[The pathogenicity of Cronobacter in the light of bacterial genomics]. / La patogenicidad de Cronobacter a la luz de la genómica bacteriana.

Introduction: Cronobacter spp. is a genus of Gram-negative bacteria belonging to the family Enterobacteriaceae. Species of the genus Cronobacter, particularly C. sakazakii, are implicated in the development of severe disease in newborns, which occurs with necrotizing enterocolitis, sepsis and meningitis. The disease has been frequently associated with powdered infant formula (PIF) and can therefore occur in the form of outbreaks. The genus Cronobacter has undergone extensive diversification in the course of its evolution, with some species being clearly pathogenic to humans while the impact of other species on human health is uncertain or unknown. Whole genome sequencing is used both in population genetic studies to identify the limited number of genotypes associated with the disease and to detect genes associated with antibiotic resistance or virulence, ultimately allowing more precise epidemiological links to be established between pediatric disease and infant foods.

Introducción: Cronobacter es un género de bacterias gramnegativas perteneciente a la familia Enterobacteriaceae. Algunas especies del género Cronobacter, en particular C. sakazakii, están implicadas en el desarrollo de infecciones neonatales graves, incluyendo meningitis, sepsis y enterocolitis necrotizante. La enfermedad se ha relacionado frecuentemente con los preparados en polvo para lactantes (PPL) y se puede presentar, por tanto, en forma de brotes. El género Cronobacter ha experimentado una amplia diversificación en el curso de su evolución, siendo algunas especies claramente patógenas para los humanos mientras que el impacto de otras especies sobre la salud humana es incierto o desconocido. La secuenciación genómica se utiliza en los estudios de genética de poblaciones tanto para identificar el limitado número de genotipos asociados a la enfermedad como para detectar los genes asociados a la virulencia, la adaptación al estrés o la resistencia a antibióticos, lo que permite, en definitiva, establecer vínculos epidemiológicos más precisos entre la enfermedad pediátrica y los alimentos infantiles.

Prevalence and Population Diversity of Listeria monocytogenes Isolated from Dairy Cattle Farms in the Cantabria Region of Spain.

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.

Lactobacillus plantarum in Dual-Species Biofilms With Listeria monocytogenes Enhanced the Anti-Listeria Activity of a Commercial Disinfectant Based on Hydrogen Peroxide and Peracetic Acid.

The aim of this work was to investigate the effect of dual-species biofilms of Listeria monocytogenes with Lactobacillus plantarum on the anti-Listeria activity of a hydrogen peroxide/peracetic acid based commercial disinfectant (P3, Oxonia) when using conditions approaching the food industry environment. Nine strains of L. monocytogenes, including eight persistent strains collected from the meat industry and one laboratory control strain, were used in mono and in dual-species biofilms with a strain of L. plantarum. Biofilms were produced on stainless steel coupons (SSCs), at 11°C (low temperature) or at 25°C (control temperature), in TSB-YE (control rich medium) or in 1/10 diluted TSB-YE (mimicking the situation of biofilm formation after a deficient industrial cleaning procedure). The biofilm forming ability of the strains was evaluated by enumeration of viable cells, and the antibiofilm activity of P3 was assessed by the log reduction of viable cells on SSC. In both nutrient conditions and at low temperature, there was no significant difference (p > 0.05) between L. monocytogenes biofilm forming ability in mono- and in dual-species biofilms. In dual-species biofilms, L. monocytogenes was the dominant species. However, it was generally more susceptible to the lower concentration of P3 0.5% (v/v) than in pure culture biofilms. The presence of L. plantarum, although without significant interference in the number of viable cells of L. monocytogenes, enhanced the efficacy of the anti-Listeria activity of P3, since dual-species biofilms were easier to control. The results presented here reinforce the importance of the investigation into co-culture biofilms produced in food industry conditions, namely at low temperatures, when susceptibility to sanitizers is being assessed.

Analysis of Benzalkonium Chloride Resistance and Potential Virulence of Listeria monocytogenes Isolates Obtained from Different Stages of a Poultry Production Chain in Spain.

ABSTRACT: Listeria monocytogenes can survive in food production facilities and can be transmitted via contamination of food during the various stages of food production. This study was conducted to compile the results of three independent previous studies on the genetic diversity of L. monocytogenes in a poultry production company in Spain and to determine the potential virulence and sanitizer resistance of the strains by using both genotype and phenotype analyses. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain. A set of 347 L. monocytogenes isolates representing 39 subtypes was obtained using pulsed-field gel electrophoresis (PFGE). A total of 28 subtypes (68%) had a full-length internalin A gene, and two subtypes had a phenotype with low potential for virulence because of a mutation in the prfA gene. A total of 32 subtypes (82%) were classified as benzalkonium chloride resistant (BAC-R) and contained the resistance determinant bcrABC (21 subtypes, 54%) or the resistance gene qacH (11 subtypes, 28%). A total of 13 persistent BAC-R subtypes (minimum of 3 months between the first and last sample from with the isolate was recovered) were identified at the abattoir and processing plant. The three production stages shared a unique subtype (PFGE type 1), which had the mutation in the prfA gene and the bcrABC resistance determinant. Whole genome sequencing revealed this subtype to be sequence type 31. Limited genetic diversity was noted in the isolates studied, including some subtypes that were persistent in the environment of the investigated facilities. Given the high prevalence of BAC-R subtypes, these results support the association between resistance to biocides and persistence of L. monocytogenes.

Inactivation of Listeria monocytogenes during dry-cured ham processing.

The effect of Serrano and Iberian dry-cured ham processing and ripening on Listeria monocytogenes inactivation at the surface of whole hams was investigated. Salted hams were surface inoculated (6.5 log CFU) with a cocktail of 4 L. monocytogenes strains isolated from environment and products of a meat industry. Serrano and Iberian hams were ripened for 16 and 24 months, respectively. A decrease of at least 4.6 log units on the surface of Serrano ham was recorded after 4 months for L. monocytogenes counts, which remained under the detection limit thereafter. L. monocytogenes declined by >5 log units on the surface of Iberian ham during the first 9 months and was not detected afterwards. The higher nitrite content of Serrano ham might have accelerated the decrease of the pathogen. This study validates the inactivation of L. monocytogenes on the surface of whole dry-hams during extended ripening.

Whole-Genome Sequences of Seven Listeria monocytogenes Strains from Different Stages of a Poultry Meat Production Chain.

Here, we present the draft genome sequences of seven Listeria monocytogenes strains isolated during three independent studies carried out in three stages of a poultry meat production chain. The genome sequences of these strains obtained from different stages can help to understand the possible transmission of L. monocytogenes.

Effect of Low Doses of Disinfectants on the Biofilm-Forming Ability of Listeria monocytogenes.

This study was intended to investigate the effect of contact with concentrations close to the minimum inhibitory concentration (MIC) (0.5, 1, and 1.5 MIC; MIC of planktonic cells was determined using a microdilution broth method) of sodium hypochlorite (SHY) or benzalkonium chloride (BZK) during the process of formation of biofilm (24 h), upon the architecture and viability of the biofilms formed by four L. monocytogenes isolates of molecular serotype 1/2a: S2-1 (BZK-susceptible strain; MICBZK = 3.0 ppm), S2-2 (BZK-resistant strain qacH positive; MICBZK = 13 ppm), CDL 69 (BZK-resistant strain bcrABC positive; MICBZK = 10 ppm), and S2BAC (BZK-resistant laboratory mutant of S2-1, with multidrug resistance phenotype; MICBZK = 9 ppm). Images were examined through confocal laser scanning microscopy after staining with SYTO 9 and Propidium Iodide. Biovolume values in the observation field (14,161 µm2) in the absence of biocides ranged from 103,928.3 ± 6,730.2 µm3 (S2BAC) to 276,030.9 ± 42,291.9 µm3 (S2-1). Exposure to SHY at 0.5 MIC reduced (p < 0.05) the biovolume of biofilms formed by S2-1 and S2BAC and did not modify (p > 0.05) the biovolume of biofilms by S2-2 and CDL 69. Exposure to sub-MICs of BZK decreased (p < 0.05; S2-1) or enhanced (p < 0.05; S2-2, CDL 69 and S2BAC) biofilm development. Exposure to biocides at 1 or 1.5 MIC inhibited biofilm formation. This study provides clear evidence that BZK at sub-MICs can enhance the biofilm-forming ability of BZK-resistant L. monocytogenes strains. Because biofilms contribute to the persistence of bacteria throughout the food chain and represent a major source of food contamination, our findings suggest the importance of avoiding sub-MICs of disinfectants in food-handling environments.

Potential Impact of the Resistance to Quaternary Ammonium Disinfectants on the Persistence of Listeria monocytogenes in Food Processing Environments.

The persistence of certain strains of Listeria monocytogenes, even after the food processing environment has been cleaned and disinfected, suggests that this may be related to phenomena that reduce the concentration of the disinfectants to subinhibitory levels. This includes (i) the existence of environmental niches or reservoirs that are difficult for disinfectants to reach, (ii) microorganisms that form biofilms and create microenvironments in which adequate concentrations of disinfectants cannot be attained, and (iii) the acquisition of resistance mechanisms in L. monocytogenes, including those that lead to a reduction in the intracellular concentration of the disinfectants. The only available data with regard to the resistance of L. monocytogenes to disinfectants applied in food production environments refer to genotypic resistance to quaternary ammonium compounds (QACs). Although there are several well-characterized efflux pumps that confer resistance to QACs, it is a low-level resistance that does not generate resistance to QACs at the concentrations applied in the food industry. However, dilution in the environment and biodegradation result in QAC concentration gradients. As a result, the microorganisms are frequently exposed to subinhibitory concentrations of QACs. Therefore, the low-level resistance to QACs in L. monocytogenes may contribute to its environmental adaptation and persistence. In fact, in certain cases, the relationship between low-level resistance and the environmental persistence of L. monocytogenes in different food production chains has been previously established. The resistant strains would have survival advantages in these environments over sensitive strains, such as the ability to form biofilms in the presence of increased biocide concentrations.

The Connection between Persistent, Disinfectant-Resistant Listeria monocytogenes Strains from Two Geographically Separate Iberian Pork Processing Plants: Evidence from Comparative Genome Analysis.

The aim of this study was to investigate the basis of the putative persistence of Listeria monocytogenes in a new industrial facility dedicated to the processing of ready-to-eat (RTE) Iberian pork products. Quaternary ammonium compounds, which included benzalkonium chloride (BAC), were repeatedly used as surface disinfectants in the processing plant. Clean and disinfected surfaces were sampled to evaluate if resistance to disinfectants was associated with persistence. Of the 14 isolates obtained from product contact and non-product contact surfaces, only five different pulsed-field gel electrophoresis (PFGE) types were identified during the 27-month study period. Two of these PFGE types (S1 and S10-1) were previously identified to be persistent and BAC-resistant (BAC(r)) strains in a geographically separate slaughterhouse belonging to the same company. The remaining three PFGE types, which were first identified in this study, were also BAC(r). Whole-genome sequencing and in silico multilocus sequence typing (MLST) analysis of five BAC(r) isolates of the different PFGE types identified in this study showed that the isolate of the S1 PFGE type belonged to MLST sequence type 31 (ST31), a low-virulence type characterized by mutations in the inlA and prfA genes. The isolates of the remaining four PFGE types were found to belong to MLST ST121, a persistent type that has been isolated in several countries. The ST121 strains contained the BAC resistance transposon Tn6188. The disinfection-resistant L. monocytogenes population in this RTE pork product plant comprised two distinct genotypes with different multidrug resistance phenotypes. This work offers insight into the L. monocytogenes subtypes associated with persistence in food processing environments.

Genome Sequences of Five Disinfectant-Resistant Listeria monocytogenes Strains from Two Iberian Pork-Processing Plants.

We announce the draft genome sequences of five Listeria monocytogenes strains from two Iberian pork-processing plants located in Spain that were distinguished by their resistance to benzalkonium chloride. These strains seem highly adapted to the meat-processing environment according to their persistence and transmission capabilities.

The influence of subminimal inhibitory concentrations of benzalkonium chloride on biofilm formation by Listeria monocytogenes.

Disinfectants, such as benzalkonium chloride (BAC), are commonly used to control Listeria monocytogenes and other pathogens in food processing plants. Prior studies have demonstrated that the resistance to BAC of L. monocytogenes was associated with the prolonged survival of three strains of molecular serotype 1/2a in an Iberian pork processing plant. Because survival in such environments is related to biofilm formation, we hypothesised that the influence of BAC on the biofilm formation potential of L. monocytogenes might differ between BAC-resistant strains (BAC-R, MIC≥10mg/L) and BAC-sensitive strains (BAC-S, MIC≤2.5mg/L). To evaluate this possibility, three BAC-R strains and eight BAC-S strains, which represented all of the molecular serotype 1/2a strains detected in the sampled plant, were compared. Biofilm production was measured using the crystal violet staining method in 96-well microtitre plates. The BAC-R strains produced significantly (p<0.05) less biofilm than the BAC-S in the absence of BAC, independent of the rate of planktonic growth. In contrast, when the biofilm values were measured in the presence of BAC, one BAC-R strain (S10-1) was able to form biofilm at 5mg/L of BAC, which prevented biofilm formation among the rest of the strains. A genetic determinant of BAC resistance recently described in L. monocytogenes (Tn6188) was detected in S10-1. When a BAC-S strain and its spontaneous mutant BAC-R derivative were compared, resistance to BAC led to biofilm formation at 5mg/L of BAC and to a significant (p<0.05) stimulation of biofilm formation at 1.25mg/L of BAC, which significantly (p<0.05) reduced the biofilm level in the parent BAC-S strain. Our results suggest that the effect of subminimal inhibitory concentrations of BAC on biofilm production by L. monocytogenes might differ between strains with different MICs and even between resistant strains with similar MICs but different genetic determinants of BAC resistance. For BAC-R strains similar to S10-1, subminimal inhibitory BAC may represent an advantage, compensating for the weak biofilm formation level that might be associated with resistance. Biofilm formation in the presence of increased subminimal inhibitory concentrations of the disinfectant may represent an important attribute among certain resistant and persistent strains of L. monocytogenes.

Antibiotic susceptibility in benzalkonium chloride-resistant and -susceptible Listeria monocytogenes strains.

This study aimed to investigate whether Listeria monocytogenes strains with resistance to a commonly used biocide display any cross-resistance to antibiotics. Using pulsed-field gel electrophoresis (PFGE), 29 different PFGE types were previously identified in an Iberian pig abattoir and processing plant. Only three PFGE types were resistant to benzalkonium chloride (BAC), but they represented a significant proportion of the PFGE types surviving in the plant after 4 years. In the present study, a subset of 29 strains, representing the 29 different PFGE types, underwent antibiotic susceptibility testing. Antibiotic susceptibility was assessed by Etest, utilizing 12 commonly prescribed antibiotics. All of the 29 strains were susceptible to all of the antibiotics tested. The study revealed that this group of different PFGE types of L. monocytogenes, including those resistant to BAC, possesses uniform sensitivity to antibiotics.

Control of Listeria monocytogenes contamination in an Iberian pork processing plant and selection of benzalkonium chloride-resistant strains.

The aims of this study were to characterize the different strains of Listeria monocytogenes collected at an Iberian pork processing plant and to investigate whether their specific characteristics were associated with prolonged survival in the plant. Using pulsed-field gel electrophoresis (PFGE), 29 PFGE types were previously identified during a three-year period. Eight of these PFGE types persisted in the plant during that period. In the present study, a subset of 29 PFGE type strains, which represented the 29 different PFGE types, was further characterized by assessing the potential virulence, and using motility, surface attachment, and antimicrobial susceptibility tests. After changing the disinfection procedures in the plant, the isolation rate of L. monocytogenes decreased, and only four of the 29 PFGE types, including three of the eight persistent PFGE types, were found the following year. These four "surviving" PFGE types included three from PCR serogroup IIa that were characterized by their low virulence mutations and low-level resistance to benzalkonium chloride (BAC). Furthermore, these PFGE types comprised the only BAC-resistant isolates found in the study, and they appear to have been selected due to the control of Listeria contamination. The resistance to increased sublethal concentrations of disinfectants may lead to prolonged survival of L. monocytogenes in food plants.

Low potential virulence associated with mutations in the inlA and prfA genes in Listeria monocytogenes isolated from raw retail poultry meat.

Packaged raw foods can represent a potential source of Listeria monocytogenes contamination when opened at home, and listeriosis is associated with the consumption of undercooked raw foods. The aim of this study was to characterize a group of L. monocytogenes strains isolated from 56 packages of raw chicken meat from a single brand in order to determine the diversity of the strains that dominate in a particular food over time, as well as their pathogenic potential. Forty (71%) samples were found to be positive for L. monocytogenes, and three isolates per sample were subjected to PCR molecular serotyping. Subtyping of 45 isolates from different manufacturing dates (n = 40) or different molecular serotype within the same sample (n = 5) identified 11 different L. monocytogenes subtypes as defined by pulsed-field gel electrophoresis and sequencing of virulence genes actA and inlA. Two of the subtypes accounted for 51% of the isolates. About 40% of isolates (three subtypes) were found to potentially present attenuated virulence because of the presence of mutations in the prfA and inlA genes.

Molecular tracking of Listeria monocytogenes in an Iberian pig abattoir and processing plant.

The environment and products from an Iberian pig abattoir and processing plant were sampled to investigate the prevalence and genetic diversity of Listeria monocytogenes. The organism was not detected in the pig carcasses prior to processing. Fresh and dry-cured pork did show detectable levels, always ranging below 100CFU per gram. A total of 163 L. monocytogenes isolates collected during one year were characterized by PCR-based serotyping and pulsed-field gel electrophoresis (PFGE) restriction analysis. Three predominant PFGE types or pulsotypes seemed to persist in the plant. The pulsotype S1 (serotype group 1/2a, 53% of the isolates) was mostly recovered from whole pieces of meat and environmental sites, while pulsotypes S2 (1/2a, 17%) and S4 (1/2b, 21%) were more frequently associated with ground pork products. The pulsotype S4 was also found in a grinding machine, suggesting a possible association of this machine with the contamination of the ground meat products.

Different enrichment procedures for recovery of Listeria monocytogenes from raw chicken samples can affect the results of detection (by chromogenic plating or real-time PCR) and lineage or strain identification.

This study aimed to evaluate the effect of different enrichment procedures on the detection of Listeria monocytogenes in food, by a comparison of subculture onto chromogenic agar with real-time PCR. Two different culture media, the primary and secondary enrichment broths of the U.S. Food Safety and Inspection Service (FSIS) method used for PCR detection of L. monocytogenes, were compared for the primary enrichment of retail ground chicken samples. L. monocytogenes was detected after the completion of each enrichment procedure in 63% (complete FSIS procedure) and 60% (plain FSIS secondary enrichment broth incubated for 48 h) of the samples by both culture and PCR, whereas a combination of the results for the two enrichment procedures revealed 86% of the samples to be positive. Most of the samples analyzed contained a mixture of lineage I and II strains, and their ratio varied for each enrichment procedure. This mixture could have a significant effect on the result of detection of L. monocytogenes for each individual sample, explaining the increase in positive samples when the results of the two enrichment procedures were combined. The use of different isolation procedures can affect the specific samples identified as positive and the specific strains isolated.

Traceback identification of an ingredient (pork dewlap) as the possible source of Listeria monocytogenes serotype 4b contamination in raw chicken products.

In surveys conducted on finished product samples from a single poultry processing plant in Spain, Listeria monocytogenes was found in 14 different uncooked products. To track contamination patterns, 77 L. monocytogenes isolates were characterized by PCR-based serotyping, pulsed-field gel electrophoresis (PFGE) restriction analysis, and PCR-based allelic analysis of the virulence gene actA. Serotyping revealed that 12 isolates (15.6%) were of the L. monocytogenes serotype 4b complex (serotype 4b or the closely related serotypes 4d and 4e). A combination of endonucleases AscI and ApaI PFGE patterns yielded 15 different pulsotypes among all 77 tested isolates. All the serotype 4b isolates belonged to one pulsotype. Sequencing of the actA gene confirmed that all serotype 4b isolates corresponded to the same allelic subtype. The subtype was recovered from five product types, but its presence was not correlated with the production line or the date of isolation, suggesting a possible association of this strain with a common ingredient. This traceback investigation established that pork dewlap, an ingredient common to all the products contaminated with this strain, was the most probable source of L. monocytogenes 4b. The same 4b strain was isolated from four samples of pork dewlap from one specific supplier. After replacement of this contaminated ingredient in the fresh products, this strain of L. monocytogenes serotype 4b was not detected. This study confirms the effectiveness of molecular subtyping to control contamination by specific strains of L. monocytogenes and the importance of testing the different ingredients added to the food products.

Evaluation of effects of primary and secondary enrichment for the detection of Listeria monocytogenes by real-time PCR in retail ground chicken meat.

A SYBR Green I based real-time PCR assay with inlA-specific oligonucleotide primers was developed for easy and rapid detection of Listeria monocytogenes in a model food that usually has a high incidence of contamination with this pathogen. Results with pure cultures and artificially contaminated chicken meat samples indicate that the PCR assay was highly specific and sensitive. The melting point analysis of the 160 bp amplified DNA fragment was different for L. monocytogenes isolates of the two major phylogenetic divisions of the species, 1 and 2. The assay was then used to survey retail ground chicken meat for contamination with L. monocytogenes. Thirty-seven samples were enriched according to the United States Department of Agriculture culture assays to detect L. monocytogenes on meat. The use and efficiency of PCR assay was examined following both primary and secondary enrichments, which were also plated on chromogenic agar for enumeration of L. monocytogenes and nonpathogenic Listeria spp. to investigate the discrepancies between culture and PCR. Overall, L. monocytogenes was detected in 75% of the samples. Primary enrichment yielded detection rates of 70% and 37% for culture and PCR, respectively. The corresponding rates for secondary enrichment were 54% and 70%, respectively. Test sensitivity is therefore influenced by the type of enrichment and is probably related not only to the limited growth of L. monocytogenes in the primary enrichment media (false-negative PCR results), but also to the high populations of nonpathogenic Listeria spp. in the secondary enrichment broths (false-negative culture results). The main challenge of rapid PCR-based detection of L. monocytogenes from food is the poor sensitivity of primary enrichment media. The improvement of enrichment conditions may help increase assay sensitivity.

Simultaneous detection of listeria monocytogenes in chicken meat enrichments by PCR and reverse-transcription PCR without DNA/RNA isolation.

Environmental and food samples can be analyzed using PCR and reverse transcription (RT)-PCR techniques to discriminate between viable and nonviable cells of bacterial pathogens. Here, we describe the use of a commercial lysis buffer, initially designed for mammalian cells, that permits the rapid extraction of bacterial DNA and RNA. The buffer is an RT-PCR-compatible lysis solution in which RNA is stable and can be frozen for later use. RT-PCR is carried out directly after DNase I treatment of crude bacterial lysates using rTth polymerase for RT-PCR in a single tube. Untreated lysate is used for standard PCR. The procedure permits the amplification of either mRNA or DNA of Listeria monocytogenes at a level similar to that obtained with purified nucleic acids. Using lysates obtained with this buffer, nested PCR and RT-PCR assays detected low numbers of L. monocytogenes cells from artificially contaminated chicken meat samples. The simplicity of this system may foster the development of similar buffers specifically designed for bacteria to improve RNA detection methods that can be performed in parallel with DNA analysis. The use of a single buffer decreases the time needed for analysis, is amenable to automation and real-time assays, and might be adaptable to all bacteria and amplification methods.

Isolation, characterization, and antifungal susceptibility of melanin-deficient mutants of Scedosporium prolificans.

Scedosporium prolificans mutants lacking the ability to synthesize melanin were selected after ultraviolet light (UV) irradiation. UV exposure of S. prolificans conidia resulted in a high frequency of melanin-deficient (mel-) mutants. Stable and non-stable morphological variants were found in the population: reversion of the mutant phenotype was always to the wild-type phenotype. Based on their morphological differences, these variants were classified into five different groups that were phenotypically characterized. The mel- mutants plus the wild-type strain were examined for in vitro susceptibility to antifungal agents with different and/or the same mechanism of action. There was no apparent difference in minimum inhibitory concentrations when comparing the wild-type and the mel- mutants. Therefore, melanin does not appear to confer protection against the more important antifungal agents in S. prolificans.