Effects of Agricultural Fungicide Use on Aspergillus fumigatus Abundance, Antifungal Susceptibility, and Population Structure.

Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is both an environmental saprobe and an opportunistic human fungal pathogen, resistance is suggested to arise from fungicide use in agriculture, as the azoles used for plant protection share the same molecular target as the frontline antifungals used clinically. However, limiting azole fungicide use on crop fields to preserve their activity for clinical use could threaten the global food supply via a reduction in yield. In this study, we clarify the link between azole fungicide use on crop fields and resistance in a prototypical human pathogen through systematic soil sampling on farms in Germany and surveying fields before and after fungicide application. We observed a reduction in the abundance of A. fumigatus on fields following fungicide treatment in 2017, a finding that was not observed on an organic control field with only natural plant protection agents applied. However, this finding was less pronounced during our 2018 sampling, indicating that the impact of fungicides on A. fumigatus population size is variable and influenced by additional factors. The overall resistance frequency among agricultural isolates is low, with only 1 to 3% of isolates from 2016 to 2018 displaying resistance to medical azoles. Isolates collected after the growing season and azole exposure show a subtle but consistent decrease in susceptibility to medical and agricultural azoles. Whole-genome sequencing indicates that, despite the alterations in antifungal susceptibility, fungicide application does not significantly affect the population structure and genetic diversity of A. fumigatus in fields. Given the low observed resistance rate among agricultural isolates as well the lack of genomic impact following azole application, we do not find evidence that azole use on crops is significantly driving resistance in A. fumigatus in this context.IMPORTANCE Antibiotic resistance is an increasing threat to human health. In the case of Aspergillus fumigatus, which is an environmental fungus that also causes life-threatening infections in humans, antimicrobial resistance is suggested to arise from fungicide use in agriculture, as the chemicals used for plant protection are almost identical to the antifungals used clinically. However, removing azole fungicides from crop fields threatens the global food supply via a reduction in yield. In this study, we survey crop fields before and after fungicide application. We find a low overall azole resistance rate among agricultural isolates, as well as a lack of genomic and population impact following fungicide application, leading us to conclude azole use on crops does not significantly contribute to resistance in A. fumigatus.

Urinary transcript quantitation of CK20 and IGF2 for the non-invasive bladder cancer detection.

PURPOSE: Cytokeratin 20 (CK20) and insulin-like growth factor 2 (IGF2) were previously proposed to be elevated in clinical samples from patients with bladder cancer (BCa). A two cohort design validation study was used to assess the relevance for BCa detection by transcript quantitation of both markers in urine samples. Their diagnostic value was assessed in comparison with voided urine cytology (VUC). METHODS: RNA isolation was carried out using cellular sediments of urine samples from 196/103 histologically positive BCa patients, as well as 97/50 control subjects for the test (TC) and validation cohort (VC), respectively. Urinary transcript levels of CK20 and IGF2 were determined by qPCR. RESULTS: Relative transcript levels were significantly elevated 3.4/11-fold for CK20 and 188/64-fold for IGF2 (p < 0.001) in urine sediments of BCa patients compared to controls in the TC and VC, respectively. In a combined analysis, the resulting sensitivity (SN) (SNTC: 77.9; SNVC: 90.3%) and specificity (SP) (SPTC: 88.0; SPVC: 84.0%) were similar to that of VUC. The sensitivity of VUC in combination with CK20 and IGF2 was considerably increased (SNTC: 94.6; SNVC: 93.2%) while specificity was reduced (SPTC: 72.0; SPVC: 82.0%) compared to VUC alone in the test and validation cohort. CONCLUSIONS: Transcript levels of IGF2 and CK20 enabled the detection of BCa with a diagnostic performance similar to VUC. Combined analysis of voided urine cytology together with altered transcript levels of CK20 and IGF2 enhanced sensitivity, but did not improve overall test performance.

Evaluation of seven X-chromosomal short tandem repeat loci located within the Xq26 region.

In this study a set of 29 X-chromosomal short tandem repeats (STRs) located within the Xq26 region was evaluated. These STRs were found within the 133.14-133.45Mb region around the HPRTB locus. Evaluation of the microsatellites was performed with regard to polymorphism, reliable amplification, and low stutter artefacts. DXS10101, DXS10102, and DXS10103 were identified as those X-STRs with highest diversity; i.e. PIC values of 0.7174-0.8933. The locus DXS10101 was the optimal candidate for the integration in the commercial available test system Mentype Argus X-8 PCR amplification kit.

DNA microarrays for hybridization detection by surface plasmon resonance spectroscopy.

We report on the development of a new platform technology for the detection of genetic variations by means of surface plasmon resonance (SPR) spectroscopy. TOPAS chips with integrated optics were exploited in combination with microfluidics. Within minutes, the detection of hybridization kinetics was achieved simultaneously at all spots of the DNA microarray. A nanoliter dispenser is used to deposit thiol-modified single-stranded probe DNA on the gold surface of the chips. We investigated the influence of different parameters on hybridization using model polymerase chain reaction (PCR) products. These PCR products comprised a single-stranded tag sequence being complementary to an anti-tag sequence of probes immobilized on the gold surface. The signals increased with increasing length of PCR products (60, 100 or 300 base pairs) as well as with their concentration. We investigated hybridizations on DNA microarrays comprising 90 spots of probe DNA with three different sequences. Furthermore, we demonstrate that sequences with possible hairpin structures significantly lower the binding rate, and thus, the SPR signals during hybridization.

Escherichia coli K-12 Suppressor-free Mutants Lacking Early Glycosyltransferases and Late Acyltransferases: minimal lipopolysaccharide structure and induction of envelope stress response.

To elucidate the minimal lipopolysaccharide (LPS) structure needed for the viability of Escherichia coli, suppressor-free strains lacking either the 3-deoxy-d-manno-oct-2-ulosonic acid transferase waaA gene or derivatives of the heptosyltransferase I waaC deletion with lack of one or all late acyltransferases (lpxL/M/P) and/or various outer membrane biogenesis factors were constructed. Delta(waaC lpxL lpxM lpxP) and waaA mutants exhibited highly attenuated growth, whereas simultaneous deletion of waaC and surA was lethal. Analyses of LPS of suppressor-free waaA mutants grown at 21 degrees C, besides showing accumulation of free lipid IV(A) precursor, also revealed the presence of its pentaacylated and hexaacylated derivatives, indicating in vivo late acylation can occur without Kdo. In contrast, LPS of Delta(waaC lpxL lpxM lpxP) strains showed primarily Kdo(2)-lipid IV(A), indicating that these minimal LPS structures are sufficient to support growth of E. coli under slow-growth conditions at 21/23 degrees C. These lipid IV(A) derivatives could be modified biosynthetically by phosphoethanolamine, but not by 4-amino-4-deoxy-l-arabinose, indicating export defects of such minimal LPS. DeltawaaA and Delta(waaC lpxL lpxM lpxP) exhibited cell-division defects with a decrease in the levels of FtsZ and OMP-folding factor PpiD. These mutations led to strong constitutive additive induction of envelope responsive CpxR/A and sigma(E) signal transduction pathways. Delta(lpxL lpxM lpxP) mutant, with intact waaC, synthesized tetraacylated lipid A and constitutively incorporated a third Kdo in growth medium inducing synthesis of P-EtN and l-Ara4N. Overexpression of msbA restored growth of Delta(lpxL lpxM lpxP) under fast-growing conditions, but only partially that of the Delta(waaC lpxL lpxM lpxP) mutant. This suppression could be alleviated by overexpression of certain mutant msbA alleles or the single-copy chromosomal MsbA-498V variant in the vicinity of Walker-box II.

Hungarian population data of eight X-linked markers in four linkage groups.

The X chromosomal STR markers DXS10135 and DXS8378 in linkage group 1, DXS7132 and DXS10074 in linkage group 2, HPRTB and DXS10101 in linkage group 3, and DXS10134 and DXS7423 in linkage group 4 were studied in the Hungarian population. After genotyping unrelated men (219) and women (165), forensic efficiency parameters were calculated. Deviations from Hardy-Weinberg equilibrium could not be detected. There were several microvariant and rare alleles were sequenced: four in locus DXS10135 (alleles 17.1, 18.1, 20.1 and 26.1), one in locus DXS10074 (alleles 11), three in locus DXS10101 (alleles 26, 34.2 and 35) and five in locus DXS10134 (alleles 35.3, 37.2, 38.2, 39.2, 41).

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit.

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135-DXS8378, DXS7132-DXS10074, HPRTB-DXS10101 and DXS7423-DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1-4. The genetic distance within the STR pairs is assumed to be <1cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.

Evaluation of haplotype discrimination capacity of 35 Y-chromosomal short tandem repeat loci.

The haplotype discrimination capacity of the 9 Y-chromosomal short tandem repeat (Y-STR) loci comprising the so called minimal haplotype together with additional 26 recently described single-copy Y-STRs was evaluated within 391 males from Germany, The Netherlands, and Turkey. The aim of this study was to identify the minimum number of Y-STRs needed in addition to the recommended 9 minimal haplotype loci or the 11 SWGDAM loci for individualizing male lineages. Highest gene diversities were shown for DYS385 loci, DYS449, DYS481, DYS570, DYS447, DYS576, DYS389-II, and DYS390 (D=0.7518-0.8746). The five Y-STRs DYS447, DYS449, DYS481, DYS570, and DYS576 comprised the smallest set of loci in addition to the previously recommended standard Y-STRs leading to the individualization of all males from each single population group. Complete resolution of the pooled population was achieved by the additional genotyping of two further loci, DYS446 or DYS505 and DYF406S1 or DYS522.

[Antibody response after immunization with a Salmonella Typhimurium live vaccine in dependence on the way of application]. / Antikörperantwort nach Impfung von Mastschweinen mit einem Salmonella-Typhimurium-Lebendimpfstoff in Abhängigkeit von der Applikationsform.

In Germany now, the recognition of Salmonella infections in pig herds is based on three different commercial tests detecting antibodies against Salmonella-derived lipopolysaccharide (LPS). However, a serious disadvantage of these tests, used so far, is the restricted detection of antibodies belonging predominantly to the immunoglobulin class g (IgG). Therefore, a new test was developed to detect three Ig classes (IgM, IgG and IgA). Different constellations between the three Ig classes allow the evaluation of the current infection status of each pig. Under field conditions, this was proved in three different vaccination trials using a commercial Salmonella Typhimurium live vaccine.

Population data on the seven short tandem repeat loci D4S2366, D6S474, D14S608, D19S246, D20S480, D21S226 and D22S689 in a German population.

Allele frequencies for the autosomal tetranucleotide short tandem repeat loci D4S2366, D6S474, D14S608, D19S246, D20S480, D21S226 and D22S689 were investigated in a sample of 189 unrelated German individuals using a multiplex polymerase chain reaction approach. The loci showed no significant deviations from Hardy-Weinberg equilibrium except for D14S608. All genotyped alleles were cloned and sequenced, and an allelic nomenclature consistent with the ISFH recommendations was defined.

New alleles and mutational events at 14 STR loci from different German populations.

The molecular origin of DNA mutations and the mutation rates were analyzed at 14 short tandem repeat (STR) loci with samples from trio cases derived from 10 different German population samples. STR loci comprised of D2S1360, D3S1744, D4S2366, D5S2500, D6S474, D7S1517, D8S1132, D10S2325, D12S391, D18S51, D19S246, D20S480, D21S226, and D22S689. In a total of 488 meioses, 16 isolated genetic inconsistencies in 8 different STRs were observed, whereas no mutations were found at the other loci. The data of five mutations suggested the presence of silent or null alleles due to sequence variation in primer binding site. This could be confirmed for four suspected cases by the use of alternative primer sets and by DNA sequence analyses. Furthermore, this study revealed nine new allelic variants at five different loci.

OpsX from Haemophilus influenzae represents a novel type of heptosyltransferase I in lipopolysaccharide biosynthesis.

The inner core region of the lipopolysaccharide (LPS) of Haemophilus influenzae is characterized by the presence of a phosphorylated 3-deoxy-alpha-D-manno-octulosonic acid (Kdo). In this study, we show that the heptosyltransferase I adding the first L-glycero-D-manno-heptose residue to this acceptor is encoded by the gene opsX, which differs in substrate specificity from the other heptosyltransferase I, known as WaaC.