Corrigendum: Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

[This corrects the article DOI: 10.3389/fmicb.2024.1334268.].

Rapid typing of Klebsiella pneumoniae and Pseudomonas aeruginosa by Fourier-transform Infrared spectroscopy informs infection control in veterinary settings.

Introduction: The emergence of multi-drug resistant (MDR) pathogens linked to healthcare-associated infections (HCAIs) is an increasing concern in modern veterinary practice. Thus, rapid bacterial typing for real-time tracking of MDR hospital dissemination is still much needed to inform best infection control practices in a clinically relevant timeframe. To this end, the IR Biotyper using Fourier-Transform InfraRed (FTIR) spectroscopy has the potential to provide fast cluster analysis of potentially related organisms with substantial cost and turnaround time benefits. Materials and methods: A collection of MDR bacterial isolates (n = 199, comprising 92 Klebsiella pneumoniae and 107 Pseudomonas aeruginosa) obtained from companion animal (i.e., dogs, cats and horses) clinical investigations, faecal and environmental screening from four veterinary facilities between 2012 and 2019 was analysed retrospectively by FTIR spectroscopy. Its performance was compared against MLST extracted from whole genomes of a subset of clustering isolates (proportionally to cluster size) for investigation of potential nosocomial transmission between patients and the surrounding hospital environments. Results: Concordance between the FTIR and MLST types was overall high for K. pneumoniae (Adjusted Rand Index [ARI] of 0.958) and poor for P. aeruginosa (ARI of 0.313). FTIR K. pneumoniae clusters (n = 7) accurately segregated into their respective veterinary facility with evidence of intra-hospital spread of K. pneumoniae between patients and environmental surfaces. Notably, K. pneumoniae ST147 intensely circulated at one Small Animal Hospital ICU. Conversely, Pseudomonas aeruginosa FTIR clusters (n = 18) commonly contained isolates of diversified hospital source and heterogeneous genetic background (as also genetically related isolates spread across different clusters); nonetheless, dissemination of some clones, such as P. aeruginosa ST2644 in the equine hospital, was apparent. Importantly, FTIR clustering of clinical, colonisation and/or environmental isolates sharing genomically similar backgrounds was seen for both MDR organisms, highlighting likely cross-contamination events that led to clonal dissemination within settings. Conclusion: FTIR spectroscopy has high discriminatory power for hospital epidemiological surveillance of veterinary K. pneumoniae and could provide sufficient information to support early detection of clonal dissemination, facilitating implementation of appropriate infection control measures. Further work and careful optimisation need to be carried out to improve its performance for typing of P. aeruginosa veterinary isolates.

Fourier transform infrared spectroscopy; can it be used as a rapid typing method of Neisseria gonorrhoeae?

BACKGROUND: Typing of Neisseria gonorrhoeae is necessary for epidemiologic surveillance, while time consuming and resource intensive. Fourier transform infrared (FTIR) spectroscopy has shown promising results when typing several bacterial species. This study investigates whether FTIR spectroscopy can be used as a rapid method for typing clinical N. gonorrhoeae isolates, comparing FTIR spectroscopy to multi locus sequence typing (MLST), N. gonorrhoeae multi antigen sequence typing (NG-MAST) and whole genome sequencing (WGS). METHODS: Sixty consecutive isolates from a venereology clinic and three isolates from an outbreak were included. Isolates were analysed with FTIR spectroscopy on the IR Biotyper system (Bruker Daltonik) with the IR Biotyper software (version 2.1) with default analysis settings (spectral range 1300-800 cm-1). Four technical replicates of each isolate were analysed in three different runs. The output was a hierarchical cluster analysis (HCA) presented as a dendrogram; a tree-like overview of how closely different isolates are related. FTIR spectroscopy was compared to MLST, NG-MAST and WGS to see if the FTIR spectroscopy-dendrogram grouped the isolates in the same clusters. RESULTS: Fifty-one out of 60 isolates, and the three outbreak isolates, produced at least one spectrum in each run and were included. No agreement between FTIR spectroscopy and MLST or NG-MAST or WGS was shown. The FTIR spectroscopy-dendrogram failed to cluster the outbreak isolates. CONCLUSION: FTIR spectroscopy (spectral range 1300-800 cm-1) is not yet suitable for epidemiologic typing of N. gonorrhoeae. Absence of a capsule as well as phase- and antigenic variation of carbohydrate surface structures of the gonococcal cell wall may contribute to our findings. Future studies should include analysis of a wider range of the spectrum recorded (4000-500 cm-1), and should also explore further mathematical analytic approaches of the similarity between spectra.

Machine learning-based typing of Salmonella enterica O-serogroups by the Fourier-Transform Infrared (FTIR) Spectroscopy-based IR Biotyper system.

BACKGROUND: Salmonella enterica is among the major burdens for public health at global level. Typing of salmonellae below the species level is fundamental for different purposes, but traditional methods are expensive, technically demanding, and time-consuming, and therefore limited to reference centers. Fourier transform infrared (FTIR) spectroscopy is an alternative method for bacterial typing, successfully applied for classification at different infra-species levels. AIM: This study aimed to address the challenge of subtyping Salmonella enterica at O-serogroup level by using FTIR spectroscopy. We applied machine learning to develop a novel approach for S. enterica typing, using the FTIR-based IR Biotyper® system (IRBT; Bruker Daltonics GmbH & Co. KG, Germany). We investigated a multicentric collection of isolates, and we compared the novel approach with classical serotyping-based and molecular methods. METHODS: A total of 958 well characterized Salmonella isolates (25 serogroups, 138 serovars), collected in 11 different centers (in Europe and Japan), from clinical, environmental and food samples were included in this study and analyzed by IRBT. Infrared absorption spectra were acquired from water-ethanol bacterial suspensions, from culture isolates grown on seven different agar media. In the first part of the study, the discriminatory potential of the IRBT system was evaluated by comparison with reference typing method/s. In the second part of the study, the artificial intelligence capabilities of the IRBT software were applied to develop a classifier for Salmonella isolates at serogroup level. Different machine learning algorithms were investigated (artificial neural networks and support vector machine). A subset of 88 pre-characterized isolates (corresponding to 25 serogroups and 53 serovars) were included in the training set. The remaining 870 samples were used as validation set. The classifiers were evaluated in terms of accuracy, error rate and failed classification rate. RESULTS: The classifier that provided the highest accuracy in the cross-validation was selected to be tested with four external testing sets. Considering all the testing sites, accuracy ranged from 97.0% to 99.2% for non-selective media, and from 94.7% to 96.4% for selective media. CONCLUSIONS: The IRBT system proved to be a very promising, user-friendly, and cost-effective tool for Salmonella typing at serogroup level. The application of machine learning algorithms proved to enable a novel approach for typing, which relies on automated analysis and result interpretation, and it is therefore free of potential human biases. The system demonstrated a high robustness and adaptability to routine workflows, without the need of highly trained personnel, and proving to be suitable to be applied with isolates grown on different agar media, both selective and unselective. Further tests with currently circulating clinical, food and environmental isolates would be necessary before implementing it as a potentially stand-alone standard method for routine use.

Classification of Salmonella enterica of the (Para-)Typhoid Fever Group by Fourier-Transform Infrared (FTIR) Spectroscopy.

Typhoidal and para-typhoidal Salmonella are major causes of bacteraemia in resource-limited countries. Diagnostic alternatives to laborious and resource-demanding serotyping are essential. Fourier transform infrared spectroscopy (FTIRS) is a rapidly developing and simple bacterial typing technology. In this study, we assessed the discriminatory power of the FTIRS-based IR Biotyper (Bruker Daltonik GmbH, Bremen, Germany), for the rapid and reliable identification of biochemically confirmed typhoid and paratyphoid fever-associated Salmonella isolates. In total, 359 isolates, comprising 30 S. Typhi, 23 S. Paratyphi A, 23 S. Paratyphi B, and 7 S. Paratyphi C, respectively and other phylogenetically closely related Salmonella serovars belonging to the serogroups O:2, O:4, O:7 and O:9 were tested. The strains were derived from clinical, environmental and food samples collected at different European sites. Applying artificial neural networks, specific automated classifiers were built to discriminate typhoidal serovars from non-typhoidal serovars within each of the four serogroups. The accuracy of the classifiers was 99.9%, 87.0%, 99.5% and 99.0% for Salmonella Typhi, Salmonella Paratyphi A, B and Salmonella Paratyphi C, respectively. The IR Biotyper is a promising tool for fast and reliable detection of typhoidal Salmonella. Hence, IR biotyping may serve as a suitable alternative to conventional approaches for surveillance and diagnostic purposes.

Evaluation of Microsatellite Typing, ITS Sequencing, AFLP Fingerprinting, MALDI-TOF MS, and Fourier-Transform Infrared Spectroscopy Analysis of Candida auris.

Candida auris is an emerging opportunistic yeast species causing nosocomial outbreaks at a global scale. A few studies have focused on the C. auris genotypic structure. Here, we compared five epidemiological typing tools using a set of 96 C. auris isolates from 14 geographical areas. Isolates were analyzed by microsatellite typing, ITS sequencing, amplified fragment length polymorphism (AFLP) fingerprint analysis, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), and Fourier-transform infrared (FTIR) spectroscopy methods. Microsatellite typing grouped the isolates into four main clusters, corresponding to the four known clades in concordance with whole genome sequencing studies. The other investigated typing tools showed poor performance compared with microsatellite typing. A comparison between the five methods showed the highest agreement between microsatellite typing and ITS sequencing with 45% similarity, followed by microsatellite typing and the FTIR method with 33% similarity. The lowest agreement was observed between FTIR spectroscopy, MALDI-TOF MS, and ITS sequencing. This study indicates that microsatellite typing is the tool of choice for C. auris outbreak investigations. Additionally, FTIR spectroscopy requires further optimization and evaluation before it can be used as an epidemiological typing method, comparable with microsatellite typing, as a rapid method for tracing nosocomial fungal outbreaks.

The Travelling Particles: Investigating microplastics as possible transport vectors for multidrug resistant E. coli in the Weser estuary (Germany).

The prevalence of multidrug-resistant Gram-negative bacteria in aquatic environments has been a long withstanding health concern, namely extended-spectrum beta-lactamase (ESBL) producing Escherichia coli. Given increasing reports on microplastic (MP) pollution in these environments, it has become crucial to better understand the role of MP particles as transport vectors for such multidrug-resistant bacteria. In this study, an incubation experiment was designed where particles of both synthetic and natural material (HDPE, tyre wear, and wood) were sequentially incubated at multiple sites along a salinity gradient from the Lower Weser estuary (Germany) to the offshore island Helgoland (German Bight, North Sea). Following each incubation period, particle biofilms and water samples were assessed for ESBL-producing E. coli, first by the enrichment and detection of E. coli using Fluorocult® LMX Broth followed by cultivation on CHROMAgar™ ESBL media to select for ESBL-producers. Results showed that general E. coli populations were present on the surfaces of wood particles across all sites but none were found to produce ESBLs. Additionally, neither HDPE nor tyre wear particles were found to harbour any E. coli. Conversely, ESBL-producing E. coli were present in surrounding waters from all sites, 64% of which conferred resistances against up to 3 other antibiotic groups, additional to the beta-lactam resistances intrinsic to ESBL-producers. This study provides a first look into the potential of MP to harbour and transport multidrug-resistant E. coli across different environments and the approach serves as an important precursor to further studies on other potentially harmful MP-colonizing species.

Analysis of Streptococcus pneumoniae using Fourier-transformed infrared spectroscopy allows prediction of capsular serotype.

Determination of the capsule type of clinical isolates of Streptococcus pneumoniae is a prerequisite for epidemiological studies and further vaccine development. The Quellung reaction for serotyping is expensive and mostly done in reference centres. We wanted to evaluate whether Fourier-transformed infrared (FT-IR) spectroscopy is suitable for capsular type analysis and prediction of pneumococcal serotypes. We used the IR-Biotyper™ (Bruker) to create a database containing the spectra of 120 strains from invasive disease. The strains covered the 24 vaccine serotypes contained in the 13-valent conjugate vaccine (PCV13) and the 23-valent polysaccharide vaccine (PSV23). Hierarchical clustering analysis was performed. Finally, two different classification sets were created (PCV13 and PSV23). They were used to predict the serotype of 168 different challenge strains (invasive and non-invasive disease) covering 48 different serotypes (vaccine and non-vaccine types). FT-IR spectra from pneumococci (1300-800 cm-1) clustered along their serotype as determined by the Quellung reaction (120 strains, 24 different serotypes). Strains with unknown serotype fell within the cluster of the correct serotype, as long as the latter was represented in the database (168 strains, 48 different serotypes). Concordance between the Quellung reaction and FT-IR spectroscopy was excellent (kappa ≥ 0.75). FT-IR spectroscopy is a fast and cost-effective method to predict the capsular serotype of pneumococci.

Typing and Species Identification of Clinical Klebsiella Isolates by Fourier Transform Infrared Spectroscopy and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Klebsiella pneumoniae and related species are frequent causes of nosocomial infections and outbreaks. Therefore, quick and reliable strain typing is crucial for the detection of transmission routes in the hospital. The aim of this study was to evaluate Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as rapid methods for typing clinical Klebsiella isolates in comparison to whole-genome sequencing (WGS), which was considered the gold standard for typing and identification. Here, 68 clinical Klebsiella strains were analyzed by WGS, FTIR, and MALDI-TOF MS. FTIR showed high discriminatory power in comparison to the WGS reference, whereas MALDI-TOF MS exhibited a low ability to type the isolates. MALDI-TOF mass spectra were further analyzed for peaks that showed high specificity for different Klebsiella species. Phylogenetic analysis revealed that the Klebsiella isolates comprised three different species: K. pneumoniae, K. variicola, and K. quasipneumoniae Genome analysis showed that MALDI-TOF MS can be used to distinguish K. pneumoniae from K. variicola due to shifts of certain mass peaks. The peaks were tentatively identified as three ribosomal proteins (S15p, L28p, L31p) and one stress response protein (YjbJ), which exhibit amino acid differences between the two species. Overall, FTIR has high discriminatory power to recognize the clonal relationship of isolates, thus representing a valuable tool for rapid outbreak analysis and for the detection of transmission events due to fast turnaround times and low costs per sample. Furthermore, specific amino acid substitutions allow the discrimination of K. pneumoniae and K. variicola by MALDI-TOF MS.

Genotyping of German and Austrian Taylorella equigenitalis isolates using repetitive extragenic palindromic (REP) PCR and pulsed-field gel electrophoresis (PFGE).

A total of 124 Taylorella (T.) equigenitalis and five T. asinigenitalis field isolates collected between 2002 and 2014 were available for genotyping using REP- (repetitive extragenic palindromic) PCR and PFGE (pulsed-field gel electrophoresis). The study comprised 79 T. equigenitalis field isolates originating from ten defined breeds of German horses and revealed a spectrum of five REP (rep-E1-E4, rep-E3a) and 15 PFGE (TE-A1-A9, TE-B1-B3, TE-C, TE-E1, and TE-E2) genotypes. T. equigenitalis field isolates (n=40) obtained from Austrian Lipizzaner horses were differentiated into three REP (rep-E1, rep-E3a, and rep-E4) and three PFGE genotypes (TE-A2, TE-A5, and TE-D); those isolated from four Austrian Trotters belonged to the REP/PFGE genotype rep-E2/TE-A1. Interestingly, a T. equigenitalis isolate recovered from a Holsteiner stallion living in South Africa revealed the REP/PFGE genotype rep-E1/TE-A5 which was otherwise exclusively present in the majority of Austrian Lipizzaner horses in our study. The type strain included in this study revealed the genotype REP/PFGE rep-E1/TE-F. Six strains of T. asinigenitalis including the type strain were separated into three REP (rep-A1-A3) and six PFGE genotypes (TA-A1, TA-A2, TA-A3, TA-B, TA-C, TA-D). Overall, the generated REP and PFGE genotypes showed a good correlation, whereas REP-PCR proved to be a suitable method for molecular epidemiological screening of T. equigenitalis and T. asinigenitalis isolates that should be differentiated in detail by genotyping using PFGE.

Corynebacterium diphtheriae in a free-roaming red fox: case report and historical review on diphtheria in animals.

OBJECTIVES: Corynebacterium diphtheriae, the classical causative agent of diphtheria, is considered to be nearly restricted to humans. Here we report the first finding of a non-toxigenic C. diphtheriae biovar belfanti strain in a free-roaming wild animal. METHODS: The strain obtained from the subcutis and mammary gland of a dead red fox (Vulpes vulpes) was characterized by biochemical and molecular methods including MALDI-TOF and Multi Locus Sequence Typing. Since C. diphtheriae infections of animals, usually with close contact to humans, are reported only very rarely, an intense review comprising also scientific literature from the beginning of the 20th century was performed. RESULTS: Besides the present case, only 11 previously reported C. diphtheriae animal infections could be verified using current scientific criteria. CONCLUSIONS: Our report is the first on the isolation of C. diphtheriae from a wildlife animal without any previous human contact. In contrast, the very few unambiguous publications on C. diphtheriae in animals referred to livestock or pet animals with close human contact. C. diphtheriae carriage in animals has to be considered as an exceptionally rare event.

Streptobacillus notomytis sp. nov., isolated from a spinifex hopping mouse (Notomys alexis Thomas, 1922), and emended description of Streptobacillus Levaditi et al. 1925, Eisenberg et al. 2015 emend.

A pleomorphic, Gram-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium was isolated in 1979 from the heart of a spinifex hopping mouse (Notomys alexis Thomas, 1922) with septicaemia and stored as Streptobacillus moniliformis in the strain collection of the Animal Health Laboratory, South Perth, Western Australia (AHL 370-1), as well as under CCUG 12425. On the basis of 16SrRNA gene sequence analyses, the strain was assigned to the genus Streptobacillus, with 99.4 % sequence similarity to the type strain of Streptobacillus moniliformis, 95.6 %sequence similarity to the type strain of Streptobacillus hongkongensis and 99.0 %sequence similarity to the type strain of Streptobacillus felis. The clear differentiation of strain AHL 370-1T from Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis was also supported by rpoB, groEL and recA nucleotide and amino acid sequence analysis. Average nucleotide identity was 87.16 % between strain AHL 370-1T and Streptobacillus moniliformis DSM 12112T. Physiological data confirmed the allocation of strain AHL 370-1T to the family Leptotrichiaceae, considering the very similar profiles of enzyme activities and fatty acids compared to closely related species. Within the genus Streptobacillus,isolate AHL 370-1T could also be separated unambiguously from the type strains of Streptobacillus moniliformis, Streptobacillus hongkongensis and Streptobacillus felis by MALDI-TOF mass spectrometry. Two further strains (KWG2 and KWG24) isolated from asymptomatic black rats in Japan were highly similar to AHL 370-1T. On the basis of these data, we propose the novel species Streptobacillus notomytis sp. nov., with the type strain AHL370-1T (=CCUG 12425T=DSM 100026T=CCM 8593T=EF 12425T).

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus.

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

Toxigenic Corynebacterium ulcerans isolated from a free-roaming red fox (Vulpes vulpes).

Corynebacterium (C.) ulcerans could be isolated from the spleen of a red fox (Vulpes vulpes) that had been found dead in the state of Baden-Württemberg, Germany. Pathohistological examination suggested that the fox had died of distemper, as was confirmed by PCR. The isolate was identified biochemically, by MALDI-TOF MS, FT-IR and by partial 16S rRNA, rpoB and tox gene sequencing. Using the Elek test the C. ulcerans isolate demonstrated diphtheria toxin production. FT-IR and sequencing data obtained from the C. ulcerans isolate from the red fox showed higher similarity to isolates from humans than to those from wild game.

Streptobacillus felis sp. nov., isolated from a cat with pneumonia, and emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis.

A pleomorphic, Gram-stain-negative, rod-shaped, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile bacterium (strain 131000547(T)) was isolated from the lungs of a cat with pneumonia. On the basis of 16S rRNA gene sequence analyses the strain was assigned to the genus Streptobacillus with 97.6% sequence similarity to the type strain of Streptobacillus moniliformis and 94.6% to that of Streptobacillus hongkongensis. The clear differentiation of strain 131000547(T) from Streptobacillus moniliformis and Streptobacillus hongkongensis was also supported by gyrB, groEL, and recA nucleotide and amino acid sequence analysis. DNA-DNA hybridization demonstrated ≤ 19.9% (reciprocal 28.7%) DNA-DNA relatedness between strain 131000547(T) and Streptobacillus moniliformis DSM 12112(T). Physiological data confirmed the allocation of strain 131000547(T) to the family Leptotrichiaceae. Strain 131000547(T) has a unique profile of enzyme activities allowing differentiation from the most closely related species. Within the genus Streptobacillus, isolate 131000547(T) could also unambiguously be separated from Streptobacillus moniliformis and Streptobacillus hongkongensis by both matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Fourier transform-infrared spectroscopy. On the basis of these data, a novel species of the genus Streptobacillus, Streptobacillus felis sp. nov., is proposed with the type strain 131000547(T) ( = DSM 29248(T) = CCUG 66203(T) = CCM 8542(T)). Emended descriptions of the genus Streptobacillus and of Streptobacillus moniliformis are also given.

Outbreak with clonally related isolates of Corynebacterium ulcerans in a group of water rats.

BACKGROUND: The zoonotic bacterium Corynebacterium ulcerans may be pathogenic both in humans and animals: toxigenic strains can cause diphtheria or diphtheria-like disease in humans via diphtheria toxin, while strains producing the dermonecrotic exotoxin phospholipase D may lead to caseous lymphadenitis primarily in wild animals. Diphtheria toxin-positive Corynebacterium ulcerans strains have been isolated mainly from cattle, dogs and cats. RESULTS: Here, we report a series of ten isolations of Corynebacterium ulcerans from a group of water rats (Hydromys chrysogaster) with ulcerative skin lesions, which were kept in a zoo. The isolates were clearly assigned to species level by biochemical identification systems, Fourier-transform infrared-spectroscopy, Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and partial rpoB sequencing, respectively. All ten isolates turned out to represent the same sequence type, strongly indicating a cluster of infections by clonally-related isolates as could be demonstrated for the first time for this species using multilocus sequence typing. Unequivocal demonstration of high relatedness of the isolates could also be demonstrated by Fourier-transform infrared-spectroscopy. All isolates were lacking the diphtheria toxin encoding tox-gene, but were phospholipase D-positive. CONCLUSIONS: Our results indicate that water rats represent a suitable new host species that is prone to infection and must be regarded as a reservoir for potentially zoonotic Corynebacterium ulcerans. Furthermore, the applied methods demonstrated persistent infection as well as a very close relationship between all ten isolates.

The cell envelope stress response mediated by the LiaFSRLm three-component system of Listeria monocytogenes is controlled via the phosphatase activity of the bifunctional histidine kinase LiaSLm.

Most members of the phylum Firmicutes harbour a two-component system (TCS), LiaSR, which is involved in the response to cell envelope stress elicited most notably by inhibitors of the lipid II cycle. In all LiaSR systems studied in detail, LiaSR-mediated signal transduction has been shown to be negatively controlled by a membrane protein, LiaF, encoded upstream of liaSR. In this study we have analysed the LiaSR orthologue of Listeria monocytogenes (LiaSR(Lm)). Whole-genome transcriptional profiling indicated that activation of LiaSR(Lm) results in a remodelling of the cell envelope via the massive upregulation of membrane-associated and extracytoplasmic proteins in the presence of inducing stimuli. As shown for other LiaSR TCSs, LiaSR(Lm) is activated by cell wall-active antibiotics. We demonstrate that the level of phosphorylated LiaR(Lm), which is required for the induction of the LiaSR(Lm) regulon, is controlled by the interplay between the histidine kinase and phosphatase activities of the bifunctional sensor protein LiaS(Lm). Our data suggest that the phosphatase activity of LiaS(Lm) is stimulated by LiaF(Lm) in the absence of cell envelope stress.

Response regulator DegU of Listeria monocytogenes controls temperature-responsive flagellar gene expression in its unphosphorylated state.

We demonstrate that in Listeria monocytogenes, temperature-responsive transcriptional control of flagellar genes does not rely on the phosphorylation of the conserved phosphorylation site (D55) in the receiver domain of response regulator DegU. Furthermore, proper control of DegU-regulated genes involved in ethanol tolerance and virulence is independent of receiver phosphorylation.

Species-specific differences in the activity of PrfA, the key regulator of listerial virulence genes.

PrfA, the master regulator of LIPI-1, is indispensable for the pathogenesis of the human pathogen Listeria monocytogenes and the animal pathogen Listeria ivanovii. PrfA is also present in the apathogenic species Listeria seeligeri, and in this study, we elucidate the differences between PrfA proteins from the pathogenic and apathogenic species of the genus Listeria. PrfA proteins of L. monocytogenes (PrfA(Lm) and PrfA*(Lm)), L. ivanovii (PrfA(Li)), and L. seeligeri (PrfA(Ls)) were purified, and their equilibrium constants for binding to the PrfA box of the hly promoter (Phly(Lm)) were determined by surface plasmon resonance. In addition, the capacities of these PrfA proteins to bind to the PrfA-dependent promoters Phly and PactA and to form ternary complexes together with RNA polymerase were analyzed in electrophoretic mobility shift assays, and their abilities to initiate transcription in vitro starting at these promoters were compared. The results show that PrfA(Li) resembled the constitutively active mutant PrfA*(Lm) more than the wild-type PrfA(Lm), whereas PrfA(Ls) showed a drastically reduced capacity to bind to the PrfA-dependent promoters Phly and PactA. In contrast, the efficiencies of PrfA(Lm), PrfA*(Lm), and PrfA(Li) forming ternary complexes and initiating transcription at Phly and PactA were rather similar, while those of PrfA(Ls) were also much lower. The low binding and transcriptional activation capacities of PrfA(Ls) seem to be in part due to amino acid exchanges in its C-terminal domain (compared to PrfA(Lm) and PrfA(Li)). In contrast to the significant differences in the biochemical properties of PrfA(Lm), PrfA(Li), and PrfA(Ls), the PrfA-dependent promoters of hly (Phly(Lm), Phly(L)(i), and Phly(L)(s)) and actA (PactA(Lm), PactA(L)(i), and PactA(L)(s)) of the three Listeria species did not significantly differ in their binding affinities to the various PrfA proteins and in their strengths to promote transcription in vitro. The allelic replacement of prfA(Lm) with prfA(Ls) in L. monocytogenes leads to low expression of PrfA-dependent genes and to reduced in vivo virulence of L. monocytogenes, suggesting that the altered properties of PrfA(Ls) protein are a major cause for the low virulence of L. seeligeri.

Necrotic death of Rhodococcus equi-infected macrophages is regulated by virulence-associated plasmids.

Rhodococcus equi is a gram-positive intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. It has been reported that advanced infection of foals is characterized by tissue necrosis, coinciding with the presence of degenerate bacteria-laden macrophages. Here, we report that the possession of the VapA-expressing plasmid, which has been previously correlated with a high level of virulence for foals and mice, strongly increases cytotoxicity of R. equi for murine macrophage-like (J774E) cells. Isolates containing different, VapB-expressing plasmids are less virulent and also have a lower cytotoxic potential. Isogenic strains lacking either plasmid are avirulent and have a very low cytotoxic potential. We show, using fluorescence-activated cell sorter analysis (annexin V/7-amino-actinomycin D and sub-G1-analysis), Western blotting [poly(ADP-ribose) polymerase processing analysis], and electron microscopy (macrophage and nucleus morphologies) that the deaths of murine macrophages are the result of necrotic rather than apoptotic events. We demonstrate that the bacteria must be alive in order to act cytotoxic. Therefore, one effect of the virulence-associated plasmids during infection with R. equi is the promotion of necrotic damage to the host.