Amplified Fragment Length Polymorphism Fingerprinting Research Articles

P257 Amplified fragment length polymorphism fingerprinting supports the absence of correlation of genotype with clinical phenotype or source of the isolate in Aspergillus flavus infections

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectives Aspergillus flavus accounts for ∼ 10% of bronchopulmonary aspergillosis and is the second leading etiological agent of invasive aspergillosis worldwide. It is the commonest cause of fungal rhinosinusitis and ocular mycoses in tropical countries including India. We report amplified fragment length polymorphism genotyping of a set of clinical and environmental isolates to unravel the genetic diversity of A. flavus in India and to further determine correlation between isolate genotype and the source/clinical phenotype of the Aspergillus infection.MethodsTwo sets of morphologically identified isolates of A. flavus from clinical (n = 71) and environmental sources (n = 22) were included in the study using a stratified random sampling method. Clinical strains were isolated from lower respiratory tract specimens (n = 22), sinus and sino-nasal biopsies (n = 25), corneal samples (n = 12), and others (n = 12). Environmental strains were isolated from different niches like air, soil, and infected crop samples. Aspergillus fumigatus F145 was used as an out-group. DNA was extracted from the fungal broth culture following the method of Lee et al. AFLP was done as per an earlier described method using HpyCH41V and Mse1 (New England BioLabs) for restriction digestion followed by selective amplification of restriction-digested products with 1 mm HpyCH4 IV primer with one selective residue (5-Flu-GTA-GACTGCGTACCCGTC-3’), 1 mm MseI primer with four selective residues (5-GATGAGTCCTGACTAATGAA). Fingerprint data was analyzed in BioNumerics software version 7.6 (Applied Maths, Belgium). The phylogenetic tree was constructed using Pearson's Correlation coefficient. An AFLP genotype was assigned to a cluster using an arbitrary cut-off value of ≥90% fingerprint similarity. The distribution of genotypes among different categories was analyzed by Fisher's exact test or Pearson's χ 2 goodness of fit test with Yates correction factor as appropriate and two-tailed P-values of <.05 were considered statistically significant.ResultsThe analyses revealed a total of 16 AFLP genotypes with 5 major clusters (≥ 5 isolates) reflecting the extent of genetic diversity in A. flavus. Genotype VIII encompassed predominantly clinical isolates (P <.01) and genotype XI with majority of isolates from environmental sources (P <.0001). The strains which were isolated from invasive and non-invasive forms or from different sites (pulmonary, sinus, and ocular) did not diverge into separate or unique clusters. Although the genotypes had an asymmetric distribution in different clinical presentations as revealed by the χ2 goodness of fit test, none of the genotypes was exclusively responsible for causing a particular infection. Isolates from the north zone of India shared genotypes with those from the southern region of the country. Three isolates formed a separate genotype XVI and diverged from the A. flavus cluster by 42% fingerprint similarity. Partial β-tubulin and calmodulin gene sequencing-based phylogeny reconstruction placed those three isolates in A. tamarii clade of the A. flavus species complex.Conclusions: This study suggests that every genotype of A. flavus has the potential of causing an allergic, non-invasive or invasive infection. Further, A. flavus sensu stricto was predominantly (97%) isolated from clinical specimens revealing the majority of infections/colonization are caused by this species compared to other members in the Flavi complex.

Candidemia among Hospitalized Pediatric Patients Caused by Several Clonal Lineages of Candida parapsilosis.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Performance comparison of two reduced-representation based genome-wide marker-discovery strategies in a multi-taxon phylogeographic framework

Multi-locus genetic data are pivotal in phylogenetics. Today, high-throughput sequencing (HTS) allows scientists to generate an unprecedented amount of such data from any organism. However, HTS is resource intense and may not be accessible to wide parts of the scientific community. In phylogeography, the use of HTS has concentrated on a few taxonomic groups, and the amount of data used to resolve a phylogeographic pattern often seems arbitrary. We explore the performance of two genetic marker sampling strategies and the effect of marker quantity in a comparative phylogeographic framework focusing on six species (arthropods and plants). The same analyses were applied to data inferred from amplified fragment length polymorphism fingerprinting (AFLP), a cheap, non-HTS based technique that is able to straightforwardly produce several hundred markers, and from restriction site associated DNA sequencing (RADseq), a more expensive, HTS-based technique that produces thousands of single nucleotide polymorphisms. We show that in four of six study species, AFLP leads to results comparable with those of RADseq. While we do not aim to contest the advantages of HTS techniques, we also show that AFLP is a robust technique to delimit evolutionary entities in both plants and animals. The demonstrated similarity of results from the two techniques also strengthens biological conclusions that were based on AFLP data in the past, an important finding given the wide utilization of AFLP over the last decades. We emphasize that whenever the delimitation of evolutionary entities is the central goal, as it is in many fields of biodiversity research, AFLP is still an adequate technique.

High genetic and morphological diversification of the Euphorbia verrucosa alliance (Euphorbiaceae) in the Balkan and Iberian peninsulas

AbstractWe explored the diversification of the southern European Euphorbia verrucosa alliance applying molecular (amplified fragment length polymorphism fingerprinting [AFLP], sequencing of the nuclear ribosomal internal transcribed spacer), karyological (relative genome size estimations, chromosome counts) and morphometric methods. The AFLP data inferred four main phylogenetic lineages corresponding to western‐southern Balkan E. montenegrina, central Balkan E. serpentini, northern Balkan‐central European‐north Italian E. verrucosa and Iberian‐southern French E. flavicoma. Genetic diversification is strongest within the Iberian and the Balkan peninsulas, suggesting Pleistocene persistence of the species in different micro‐refugia. In contrast, weak genetic structure in E. verrucosa suggests Holocene (after last glacial maximum) expansion to central and western Europe, likely from a northern Balkan refugium. Karyological data provide evidence for tetraploidisation events in E. flavicoma and E. montenegrina, but not in E. verrucosa and E. serpentini. By integrating phylogenetic data with multivariate morphometric analyses, we propose a new taxonomic treatment for this group, mainly by recognising the Balkan endemics E. montenegrina and E. serpentini as independent species and by redefining the distributions of E. flavicoma and E. verrucosa. Our study underlines the importance of the Balkan and Iberian peninsulas as major Pleistocene refugia.

Strain improvement of Trichoderma spp. through two-step protoplast fusion for cellulase production enhancement.

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography- mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.

Macroscopic-microscopic characteristics and AFLP fingerprint for identification of Erythroxylum novogranatense, E. cambodianum and E. cuneatum endemic to Thailand

Erythroxylum novogranatense (Morris) Hieron, E. cambodianum Pierre and E. cuneatum (Miq.) Kurz in family Erythroxylaceae was traditionally used as an antipyretic, general stimulant and gastrointestinal diseases. Due to their morphological similarity, the correct identification was necessary for the quality control in herbal medicine. E. novogranatense (Morris) Hieron, E. cambodianum Pierre and E. cuneatum (Miq.) Macroscopic and microscopic characteristics evaluated Kurz endemic to Thailand according to WHO standard guideline and amplified fragment length polymorphism (AFLP) fingerprint. Morphological characters of E. novogranatense, E. cambodianum and E. cuneatum were similar in their flower, fruit and seed but different in stem and leaf. Microscopic characteristics from these three species, including constant leaf numbers, showed individual values. The stomata were classified as paracytic type. The midrib transverse section showed distinct characters of the epidermis, palisade cell, stomata, spongy cell, parenchyma, xylem vessel, phloem tissue and collenchyma. AFLP fingerprint showed highly polymorphisms 97.42% with the number of bands (349 bands) ranging between 50-750 bands. Primer E+ACG/M+CTT had the highest number of AFLP band (91 bands). The dendrogram generated from UPGMA could separate these three species. In summary, the combination of morphological characteristics, microscopic investigation and AFLP fingerprinting can be used to identify plant species and determine the genetic relationship among three Erythroxylum species.

Genome-wide mapping using new AFLP markers to explore intraspecific variation among pathogenic Sporothrix species.

Sporotrichosis is a chronic subcutaneous mycosis caused by Sporothrix species, of which the main aetiological agents are S. brasiliensis, S. schenckii, and S. globosa. Infection occurs after a traumatic inoculation of Sporothrix propagules in mammals' skin and can follow either a classic route through traumatic inoculation by plant debris (e.g., S. schenckii and S. globosa) or an alternative route through zoonotic transmission from animals (e.g., S. brasiliensis). Epizootics followed by a zoonotic route occur in Brazil, with Rio de Janeiro as the epicenter of a recent cat-transmitted epidemic. DNA-based markers are needed to explore the epidemiology of these Sporothrix expansions using molecular methods. This paper reports the use of amplified-fragment-length polymorphisms (AFLP) to assess the degree of intraspecific variability among Sporothrix species. We used whole-genome sequences from Sporothrix species to generate 2,304 virtual AFLP fingerprints. In silico screening highlighted 6 primer pair combinations to be tested in vitro. The protocol was used to genotype 27 medically relevant Sporothrix. Based on the overall scored AFLP markers (97-137 fragments), the values of polymorphism information content (PIC = 0.2552-0.3113), marker index (MI = 0.002-0.0039), effective multiplex ratio (E = 17.8519-35.2222), resolving power (Rp = 33.6296-63.1852), discriminating power (D = 0.9291-0.9662), expected heterozygosity (H = 0.3003-0.3857), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Sporothrix. AFLP markers revealed cryptic diversity in species previously thought to be the most prevalent clonal type, such as S. brasiliensis, responsible for cat-transmitted sporotrichosis, and S. globosa responsible for large sapronosis outbreaks in Asia. Three combinations (#3 EcoRI-FAM-GA/MseI-TT, #5 EcoRI-FAM-GA/MseI-AG, and #6 EcoRI-FAM-TA/MseI-AA) provide the best diversity indices and lowest error rates. These methods make it easier to track routes of disease transmission during epizooties and zoonosis, and our DNA fingerprint assay can be further transferred between laboratories to give insights into the ecology and evolution of pathogenic Sporothrix species and to inform management and mitigation strategies to tackle the advance of sporotrichosis.

Phylotype and sequevar determination and AFLP fingerprinting of Ralstonia solanacearum strains causing bacterial wilt of potato in southeastern Iran

Bacterial wilt of potato caused by Ralstonia solanacearum is a severe threat to potato cultivation in Kerman province, located in southeastern Iran and the third largest potato producer in the country. The present study intends to determine the precise phylogenetic position, sequevar, and genetic diversity of R. solanacearum strains associated with the disease. To this end, the major potato-growing regions of the province (Jiroft and Laleh Zaar) were surveyed to collect tuber samples from affected potato plants showing typical wilt symptoms. Twenty-three strains were collectively purified and subjected to phenotypic and genotypic characterization. Biochemical tests showed that all the strains belonged to R. solanacearum biovar 2A. Furthermore, phylotype-specific multiplex polymerase chain reaction results indicated that the strains belonged to phylotype II. For sequevar typing as well as the determination of the phylotype subcluster (IIA/IIB), polymerase chain reaction amplicons of the endoglucanase precursor gene of 11 representative strains were sequenced. In phylogenetic analysis, the strains were grouped with the reference strains of phylotype IIB-1 retrieved from the GenBank. Then, to assess genetic diversity, 19 selected strains were subjected to genomic fingerprinting, i.e., amplified fragment length polymorphism (AFLP) using seven primer pair combinations. The AFLP data showed heterogeneity among the strains of R. solanacearum and were grouped into four clusters. With our knowledge, this is the first report of the phylotype, and sequevar determination (i.e., IIB-1) in addition to AFLP-based genomic fingerprinting of R. solanaceraum strains causing potato wilt in southeastern Iran.

Assessment of phylogenetic relationship among twenty Curcuma species in Thailand using amplified fragment length polymorphism marker.

Plants in the genus Curcuma are a rhizomatous perennial herb which is widely distributed in Thailand. It has long been known for their uses as folk medicines, foods, spices, and cosmetics. However, the identification of plants in the genus Curcuma is very difficult due to morphological similarity in the early flowering stage. Recently, the molecular technique is one of the reliable and powerful tools for plant identification. In this study, the genetic relationship among twenty Curcuma species from Thailand was accessed by the amplified fragment length polymorphism (AFLP) method. AFLP fingerprint showed 98.54% highly polymorphisms with the number of bands (617 bands) ranging between 48 and 80 bands. The dendrogram generated from the unweighted pair group method of the arithmetic average could separate these Curcuma species into three major clusters. Cluster I can be subdivided into IA, which composed of Curcuma parviflora, Curcuma sparganiifolia, Curcuma alismatifolia, Curcuma larsenii, Curcuma Gracillima, and Curcuma rhabdota with similarity index (SI) 0.7926–0.9358 and IB composed of Curcuma petiolata and Curcuma rubrobracteata with the SI 0.9240. Cluster II can be subdivided into IIA being composed of Curcuma longa, Curcuma Zedoaria, and Curcuma aromatica with the SI 0.8989–0.9071, whereas Cluster IIB was composed of Curcuma leucorrhiza, Curcuma aeruginosa, Curcuma comosa, Curcuma mangga, Curcuma angustifolia, Curcuma amada, Curcuma sessilis, and Curcuma albicoma with the SI 0.8236–0.9500. Cluster III belongs to Curcuma singularis and Alpinia galanga (outgroup plant), which clearly separated into different clusters from twenty Curcuma species. In summary, the ten successful AFLP primer combinations could be used to determine the genetic relationship among closely related twenty Curcuma species in Thailand.

Antifungal susceptibility, genotyping, resistance mechanism, and clinical profile of Candida tropicalis blood isolates.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014–2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2, and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 μg/ml, 2/64], itraconazole (MIC > 0.5 μg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 μg/ml, 4/64), and voriconazole (MIC ≥ 1 μg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission.

Is there a need for accepting paraphyletic taxa? A case study in the Sardinian endemic Cymbalaria muelleri (Plantaginaceae)

AbstractEvolution does not always result in dichotomous phylogenetic trees. For instance, in anacladogenetic speciation, where a new species originates by budding, the ancestral taxon is often initially paraphyletic. Here we study Cymbalaria muelleri (Plantaginaceae), a chasmophytic species endemic to Sardinia, a major island in the Mediterranean Basin. Its distribution range is divided into two well-delimited geographical groups with some morphological differences. Using a combination of morphology, molecular data (amplified fragment length polymorphism fingerprinting) and relative genome size, we found that the two geographical groups should be considered two separate taxa, which split through anacladogenesis. Accordingly, we formally describe the new paraphyletic subspecies C. muelleri subsp. villosa as the ancestor, from which C. muelleri subsp. muelleri originated by budding. Morphological analyses support the differentiation of the two subspecies, and there are strong diagnostic characters to differentiate them. In addition to morphology and genetics, slightly divergent habitat preferences and the disjunct distribution of the two subspecies also support the recognition of two taxa. Genome size data obtained for the two subspecies are consistent with the previously established hexaploidy of C. muelleri.

Divergent selection and genetic structure of Sideritis scardica populations from southern Balkan Peninsula as revealed by AFLP fingerprinting

Sideritis scardica Giseb. is a subalpine/alpine plant species endemic to the central part of the Balkan Peninsula. In this study, we combined Amplified Fragment Length Polymorphism (AFLP) and environmental data to examine the adaptive genetic variations in S. scardica natural populations sampled in contrasting environments. A total of 226 AFLP loci were genotyped in 166 individuals from nine populations. The results demonstrated low gene diversity, ranging from 0.095 to 0.133 and significant genetic differentiation ranging from 0.115 to 0.408. Seven genetic clusters were revealed by Bayesian clustering methods as well as by Discriminant Analysis of Principal Components and each population formed its respective cluster. The exception were populations P02 Mt. Shara and P07 Mt. Vermio, that were admixed between two clusters. Both landscape genetic methods Mcheza and BayeScan identified a total of seven (3.10%) markers exhibiting higher levels of genetic differentiation among populations. The spatial analysis method Samβada detected 50 individual markers (22.12%) associated with bioclimatic variables, among them seven were identified by both Mcheza and BayeScan as being under directional selection. Four bioclimatic variables associated with five out of seven outliers were related to precipitation, suggesting that this variable is the key factor affecting the adaptive variation of S. scardica.

Numerical analysis of phenotypic properties, genomic fingerprinting, and multilocus sequence analysis of Bradyrhizobium strains isolated from root nodules of Lembotropis nigricans of the tribe Genisteae

PurposeThe aim of this study was to estimate the level of genomic and phenotypic diversity as well as the genus and species position of bacterial strains isolated from root nodules of Lembotropis nigricans (family Fabaceae).MethodsThe genomic diversity of studied L. nigricans nodule symbionts was examined by using BOX-PCR and AFLP (amplified fragment length polymorphism) fingerprinting techniques. To assign bacteria to the genus, numerical analysis of phenotypic features and comparative analysis of 16S rDNA sequences were performed. The comparative analysis of combined atpD, dnaK, gyrB, and rpoB gene sequences (multilocus sequence analysis, MLSA) was used to determine the most closely related species to the studied bacteria.ResultsBoth BOX-PCR and AFLP techniques revealed a high level of genomic heterogeneity of L. nigricans nodulators. Among 33 studied bacteria, 32 genotypes were delineated by the AFLP method and 27 genotypes were identified by the BOX-PCR fingerprinting. The numerical analysis of 86 phenotypic characteristics of L. nigricans nodule isolates and reference rhizobia showed that studied bacteria belong to the genus Bradyrhizobium. Affiliation of L. nigricans nodule isolates to the genus Bradyrhizobium was supported by comparative analysis of 16S rDNA sequences and the concatenation of atpD, dnaK, gyrB, and rpoB gene sequences. MLSA indicated also that L. nigricans microsymbionts are members of Bradyrhizobium japonicum.ConclusionL. nigricans root nodule symbionts are members of Bradyrhizobium japonicum and exhibit high phenotypic and genomic diversity important for their survival in soil.

Molecular epidemiology and in vitro antifungal susceptibility of Trichophyton schoenleinii, agent of tinea capitis favosa.

Trichophyton schoenleinii is an anthropophilic dermatophyte usually causing tinea favosa. Only few studies have provided data on molecular epidemiology and antifungal profiles of this fungus due to its limited prevalence after 1950s. Forty-nine strains from Asia (n=27), Africa (n=10), Europe (n=10) and from unknown regions (n=2) were analysed with amplified fragment length polymorphism fingerprinting (AFLP) to reveal intraspecific genetic diversity in this dataset. Amplified fragment length polymorphism fingerprinting genotyping revealed five clusters which did not correspond to geographic origins or clinical characteristics. Additionally, in vitro antifungal susceptibility to seven antifungals was provided for all strains. Terbinafine, ketoconazole, miconazole and itraconazole proved to be the most effective drugs, followed by griseofulvin. No correlation between genotypes and differences in antifungal susceptibility was observed. It is concluded that the AFLP groups are lineages within a single species.

Influence of Dehydrated Wheat/Rice Cereal Matrices on Probiotic Activity of Bifidobacterium animalis ssp. lactis BB-12®§.

SUMMARYThree novel dehydrated wheat/rice cereal functional products with an addition of well documented probiotic Bifidobacterium animalis ssp. lactis BB-12® (BB-12®) were developed in Podravka factory for the infants older than 4 months: instant rice cereal, instant rice cereal with fruits and instant wheat cereal with vanilla. Notably, the number of viable BB-12® cells in each of the novel products was higher than the required minimal number of probiotic cells per gram of product (106 CFU/g) during the storage period of 106 weeks. Therefore, BB-12® strain recovery and genome stability were evaluated by strain-specific polimerase chain reaction and amplified fragment length polymorphism fingerprinting analysis. Further aim was to evaluate the influence of these three different cereal food matrices on specific probiotic properties of BB-12® strain in vitro. Applied food matrices positively influenced the survival in the simulated conditions of the gastrointestinal tract and antagonistic activity against undesirable microorganisms, while no influence on auto- and coaggregation ability of B. animalis ssp. lactis BB-12® was observed. Adhesion to extracellular matrix proteins and intestinal epithelial Caco-2 cells together with antibacterial activity emphasized competitive pathogen exclusion from Caco-2 cells by probiotic strain BB-12®.

The genetic relatedness of &lt;i&gt;E. coli&lt;/i&gt; associated with post- collection drinking water contamination in rural households

Rural households are often dependent on rivers, springs, boreholes or standpipes some distance from their homes for their daily water requirements. Water drinking and domestic use is consequently stored in containers in-house which are prone to post-collection contamination. The objective off the study was to determine the most likely origin or place off introduction off E. coli associated with post-collection contamination in rural households, by assessing the degree off genetic relatedness off E. coli present in the stored water and other environmental samples. E. coli isolates were obtained using either mFC agar with confirmation of indole production (44 isolates) or Colilert(R)-18 (52 isolates). Amplified fragment length polymorphism (AFLP) fingerprinting was applied to determine the genetic relatedness of E. coli isolated in-house storage containers, drinking cups, hand-swab samples, cattle dung and from the source water (spring water). DNA fingerprints of E. coli produced a number of clusters (>85% similarity scores calculated with the cosine coefficient). Identical E. coli genetic patterns were observed at closely linked points within the domestic pathway off water handling, such as between hand-swab and drinking-cup samples, between storage container and source isolates, and between drinking cups, source water and storage containers. The results indicated that AFLP fingerprinting could be applied to determine the genetic relatedness of E. coli isolated closely linked points within the domestic pathway off water use within a household. However, the high genetic diversity observed E. coli bacteria isolated the different water and environmental samples tested in this study, hampered the identification of post collection points of contamination.

An outbreak due to Candida auris with prolonged colonisation and candidaemia in a tertiary care European hospital.

Multidrug-resistant Candida auris has emerged as a cause of insidious hospital outbreaks and complicated infections. We present the analysis of an ongoing C.auris outbreak including the largest published series of C.auris bloodstream infection. All C.auris-positive patients from April-2016 to January-2017 were included. Environmental, clinical and microbiological data were recorded. Definitive isolate identification was performed by ITS-rDNA sequencing, and typing by amplified fragment length polymorphism fingerprinting. One hundred and forty patients were colonised by C.auris during the studied period (68% from surgical intensive care). Although control measures were implemented, we were not able to control the outbreak. Forty-one invasive bloodstream infections (87.8% from surgical intensive care) were included. Clinical management included prompt intravascular catheter removal and antifungal therapy with echinocandins. All isolates were fluconazole- and voriconazole-resistant, but echinocandin- and amphotericin B-susceptible. Thirty-day mortality rate was 41.4%, and severe septic metastasis as spondylodiscitis and endocarditis were observed in 5 patients (12%). C.auris was also recovered from inanimate patient surroundings and medical equipment. Despite antifungal treatment, high mortality and late complication rates were recorded. Molecular typing suggested a clonal outbreak different from those previously published.

Molecular characterization and antifungal susceptibility testing of Cryptococcus neoformans sensu stricto from southern Brazil.

Cryptococcosis is acquired from the environment by the inhalation of Cryptococcus cells and may establish from an asymptomatic latent infection into pneumonia or meningoencephalitis. The genetic diversity of a Cryptococcus neoformans species complex has been investigated by several molecular tools, such as multi-locus sequence typing, amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism and microsatellite analysis. This study aimed to investigate the genotype distributions and antifungal susceptibility profiles of C. neoformans sensu lato isolates from southern Brazil. We studied 219 C. neoformans sensu lato isolates with mating- and serotyping, AFLP fingerprinting, microsatellite typing and antifungal susceptibility testing.Results/Key findings. Among the isolates, 136 (69 %) were from HIV-positive patients. Only C. neoformans mating-type α and serotype A were observed. AFLP fingerprinting analysis divided the isolates into AFLP1/VNI (n=172; 78.5 %), AFLP1A/VNII (n=19; 8.7 %), AFLP1B/VNII (n=4; 1.8 %) and a new AFLP pattern AFLP1C (n=23; 10.5 %). All isolates were susceptible to tested antifungals and no correlation between antifungal susceptibility and genotypes was observed. Through microsatellite analysis, most isolates clustered in a major microsatellite complex and Simpson's diversity index of this population was D=0.9856. The majority of C. neoformans sensu stricto infections occurred in HIV-positive patients. C. neoformans AFLP1/VNI was the most frequent genotype and all antifungal drugs had high in vitro activity against this species. Microsatellite analyses showed a high genetic diversity within the regional C. neoformans sensu stricto population, and correlation between environmental and clinical isolates, as well as a temporal and geographic relationship.

Molecular diversity and allergenic profiles of Alternaria spp. from desert environments in Arizona

This study examined the genetic diversity of small-spored Alternaria species in the southwest desert of the USA by sampling 552 isolates from different habitats (soil and plant debris) in different locations (urban and an undisturbed desert). To estimate the genetic diversity, Amplified Fragment Length Polymorphism (AFLP) fingerprinting analysis was performed for all isolates. Strains representative of the sampled genotypic diversity (n = 125) were further characterized according their sporulation pattern and the capability to produce allergens. Morphological characterization assigned the majority of the strains to the Alternaria alternata and Alternaria tenuissima morpho-groups with only two isolates assigned to the Alternaria arborescens morpho-group. AFLP fingerprinting differentiated the A. arborescens morpho-groups, but could not distinguish between the A. alternata and A. tenuissima morpho-groups. Western blot analysis showed that a large number of allergenic proteins were produced by strains. These proteins were not specific for any morpho-group nor source of isolation. A hierarchical analysis of molecular variance was performed on the AFLP data to quantify molecular variation and partition this variation among sampled locations and habitat. No statistically significant differentiation among locations and habitat was detected indicating a lack of population structure across environments.

Breakthrough candidemia after the introduction of broad spectrum antifungal agents: A 5-year retrospective study.

Candidemia is the main invasive fungal disease among hospitalized patients. Several breakthrough candidemia (BrC) cases have been reported, but few studies evaluate the epidemiology, risk factors, molecular characterization, antifungal susceptibility profile and outcome of those patients, especially in developing countries and including patients using broad spectrum antifungals. We conducted a retrospective study from 2011 to 2016, including patients aged 12 years or older with candidemia. Epidemiological characteristics and risk factors for candidemia were evaluated and compared with patients with BrC using univariate and multivariate analysis. Sequential Candida isolates from BrC were identified by internal transcribed spacer sequencing, genotyped with amplified fragment length polymorphism fingerprinting (AFLP), and tested for antifungal susceptibility. From 148 candidemia episodes, 27 breakthrough episodes (18%) were identified, with neutropenia and mucositis being independent risk factors for BrC. Candida non-albicans was more frequent in the BrC group (P < .001). AFLP showed high correlation with conventional methods of identification among breakthrough isolates and a high genetic similarity among isolates from the same patient was observed. C. albicans was the most susceptible species with low MIC values for all antifungal agents tested. In contrast, we found isolates of C. glabrata, C. parapsilosis and C. tropicalis resistant to triazoles and echinocandins. In conclusion, BrC occurred mainly in severely immunosuppressed patients, with neutropenia and mucositis. Mortality did not differ between the groups. Candida non-albicans species were more recovered from BrC, with C. albicans being the most susceptible to antifungals.