli and a greater tolerance to the antibiotic agents. In dual-species biofilms exposed to antibiotics, E. coli was able to dominate the microbial consortia in spite of being the most sensitive strain. This is the first study demonstrating the protective effect of less common species over E. coli under adverse conditions imposed by the use of antibiotic agents.

Efficacy of antimicrobials against biofilms of Achromobacter and Pseudomonas.

PURPOSE: Achromobacter xylosoxidans and Pseudomonas aeruginosa biofilms can develop in ophthalmic products and accessories such as contact lens cases, leading to the development of ocular infections. This study evaluated the efficacy of the antimicrobials polyaminopropyl biguanide (PAPB) and benzalkonium chloride (BAK) against A. xylosoxidans and P. aeruginosa biofilms. METHODS: Biofilms of A. xylosoxidans and P. aeruginosa used as a comparative control were formed by incubating the bacteria on contact lens cases and on coverslips in phosphate-buffered saline. The biofilms were then exposed to PAPB and BAK for 5 minutes and 4 hours. After exposure, alginate swabs were used to remove the biofilms from the lens cases and the bacteria were plated on tryptic soy agar for determination of survivors. Also, after exposure to these disinfectants, the A. xylosoxidans and P. aeruginosa biofilms were stained with SYTO 9 and propidium iodide. Using a confocal microscope with a 488-nm laser, the number of cells with damaged cell membranes was determined. RESULTS: After 5 minutes of exposure to BAK or PAPB, A. xylosoxidans biofilms were more resistant to the antimicrobial effects of these disinfectants than P. aeruginosa biofilms. After 4 hours, both organisms were reduced by more than 3 logs after exposure to either BAK or PAPB. Confocal microscopy studies revealed that BAK was more effective at damaging A. xylosoxidans and P. aeruginosa cell membranes than PAPB at the concentrations used in ophthalmic products. CONCLUSIONS: Biofilms of the emerging pathogen A. xylosoxidans were more resistant to the disinfectants PAPB and BAK than biofilms of P. aeruginosa. Because of the emergence of A. xylosoxidans and the demonstrated greater resistance to the common ophthalmic preservatives BAK and PAPB than the standard Gram-negative organism P. aeruginosa, A. xylosoxidans biofilms should be assessed in antimicrobial challenge tests to assure the safety of multiuse ophthalmic products.

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms.

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84-7.25 CFU cm(-2) at 192 h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.

Enhanced biodegradation of total polycyclic aromatic hydrocarbons (TPAHs) by marine halotolerant Achromobacter xylosoxidans using Triton X-100 and ß-cyclodextrin--a microcosm approach.

Ability of Achromobacter xylosoxidans, a chrysene degrading marine halotolerant bacterium to degrade polycyclic aromatic hydrocarbons (PAHs) using a cost effective laboratory microcosm approach, was investigated. Effect of variables as chrysene, glucose as a co-substrate, Triton X-100 as a non-ionic surfactant and ß-cyclodextrin as a PAHs solubilizer was examined on degradation of low molecular weight (LMW) and high molecular weight (HMW) PAHs. A total of eleven PAHs detected from polluted saline soil were found to be degraded. Glucose, in combination with Triton X-100 and ß-cyclodextrin resulted in 2.8 and 1.4-fold increase in degradation of LMW PAHs and 7.59 and 2.23-fold increase in degradation of HMW PAHs, respectively. Enhanced biodegradation of total PAHs (TPAHs) by amendments with Triton X-100 and ß-cyclodextrin using Achromobacter xylosoxidans can prove to be promising approach for in situ bioremediation of marine sites contaminated with PAHs.

Bioremediation assessment of diesel-biodiesel-contaminated soil using an alternative bioaugmentation strategy.

This study investigated the effectiveness of successive bioaugmentation, conventional bioaugmentation, and biostimulation of biodegradation of B10 in soil. In addition, the structure of the soil microbial community was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis. The consortium was inoculated on the initial and the 11th day of incubation for successive bioaugmentation and only on the initial day for bioaugmentation and conventional bioaugmentation. The experiment was conducted for 32 days. The microbial consortium was identified based on sequencing of 16S rRNA gene and consisted as Pseudomonas aeruginosa, Achromobacter xylosoxidans, and Ochrobactrum intermedium. Nutrient introduction (biostimulation) promoted a positive effect on microbial populations. The results indicate that the edaphic community structure and dynamics were different according to the treatments employed. CO2 evolution demonstrated no significant difference in soil microbial activity between biostimulation and bioaugmentation treatments. The total petroleum hydrocarbon (TPH) analysis indicated a biodegradation level of 35.7 and 32.2 % for the biostimulation and successive bioaugmentation treatments, respectively. Successive bioaugmentation displayed positive effects on biodegradation, with a substantial reduction in TPH levels.

Complete genome sequence of the cystic fibrosis pathogen Achromobacter xylosoxidans NH44784-1996 complies with important pathogenic phenotypes.

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-ß-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.

Heterotrophic nitrification by Achromobacter xylosoxidans S18 isolated from a small-scale slaughterhouse wastewater.

Heterotrophic carbon utilizing microbes were acclimatized in the laboratory by inoculating sludge collected from the waste discharge pond of a small-scale rural abattoir in India in a nutrient solution intermittently fed with glucose and ammonium chloride. Cultures of 10 well-developed isolates were selected and grown in a basal medium containing glucose and ammonium chloride. Culture supernatants were periodically analyzed for ammonium nitrogen (NH(4)(+)-N) and chemical oxygen demand (COD). Polyphasic taxonomic study of the most active nitrifier (S18) was done. Half saturation concentration (K(s)), maximum rate of substrate utilization (k), yield coefficient (Y) and decay coefficient (K(d)) were determined from the Lineweaver-Burk plot using the modified Monod equation. S18 was able to remove 97 ± 2% of (NH(4)(+)-N) and 88 ± 3% of COD. Molecular phylogenetic study supported by physiological and biochemical characteristics assigned S18 as Achromobacter xylosoxidans. Nitrification activity of A. xylosoxidans was demonstrated for the first time, while interestingly, the distinctive anaerobic denitrification property was preserved in S18. K (s) values were determined as 232.13 ± 1.5 mg/l for COD reduction and 2.131 ± 1.9 mg/l for NH(4)(+)-N utilization. Yield coefficients obtained were 0.4423 ± 0.1134 mg of MLVSS/mg of COD and 0.2461 ± 0.0793 mg of MLVSS/mg of NH(4)(+)-N while the decay coefficients were 0.0627 ± 0.0013 per day and 0.0514 ± 0.0008 per day, respectively. After a contact period of 24 h, 650 ± 5 mg/l solids were produced when the initial concentration of COD and NH(4)(+)-N were 1820 ± 10 mg/l and 120 ± 5.5 mg/l, respectively. This is the first report on the kinetic coefficients for carbon oxidation and nitrification by a single bacterium isolated from slaughterhouse wastewater.

A case of meningitis due to Achromobacter xylosoxidans denitrificans 60 years after a cranial trauma.

BACKGROUND: Achromobacter xylosoxidans (AX) is a non-fermentative aerobic gram-negative bacillus. It is an opportunistic pathogen and the causative agent of various infections. We report an original case of late posttraumatic meningitis due to AX denitrificans. CASE REPORT: An 83-year-old man was hospitalized for acute headache, nausea and vomiting. The emergency brain computer tomography (CT) scan did not reveal any anomaly. In his medical history, there was an auditory injury due to a cranial trauma incurred in a skiing accident 60 years earlier. Cytobiochemical analysis of the cerebrospinal fluid (CSF) revealed increased levels of neutrophils and proteins. The CSF bacterial culture was positive: the Gram stain showed a gram-negative bacillus, oxidase + and catalase +, and the biochemical pattern using the API 20 NE strip revealed AX dentrificans. Late posttraumatic meningitis on a possible osteomeningeal breach was diagnosed even though the breach was not confirmed because the patient declined a second brain CT scan. The patient was successfully treated with meropenem. CONCLUSIONS: This report demonstrates the importance of searching for unusual or atypical organisms when the clinician encounters meningitis in a particular context, as well as the importance of adequate follow-up of craniofacial traumas.

Endophytic bacteria improve seedling growth of sunflower under water stress, produce salicylic acid, and inhibit growth of pathogenic fungi.

Endophytic bacterial strains SF2 (99.9% homology with Achromobacter xylosoxidans), and SF3 and SF4 (99.9% homology with Bacillus pumilus) isolated from sunflower grown under irrigation or drought were selected on the basis of plant growth-promoting bacteria (PGPB) characteristics. Aims of the study were to examine effects of inoculation with SF2, SF3, and SF4 on sunflower cultivated under water stress, to evaluate salicylic acid (SA) production by these strains in control medium or at Ψa = -2.03 MPa, and to analyze effects of exogenously applied SA, jasmonic acid (JA), bacterial pellets, and bacterial supernatants on growth of pathogenic fungi Alternaria sp., Sclerotinia sp., and Verticillum sp. Growth response to bacterial inoculation was studied in two inbred lines (water stress-sensitive B59 and water stress-tolerant B71) and commercial hybrid Paraiso 24. Under both water stress and normal conditions, plant growth following inoculation was more strongly enhanced for Paraiso 24 and B71 than for B59. All three strains produced SA in control medium; levels for SF3 and SF4 were higher than for SF2. SA production was dramatically higher at Ψa = -2.03 MPa. Exogenously applied SA or JA caused a significant reduction of growth for Sclerotinia and a lesser reduction for Alternaria and Verticillum. Fungal growth was more strongly inhibited by bacterial pellets than by bacterial supernatants. Our findings indicate that these endophytic bacteria enhance growth of sunflower seedlings under water stress, produce SA, and inhibit growth of pathogenic fungi. These characteristics are useful for formulation of inoculants to improve growth and yield of sunflower crops.

Multiple drug-resistant Alcaligenes xylosoxidans keratitis in a sanitation worker.

PURPOSE: To report a case of daily wear soft contact lens-associated keratitis caused by Alcaligenes xylosoxidans in a portable toilet sanitation worker. METHODS: A previously healthy 30-year-old man presented with bilateral decreased vision, redness, and irritation, beginning 1 week earlier. The patient had been treated with moxifloxocin for 1 week before presentation without improvement. Bacterial staining cultures were performed from corneal scrapings, contact lenses, contact lens case, and solution. Preliminary culture results and antibiotic sensitivities were generated using an automated identification system. Positive results from the contact lens, case, and solution were confirmed using polymerase chain reaction (PCR). RESULTS: The contact lenses, contact lens case, and solution cultures revealed heavy growth of A. xylosoxidans. These findings were confirmed by PCR. The organism was found to be resistant to aminoglycosides, fluoroquinolones, and cephalosporins. The patient was started on polymyxin B or trimethoprim. After 21 days of treatment, the infection completely resolved with a final spectacle-corrected visual acuity of 20/25. CONCLUSIONS: To our knowledge, this is the first case report of a corneal infection caused by A. xylosoxidans, which was isolated and identified from a contact lens, case, and solution using culture and PCR technique as well the first description of ocular surface disease resolution after the treatment with topical polymyxin B or trimethoprim. A. xylosoxidans should be considered as uncommon but potential pathogen capable of infectious spread through contaminated contact lens solution.

Epidemiological typing of clinical isolates of Achromobacter xylosoxidans: comparison of phenotypic and genotypic methods.

The purpose of this paper is to evaluate the utility of different typing methods for Achromobacter xylosoxidans clinical isolates. Ninety-two blood culture isolates of A. xylosoxidans subsp. xylosoxidans were collected over a 25-month period. The typeability, discriminatory power and reproducibility of commonly used phenotypic and genotypic methods, such as resistotyping, plasmid profiling, whole-cell protein fingerprinting, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE), were compared. All 92 isolates were typeable by all of the methods used, with comparable reproducibility. PFGE showed the highest discriminatory power (98.9%), but whole-cell protein profiling showed better correlation with epidemiological data without significant loss in discriminatory power (94%). Whole-cell protein profiling is a reliable epidemiological tool for the analysis of A. xylosoxidans; PFGE is the most discriminatory.

Isolation of microorganisms and substances inhibitory to aflatoxin production.

Aflatoxins are toxic and carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The contamination of crops, feeds, and foods with aflatoxins can have serious effects on the health of humans and animals. Although many studies have been done to develop aflatoxin-control strategies, most are limited in their effectiveness. As part of an effort to develop control procedures, we have devised simple and safe methods that are useful for identifying microorganisms that effectively inhibit aflatoxin production by fungi. These include the microtitre agar plate assay using norsolorinic acid-accumulating mutant fungi, the ultraviolet light photography method using an instant film, the tip culture method, a convenient RNA extraction method for reverse transcription-polymerase chain reaction (RT-PCR) analysis, and other methods. Results of a recent trial have shown that Achromobacter xylosoxidans significantly inhibited aflatoxin production by A. parsiticus, and that the main inhibitory substance produced by the bacterium was cyclo(L-leucyl-L-prolyl). This result confirms that the methods described herein are useful for identifying microorganisms that inhibit aflatoxin production by fungi and could contribute to the development of methods to reduce aflatoxin contamination in commodities.