European multi-centre study to establish MIC and zone diameter epidemiological cut-off values for Bacillusanthracis.

OBJECTIVES: Bacillus anthracis clinical breakpoints, representing a systematic approach to guide clinicians in selecting the most appropriate antimicrobial treatments, are not part of the guidance from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). This is because defined distributions of MIC values and of epidemiological cut-off values (ECOFFs) have been lacking. In this study, a Europe-wide network of laboratories in collaboration with EUCAST, aimed at establishing standardized antimicrobial susceptibility testing methods, wild-type MIC distributions, and ECOFFs for ten therapeutically relevant antimicrobials. METHODS: About 335 B. anthracis isolates were tested by broth microdilution and disc diffusion methodologies. MIC and inhibition zone diameters were curated according to EUCAST SOP 10.2 and the results were submitted to EUCAST for ECOFFs and clinical breakpoint determination. RESULTS: Broth microdilution and disc diffusion data distributions revealed putative wild-type distributions for the tested agents. For each antimicrobial agent, ECOFFs were defined. Three highly resistant strains with MIC values of 32 mg/L benzylpenicillin were found. MIC values slightly above the defined ECOFFs were observed in a few isolates, indicating the presence of resistance mechanisms to doxycycline, tetracycline, and amoxicillin. DISCUSSION: B. anthracis antimicrobial susceptibility testing results were used by EUCAST to determine ECOFFs for ten antimicrobial agents. The MIC distributions were used in the process of determining clinical breakpoints. The ECOFFs can be used for the sensitive detection of isolates with resistance mechanisms, and for monitoring resistance development. Genetic changes causing phenotypic shifts in isolates displaying slightly elevated MICs remain to be investigated.

Machine Learning Algorithms for Classification of MALDI-TOF MS Spectra from Phylogenetically Closely Related Species Brucella melitensis, Brucella abortus and Brucella suis.

(1) Background: MALDI-TOF mass spectrometry (MS) is the gold standard for microbial fingerprinting, however, for phylogenetically closely related species, the resolution power drops down to the genus level. In this study, we analyzed MALDI-TOF spectra from 44 strains of B. melitensis, B. suis and B. abortus to identify the optimal classification method within popular supervised and unsupervised machine learning (ML) algorithms. (2) Methods: A consensus feature selection strategy was applied to pinpoint from among the 500 MS features those that yielded the best ML model and that may play a role in species differentiation. Unsupervised k-means and hierarchical agglomerative clustering were evaluated using the silhouette coefficient, while the supervised classifiers Random Forest, Support Vector Machine, Neural Network, and Multinomial Logistic Regression were explored in a fine-tuning manner using nested k-fold cross validation (CV) with a feature reduction step between the two CV loops. (3) Results: Sixteen differentially expressed peaks were identified and used to feed ML classifiers. Unsupervised and optimized supervised models displayed excellent predictive performances with 100% accuracy. The suitability of the consensus feature selection strategy for learning system accuracy was shown. (4) Conclusion: A meaningful ML approach is here introduced, to enhance Brucella spp. classification using MALDI-TOF MS data.

Proteome Analysis of a M. avium Mutant Exposes a Novel Role of the Bifunctional Protein LysX in the Regulation of Metabolic Activity.

Lysyl-phosphatidylglycerol is one of the components of the mycobacterial membrane that contributes to the resistance to cationic antimicrobial peptides, a host-induced frontline defense against invading pathogens. Its production is catalyzed by LysX, a bifunctional protein with lysyl transferase and lysyl transfer RNA synthetase activity. Comparative proteome analysis of a lysX mutant of Mycobacterium avium strain 104 and the wild type indicated that the lysX mutant strain undergoes a transition in phenotype by switching the carbon metabolism to ß-oxidation of fatty acids, along with accumulation of lipid inclusions. Surprisingly, proteins associated with intracellular survival were upregulated in the lysX mutant, even during extracellular growth, preparing bacteria for the conditions occurring inside host cells. In line with this, the lysX mutant exhibited enhanced intracellular growth in human-blood-derived monocytes. Thus, our study exposes the significance of lysX in the metabolism and virulence of the environmental pathogen M. avium hominissuis.

Genome Sequence of Bacillus safensis Strain Ingolstadt Isolated from the Pectoralis Pouch of a Patient with Defibrillator-Related Surgery.

We report the draft genome sequence of clindamycin-resistant Bacillus safensis strain Ingolstadt isolated from a patient with bacterial colonization after heart surgery. The draft genome comprises 3.75 Mbp and harbors 3,793 predicted protein-encoding genes and a small plasmid.

Metabolic phenotype of clinical and environmental Mycobacterium avium subsp. hominissuis isolates.

BACKGROUND: Mycobacterium avium subsp. hominissuis (MAH) is an emerging opportunistic human pathogen. It can cause pulmonary infections, lymphadenitis and disseminated infections in immuno-compromised patients. In addition, MAH is widespread in the environment, since it has been isolated from water, soil or dust. In recent years, knowledge on MAH at the molecular level has increased substantially. In contrast, knowledge of the MAH metabolic phenotypes remains limited. METHODS: In this study, for the first time we analyzed the metabolic substrate utilization of ten MAH isolates, five from a clinical source and five from an environmental source. We used BIOLOG Phenotype MicroarrayTM technology for the analysis. This technology permits the rapid and global analysis of metabolic phenotypes. RESULTS: The ten MAH isolates tested showed different metabolic patterns pointing to high intra-species diversity. Our MAH isolates preferred to use fatty acids such as Tween, caproic, butyric and propionic acid as a carbon source, and L-cysteine as a nitrogen source. Environmental MAH isolates resulted in being more metabolically active than clinical isolates, since the former metabolized more strongly butyric acid (p = 0.0209) and propionic acid (p = 0.00307). DISCUSSION: Our study provides new insight into the metabolism of MAH. Understanding how bacteria utilize substrates during infection might help the developing of strategies to fight such infections.

A hypervariable genomic island identified in clinical and environmental Mycobacterium avium subsp. hominissuis isolates from Germany.

Mycobacterium avium subsp. hominissuis (MAH) is an opportunistic human pathogen widespread in the environment. Genomic islands (GI)s represent a part of the accessory genome of bacteria and influence virulence, drug-resistance or fitness and trigger bacterial evolution. We previously identified a novel GI in four MAH genomes. Here, we further explored this GI in a larger collection of MAH isolates from Germany (n=41), including 20 clinical and 21 environmental isolates. Based on comparative whole genome analysis, we detected this GI in 39/41 (95.1%) isolates. Although all these GIs integrated in the same insertion hotspot, there is high variability in the genetic structure of this GI: eight different types of GI have been identified, designated A-H (sized 6.2-73.3kb). These GIs were arranged as single GI (23/41, 56.1%), combination of two different GIs (14/41, 34.1%) or combination of three different GIs (2/41, 4.9%) in the insertion hotspot. Moreover, two GI types shared more than 80% sequence identity with sequences of M. canettii, responsible for Tuberculosis. A total of 253 different genes were identified in all GIs, among which the previously documented virulence-related genes mmpL10 and mce. The diversity of the GI and the sequence similarity with other mycobacteria suggests cross-species transfer, involving also highly pathogenic species. Shuffling of potential virulence genes such as mmpL10 via this GI may create new pathogens that can cause future outbreaks.

Correlation between the genomic o454-nlpD region polymorphisms, virulence gene equipment and phylogenetic group of extraintestinal Escherichia coli (ExPEC) enables pathotyping irrespective of host, disease and source of isolation.

BACKGROUND: The mutS-rpoS intergenic region in E. coli displays a mosaic structure which revealed pathotype specific patterns. To assess the importance of this region as a surrogate marker for the identification of highly virulent extraintestinal pathogenic E. coli (ExPEC) strains we aimed to: (i) characterize the genetic diversity of the mutS gene and the o454-nlpD genomic region among 510 E. coli strains from animals and humans; (ii) delineate associations between the polymorphism of this region and features such as phylogenetic background of E. coli, pathotype, host species, clinical condition, serogroup and virulence associated genes (VAG)s; and (iii) identify the most important VAGs for classification of the o454-nlpD region. METHODS: Size variation in the o454-nlpD region was investigated by PCR amplification and sequencing. Phylogenetic relationships were assessed by Ecor- and Multilocus sequence- typing (MLST), and a comparative analysis between mutS gene phylogenetic tree obtained with RAxML and the MLST grouping method was performed. Correlation between o454-nlpD patterns and the features described above were analysed. In addition, the importance of 47 PCR-amplified ExPEC-related VAGs for classification of o454-nlpD patterns was investigated by means of Random Forest algorithm. RESULTS: Four main structures (patterns I-IV) of the o454-nlpD region among ExPEC and commensal E. coli strains were identified. Statistical analysis showed a positive and exclusive association between pattern III and the ExPEC strains. A strong association between pattern III and either the Ecor group B2 or the sequence type complexes known to represent the phylogenetic background of highly virulent ExPEC strains (such as STC95, STC73 and STC131) was found as well. RF analyses determined five genes (csgA, malX, chuA, sit, and vat) to be suitable to predict pattern III strains. CONCLUSION: The significant association between pattern III and group B2 strains suggested the o454-nlpD region to be of great value in identifying highly virulent strains among the mixed population of E. coli promising to be the basis of a future typing tool for ExPEC and their gut reservoir. Furthermore, top-ranked VAGs for classification and prediction of pattern III were identified. These data are most valuable for defining ExPEC pathotype in future in vivo assays.

Multitrophic interactions among Western Corn Rootworm, Glomus intraradices and microbial communities in the rhizosphere and endorhiza of maize.

The complex interactions among the maize pest Western Corn Rootworm (WCR), Glomus intraradices (GI-recently renamed Rhizophagus intraradices) and the microbial communities in both rhizosphere and endorhiza of maize have been investigated in view of new pest control strategies. In a greenhouse experiment, different maize treatments were established: C (control plants), W (plants inoculated with WCR), G (plants inoculated with GI), GW (plants inoculated with GI and WCR). After 20 days of WCR root feeding, larval fitness was measured. Dominant arbuscular mycorrhizal fungi (AMF) in soil and maize endorhiza were analyzed by cloning of 18S rRNA gene fragments of AMF, restriction fragment length polymorphism and sequencing. Bacterial and fungal communities in the rhizosphere and endorhiza were investigated by denaturing gradient gel electrophoresis of 16S rRNA gene and ITS fragments, PCR amplified from total community DNA, respectively. GI reduced significantly WCR larval development and affected the naturally occurring endorhiza AMF and bacteria. WCR root feeding influenced the endorhiza bacteria as well. GI can be used in integrated pest management programs, rendering WCR larvae more susceptible to predation by natural enemies. The mechanisms behind the interaction between GI and WCR remain unknown. However, our data suggested that GI might act indirectly via plant-mediated mechanisms influencing the endorhiza microbial communities.

Microbial communities associated with the larval gut and eggs of the Western corn rootworm.

BACKGROUND: The western corn rootworm (WCR) is one of the economically most important pests of maize. A better understanding of microbial communities associated with guts and eggs of the WCR is required in order to develop new pest control strategies, and to assess the potential role of the WCR in the dissemination of microorganisms, e.g., mycotoxin-producing fungi. METHODOLOGY/PRINCIPAL FINDINGS: Total community (TC) DNA was extracted from maize rhizosphere, WCR eggs, and guts of larvae feeding on maize roots grown in three different soil types. Denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene and ITS fragments, PCR-amplified from TC DNA, were used to investigate the fungal and bacterial communities, respectively. Microorganisms in the WCR gut were not influenced by the soil type. Dominant fungal populations in the gut were affiliated to Fusarium spp., while Wolbachia was the most abundant bacterial genus. Identical ribosomal sequences from gut and egg samples confirmed a transovarial transmission of Wolbachia sp. Betaproteobacterial DGGE indicated a stable association of Herbaspirillum sp. with the WCR gut. Dominant egg-associated microorganisms were the bacterium Wolbachia sp. and the fungus Mortierella gamsii. CONCLUSION/SIGNIFICANCE: The soil type-independent composition of the microbial communities in the WCR gut and the dominance of only a few microbial populations suggested either a highly selective environment in the gut lumen or a high abundance of intracellular microorganisms in the gut epithelium. The dominance of Fusarium species in the guts indicated WCR larvae as vectors of mycotoxin-producing fungi. The stable association of Herbaspirillum sp. with WCR gut systems and the absence of corresponding sequences in WCR eggs suggested that this bacterium was postnatally acquired from the environment. The present study provided new insights into the microbial communities associated with larval guts and eggs of the WCR. However, their biological role remains to be explored.

Multitrophic interaction in the rhizosphere of maize: root feeding of Western corn rootworm larvae alters the microbial community composition.

BACKGROUND: Larvae of the Western Corn Rootworm (WCR) feeding on maize roots cause heavy economical losses in the US and in Europe. New or adapted pest management strategies urgently require a better understanding of the multitrophic interaction in the rhizosphere. This study aimed to investigate the effect of WCR root feeding on the microbial communities colonizing the maize rhizosphere. METHODOLOGY/PRINCIPAL FINDINGS: In a greenhouse experiment, maize lines KWS13, KWS14, KWS15 and MON88017 were grown in three different soil types in presence and in absence of WCR larvae. Bacterial and fungal community structures were analyzed by denaturing gradient gel electrophoresis (DGGE) of the 16S rRNA gene and ITS fragments, PCR amplified from the total rhizosphere community DNA. DGGE bands with increased intensity were excised from the gel, cloned and sequenced in order to identify specific bacteria responding to WCR larval feeding. DGGE fingerprints showed that the soil type and the maize line influenced the fungal and bacterial communities inhabiting the maize rhizosphere. WCR larval feeding affected the rhiyosphere microbial populations in a soil type and maize line dependent manner. DGGE band sequencing revealed an increased abundance of Acinetobacter calcoaceticus in the rhizosphere of several maize lines in all soil types upon WCR larval feeding. CONCLUSION/SIGNIFICANCE: The effects of both rhizosphere and WCR larval feeding seemed to be stronger on bacterial communities than on fungi. Bacterial and fungal community shifts in response to larval feeding were most likely due to changes of root exudation patterns. The increased abundance of A. calcoaceticus suggested that phenolic compounds were released upon WCR wounding.

Identification of Race 1 of Fusarium oxysporum f. sp. lactucae on Lettuce by Inter-Retrotransposon Sequence-Characterized Amplified Region Technique.

ABSTRACT Fusarium wilt of lettuce, caused worldwide by Fusarium oxysporum f. sp. lactucae, is an emerging seed-transmitted disease on Lactuca sativa. In order to develop a molecular diagnostic tool for identifying race 1 (VCG0300) of the pathogen on vegetable samples, an effective technique is presented. Inter-retrotransposon amplified polymorphism polymerase chain reaction (PCR), a technique based on the amplification of genomic regions between long terminal repeats, was applied. It was shown to be useful for grouping F. oxysporum f. sp. lactucae race 1 isolates. Inter-retrotransposon sequence-characterized amplified regions (IR-SCAR) was used to develop a specific set of PCR primers to be utilized for differentiating F. oxysporum f. sp. lactucae isolates from other F. oxysporum isolates. The specific primers were able to uniquely amplify fungal genomic DNA from race 1 isolates obtained in Italy, Portugal, the United States, Japan, and Taiwan. The primers also were specific to pathogen DNA obtained from artificially infected lettuce seed and naturally and artificially infected plants.

Vegetative Compatibility Groups of Fusarium oxysporum f. sp. lactucae from Lettuce.

Fusarium oxysporum f. sp. lactucae, the causal agent of Fusarium wilt of lettuce, has been reported in three continents in the last 10 years. Forty-seven isolates obtained from infected plants and seed in Italy, the United States, Japan, and Taiwan were evaluated for pathogenicity and vegetative compatibility. Chlorate-resistant, nitrate-nonutilizing mutants were used to determine genetic relatedness among isolates from different locations. Using the vegetative compatibility group (VCG) approach, all Italian and American isolates, type 2 Taiwanese isolates, and a Japanese race 1 were assigned to the major VCG 0300. Taiwanese isolates type 1 were assigned to VCG 0301. The hypothesis that propagules of Fusarium oxysporum f. sp. lactucae that caused epidemics on lettuce in 2001-02 in Italian fields might have spread via import and use of contaminated seeds is discussed.