Species- and strain-specific differences in the phagocytosis of Prototheca: insights from live-cell imaging.

The genus Prototheca is an extremely unusual group of achlorophyllic, obligately heterotrophic algae. Six species have been identified as pathogens of vertebrates, including cattle and humans. In cattle, P. bovis is the main infectious pathogen and is associated with bovine mastitis. In contrast, human infections typically involve P. wickerhamii and are associated with a spectrum of varying clinical presentations. Prototheca spp. enter the host from the environment and are therefore likely to be initially recognized by cells of the innate immune system. However, little is known about the nature of the interaction between Prototheca spp. and host phagocytes. In the present study, we adopt a live-cell imaging approach to investigate these interactions over time. Using environmental and clinical strains, we show that P. bovis cells are readily internalized and processed by macrophages, whereas these immune cells struggle to internalize P. wickerhamii. Serum opsonization of P. wickerhamii only marginally improves phagocytosis, suggesting that this species (but not P. bovis) may have evolved mechanisms to evade phagocytosis. Furthermore, we show that inhibition of the kinases Syk or PI3K, which are both critical for innate immune signaling, drastically reduces the uptake of P. bovis. Finally, we show that genetic ablation of MyD88, a signaling adaptor critical for Toll-like receptor signaling, has little impact on uptake but significantly prolongs phagosome maturation once P. bovis is internalized. Together, our data suggest that these two pathogenic Prototheca spp. have very different host-pathogen interactions which have potential therapeutic implications for the treatment of human and animal disease.

Drug Susceptibility Profiling of Prototheca Species Isolated from Cases of Human Protothecosis.

Prototheca are unicellular, achlorophyllous, yeast-like microalgae that occur in a wide range of natural habitats. At least five species have been implicated as the causative agents of opportunistic infections of men. Human protothecosis typically manifests as cutaneous, articular, or systemic disease. Treatment is largely empirical with poorly predictable and often unsuccessful outcomes. This is largely due to the frequently observed resistance of Prototheca species to conventional antimicrobial agents. This work is the first to perform drug susceptibility profiling exclusively on isolates from human cases of protothecosis. A total of 23 such isolates were tested against amphotericin B and 9 azoles, including efinaconazole and luliconazole, whose activities against Prototheca have never been studied before. Efinaconazole was the most active, with median minimum inhibitory concentration (MIC) and minimum algicidal concentration (MAC) values of 0.031 mg/L and 0.063 mg/L, respectively. Fluconazole and luliconazole had the lowest activity, with median MIC and MAC values of 128 mg/L. To conclude, amphotericin B and most of the azoles showed in vitro activity, with an algicidal rather than algistatic effect, against Prototheca. Still, the activity of individual drugs differed significantly between the species and even between strains of the same species. These differences can be attributed to a species-specific potential for acquiring drug resistance, which, in turn, might be linked to the treatment history of the patient from whom the strain was recovered. The results of this study underscore the potential clinical utility of efinaconazole as a promising therapeutic agent for the treatment of human protothecosis.

Intralesional amphotericin B in a cat with cutaneous protothecosis.

A domestic cat was presented with nodular lesions on the nose/muzzle and pinnae. Protothecosis was diagnosed through cytological and histopathological examination, and culture. Molecular identification confirmed Prototheca wickerhamii infection. Intralesional application of amphotericin B in conjunction with oral terbinafine resulted in a significant reduction of the nasal lesion and complete resolution of the pinnal lesion, without adverse effects.

Un chat domestique est présenté avec des lésions nodulaires sur le nez/museau et le pavillon auriculaire. La protothécose est diagnostiquée par un examen cytologique et histopathologique, ainsi que par une culture. L'identification moléculaire confirme l'infection par Prototheca wickerhamii. L'application intralésionnelle d'amphotéricine B, associée à la terbinafine orale, permet une réduction significative de la lésion nasale et une résolution complète de la lésion du pavillon auriculaire, sans effets indésirables.

Um gato doméstico foi apresentado com lesões nodulares no nariz/focinho e pavilhões auriculares. Prototecose foi diagnosticada por exame citológico e histopatológico, e cultura. A identificação molecular confirmou a infecção por Prototheca wickerhamii. Aplicação intralesional de anfotericina B associada à terbinafina por via oral resultou em redução significativa da lesão nasal e resolução total da lesão na orelha, sem efeitos adversos.

Un gato doméstico se presentó con lesiones nodulares en la nariz/hocico y orejas. Se diagnosticó prototecosis mediante examen citológico, histopatológico y cultivo. La identificación molecular confirmó la infección por Prototheca wickerhamii. La aplicación intralesional de anfotericina B junto con terbinafina oral dio como resultado una reducción significativa de la lesión nasal y una resolución completa de la lesión auricular, sin efectos adversos.

Occurrence of Prototheca Microalgae in Aquatic Ecosystems with a Description of Three New Species, Prototheca fontanea, Prototheca lentecrescens, and Prototheca vistulensis.

Prototheca species are unicellular, nonphotosynthetic, saprophytic, and occasionally pathogenic, microalgae, with an extensive environmental reservoir. This study explores, for the first time, the occurrence of Prototheca in aquatic ecosystems by using a molecular profiling approach. A total of 362 samples were collected from 80 natural and artificial waterbodies at 88 sampling sites in 26 localities across Poland during a 1.5-year period. The overall isolation rate of Prototheca from water environments was 14.1%. Prototheca were most prevalent in rivers of urbanized areas, indicating that the algae are primarily adapted to lotic ecosystems with a high input of organic matter. Interestingly, it is not the amount of organic matter per se but its quality that seems to shape the habitat potential of the protothecae. The two most frequently isolated species were P. wickerhamii and P. pringsheimii, representing a third and a fourth of the strains, respectively. Additionally, three novel species were described, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The high species diversity of the genus Prototheca may reflect the complexity of water ecosystems along with ecological and functional adaptations of the algae to such environments. For further investigations, the study provides a revised scheme for identification of all 18 Prototheca species currently recognized. IMPORTANCE The study investigates the occurrence of very rare and poorly studied microalgae of the genus Prototheca, potentially pathogenic to humans and animals, in different water environments. Given the potential hazard to human and animal health from exposure to water-inhabiting protothecae, the prevalence of the algae in aquatic habitats deserves an insightful examination. The study is the first since the 1980s to explore the aquatic habitat of Prototheca spp. and the first ever performed to do this by molecular methods. Although the Prototheca isolation rate was low, a high species diversity was observed. The algae appear to represent allochthonous microflora, brought into waterbodies from various anthropogenic sources. Large rivers of urbanized areas were the most Prototheca-abundant. The study provides a description of three new Prototheca species, namely, P. fontanea, P. lentecrescens, and P. vistulensis. The study also delivers a new identification scheme for all Prototheca species currently recognized.

A first insight into the genome of Prototheca wickerhamii, a major causative agent of human protothecosis.

BACKGROUND: Colourless microalgae of the Prototheca genus are the only known plants that have consistently been implicated in a range of clinically relevant opportunistic infections in both animals and humans. The Prototheca algae are emerging pathogens, whose incidence has increased importantly over the past two decades. Prototheca wickerhamii is a major human pathogen, responsible for at least 115 cases worldwide. Although the algae are receiving more attention nowadays, there is still a substantial knowledge gap regarding their biology, and pathogenicity in particular. Here we report, for the first time, the complete nuclear genome, organelle genomes, and transcriptome of the P. wickerhamii type strain ATCC 16529. RESULTS: The assembled genome size was of 16.7 Mbp, making it the smallest and most compact genome sequenced so far among the protothecans. Key features of the genome included a high overall GC content (64.5%), a high number (6081) and proportion (45.9%) of protein-coding genes, and a low repetitive sequence content (2.2%). The vast majority (90.6%) of the predicted genes were confirmed with the corresponding transcripts upon RNA-sequencing analysis. Most (93.2%) of the genes had their putative function assigned when searched against the InterProScan database. A fourth (23.3%) of the genes were annotated with an enzymatic activity possibly associated with the adaptation to the human host environment. The P. wickerhamii genome encoded a wide array of possible virulence factors, including those already identified in two model opportunistic fungal pathogens, i.e. Candida albicans and Trichophyton rubrum, and thought to be involved in invasion of the host or elicitation of the adaptive stress response. Approximately 6% of the P. wickerhamii genes matched a Pathogen-Host Interaction Database entry and had a previously experimentally proven role in the disease development. Furthermore, genes coding for proteins (e.g. ATPase, malate dehydrogenase) hitherto considered as potential virulence factors of Prototheca spp. were demonstrated in the P. wickerhamii genome. CONCLUSIONS: Overall, this study is the first to describe the genetic make-up of P. wickerhamii and discovers proteins possibly involved in the development of protothecosis.

Protothecosis in Dogs and Cats-New Research Directions.

Protothecosis refers to disease of humans and animals caused by infection with fungus-like, colourless microalgae of the genus Prototheca. Although protothecosis remains an uncommon infection, increasing numbers of human and animal cases are being diagnosed worldwide. This review summarises major new findings in basic science (sequencing analyses of sterol 14α-demethylase (CYP51/ERG11) genes and organelle genomes of Prototheca wickerhamii) to elucidate taxonomic features of this pathogen. Furthermore, this review updates and summarises the clinical features, diagnosis and treatment of protothecosis in dogs and cats. This content of this review is based on information presented at the medical phycology symposium held in the 20th Congress of the International Society for Human and Animal Mycology ( https://www.isham.org/ ).

The first outbreak of methicillin-resistant Staphylococcus aureus in dairy cattle in Poland with evidence of on-farm and intrahousehold transmission.

Staphylococcus aureus is a widely recognized pathogen responsible for many serious diseases in both humans and animals. It is also one of the major causative agents of bovine mastitis. Methicillin-resistant S. aureus (MRSA), although relatively rare in this pathology, has been increasingly reported in livestock animals, mainly in pigs, but also cattle, sheep, and poultry. The recent emergence of livestock-associated (LA-)MRSA is cause for an immediate public health concern due to the risk of zoonotic transmission to humans, and is of particular concern for people who work in animal husbandry or have prolonged contact with livestock animals. This study reports on the first LA-MRSA outbreak in dairy cattle and the first probable case of MRSA transmission between humans and cows in Poland. A single dairy farm located in Eastern Poland was monitored on a regular basis for the occurrence of mastitis. Over a 1-yr study period, 717 quarter-milk samples from 583 cows were collected and examined microbiologically. A total of 5 MRSA isolates from as many cows with subclinical mastitis were cultured. They all belonged to the same outbreak, given a 2-mo time window in which they were identified. During the outbreak, 24 oral and nasal swabs were voluntarily taken from 6 people: a milker, a veterinarian, and 4 members of the veterinarian's family. Eight swabs from a milker, veterinarian, and 2 family members yielded positive MRSA cultures. All MRSA isolates were genotyped with a combination of multiple-locus variable number tandem repeat analysis, multilocus sequence typing, and staphylococcal protein A gene (spa) typing. Eleven bovine (n = 5; 5 cases) and human (n = 6; 4 cases) isolates showed an identical drug-susceptibility profile and were indistinguishable upon multiple-locus variable number tandem repeat analysis (pattern A), multilocus sequence typing (ST398) and spa (t034) typing. The results of this study provide the evidence of transmission of MRSA between humans and cows, and between humans in the family setting. This work, despite being a preliminary investigation, underscores the risk of intra- and interspecies transmission of LA-MRSA and urges enhancement of the existing biosecurity measures aimed at preventing MRSA (and other milk pathogens) spread at both the farm- and household levels.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera.

Fungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based on cytochrome c oxidase subunit 1 and ß-tubulin genes. The assays resulted in 2-10 restriction patterns, depending on the gene amplified and restriction enzyme applied. Pooled analysis of the patterns allowed to propose an algorithm, that can be successfully used for an accurate species-specific identification of 21 species of the Scopulariopsis-like fungi.

A survey on the incidence of Prototheca mastitis in dairy herds in Lublin province, Poland.

Prototheca mastitis has recently become an emerging disease; although its incidence is increasing steadily, its epidemiology remains largely understudied. The aim of this work was to investigate the prevalence of Prototheca spp. in dairy cows and their environment in Lublin province, covering most of southeastern Poland. Between December 2015 and July 2016, a total of 172 milking cows from 10 dairy farms were inspected for mastitis using clinical examination and the California Mastitis Test (CMT). Quarter milk samples (QMS, n = 179) and body site swabs (n = 151) from CMT-positive cows were collected for microbiological culture. In addition, we evaluated QMS and body site swabs from 23 healthy cows, along with 91 environmental samples. Of 100 CMT-positive cows, 71 had at least one QMS positive for microbial growth. In 8 (11.3%) of these cows, originating from 7 dairy farms, Prototheca spp. were cultured. The average somatic cell count of the Prototheca-containing milk was 4.02 × 106 cells/mL compared with 0.13 × 106 cells/mL of the Prototheca-free milk (collected from control animals). No significant differences were observed between mastitis and control cows with respect to counts of total white blood cells, lymphocytes, neutrophils, and eosinophils. Half of the cows with Prototheca spp. in their milk did not yield the algae from other anatomical sites. Eight cows were negative for the presence of Prototheca spp. in their milk but positive for the algae in swabs from anatomical sites. Among the environmental sources that were positive for Prototheca growth were watering troughs, manure, feed, and mud. All (45) Prototheca isolates recovered in this study were subjected to species- and genotype-level molecular identification. All QMS and most of the animal swabs (90%) yielded Prototheca zopfii genotype (gen.) 2. Of the animal samples, P. zopfii gen. 1 and Prototheca blaschkeae were isolated only from feces and rectum. Environmental samples grew either P. zopfii gen. 2 (67%) or P. zopfii gen. 1 (33%). This study demonstrates that P. zopfii gen. 2 is the third most common pathogen of mastitis in cattle in southeast Poland, with an overall incidence of 4.6%. Finding Prototheca spp., including P. zopfii gen. 1 and 2 and P. blaschkeae, in stool and rectal swabs from healthy animals may suggest their role as nonpathogenic microflora of bovine gut.

Drug Susceptibility Profiling and Genetic Determinants of Drug Resistance in Mycobacterium kansasii.

Very few studies have examined drug susceptibility of Mycobacterium kansasii, and they involve a limited number of strains. The purpose of this study was to determine drug susceptibility profiles of M. kansasii isolates representing a spectrum of species genotypes (subtypes) with two different methodologies, i.e., broth microdilution and Etest assays. To confirm drug resistance, drug target genes were sequenced. A collection of 85 M. kansasii isolates, including representatives of eight different subtypes (I to VI, I/II, and IIB) from eight countries, was used. Drug susceptibility against 13 and 8 antimycobacterial agents was tested by using broth microdilution and Etest, respectively. For drug-resistant or high-MIC isolates, eight structural genes (rrl, katG, inhA, embB, rrs, rpsL, gyrA, and gyrB) and one regulatory region (embCA) were PCR amplified and sequenced in the search for resistance-associated mutations. All isolates tested were susceptible to rifampin (RIF), amikacin (AMK), co-trimoxazole (SXT), rifabutin (RFB), moxifloxacin (MXF), and linezolid (LZD) according to the microdilution method. Resistance to ethambutol (EMB), ciprofloxacin (CIP), and clarithromycin (CLR) was found in 83 (97.7%), 17 (20%), and 1 (1.2%) isolate, respectively. The calculated concordance between the Etest and dilution method was 22.6% for AMK, 4.8% for streptomycin (STR), 3.2% for CLR, and 1.6% for RIF. For EMB, INH, and SXT, not even a single MIC value determined by one method equaled that by the second method. The only mutations disclosed were A2266C transversion at the rrl gene (CLR-resistant strain) and A128G transition at the rpsL gene (strain with STR MIC of >64 mg/liter). In conclusion, eight drugs, including RIF, CLR, AMK, SXT, RFB, MXF, LZD, and ethionamide (ETO), showed high in vitro activity against M. kansasii isolates. Discrepancies of the results between the reference microdilution method and Etest preclude the use of the latter for drug susceptibility determination in M. kansasii Drug resistance in M. kansasii may have different genetic determinants than resistance to the same drugs in M. tuberculosis.

Characterization of Mutations Conferring Resistance to Rifampin in Mycobacterium tuberculosis Clinical Strains.

Resistance of Mycobacterium tuberculosis to rifampin (RMP), mediated by mutations in the rpoB gene coding for the beta-subunit of RNA polymerase, poses a serious threat to the efficacy of clinical management and, thus, control programs for tuberculosis (TB). The contribution of many individual rpoB mutations to the development and level of RMP resistance remains elusive. In this study, the incidence of mutations throughout the rpoB gene among 115 Mycobacterium tuberculosis clinical isolates, both resistant and susceptible to RMP, was determined. Of the newly discovered rpoB mutations, the role of three substitutions in the causation of RMP resistance was empirically tested. The results from in vitro mutagenesis experiments were combined with the assessment of the prevalence of rpoB mutations, and their reciprocal co-occurrences, across global M. tuberculosis populations. Twenty-two different types of mutations in the rpoB gene were identified and distributed among 58 (89.2%) RMP-resistant strains. The MICs of RMP were within the range of 40 to 800 mg/liter, with MIC50 and MIC90 values of 400 and 800 mg/liter, respectively. None of the mutations (Gln429His, Met434Ile, and Arg827Cys) inspected for their role in the development of RMP resistance produced an RMP-resistant phenotype in isogenic M. tuberculosis H37Rv strain-derived mutants. These mutations are supposed to compensate for fitness impairment incurred by other mutations directly associated with drug resistance.

cytb as a New Genetic Marker for Differentiation of Prototheca Species.

Achlorophyllous unicellular microalgae of the genus Prototheca (Trebouxiophyceae, Chlorophyta) are the only known plants that cause infections in both humans and animals, collectively referred to as protothecosis. Human protothecosis, most commonly manifested as cutaneous, articular, and disseminated disease, is primarily caused by Protothecawickerhamii, followed by Protothecazopfii and, sporadically, by Protothecacutis and Protothecamiyajii In veterinary medicine, however, P. zopfii is a major pathogen responsible for bovine mastitis, which is a predominant form of protothecal disease in animals. Historically, identification of Prototheca spp. has relied upon phenotypic criteria; these were later replaced by molecular typing schemes, including DNA sequencing. However, the molecular markers interrogated so far, mostly located in the ribosomal DNA (rDNA) cluster, do not provide sufficient discriminatory power to distinguish among all Prototheca spp. currently recognized. Our study is the first attempt to develop a fast, reliable, and specific molecular method allowing identification of all Prototheca spp. We propose the mitochondrial cytb gene as a new and robust marker for diagnostics and phylogenetic studies of the Prototheca algae. The cytb gene displayed important advantages over the rDNA markers. Not only did the cytb gene have the highest discriminatory capacity for resolving all Prototheca species, but it also performed best in terms of technical feasibility, understood as ease of amplification, sequencing, and multiple alignment analysis. Based on the species-specific polymorphisms in the partial cytb gene, we developed a fast and straightforward PCR-restriction fragment length polymorphism (RFLP) assay for identification and differentiation of all Prototheca species described so far. The newly proposed method is advocated to be a new gold standard in diagnostics of protothecal infections in human and animal populations.

Methodological and Clinical Aspects of the Molecular Epidemiology of Mycobacterium tuberculosis and Other Mycobacteria.

Molecular typing has revolutionized epidemiological studies of infectious diseases, including those of a mycobacterial etiology. With the advent of fingerprinting techniques, many traditional concepts regarding transmission, infectivity, or pathogenicity of mycobacterial bacilli have been revisited, and their conventional interpretations have been challenged. Since the mid-1990s, when the first typing methods were introduced, a plethora of other modalities have been proposed. So-called molecular epidemiology has become an essential subdiscipline of modern mycobacteriology. It serves as a resource for understanding the key issues in the epidemiology of tuberculosis and other mycobacterial diseases. Among these issues are disclosing sources of infection, quantifying recent transmission, identifying transmission links, discerning reinfection from relapse, tracking the geographic distribution and clonal expansion of specific strains, and exploring the genetic mechanisms underlying specific phenotypic traits, including virulence, organ tropism, transmissibility, or drug resistance. Since genotyping continues to unravel the biology of mycobacteria, it offers enormous promise in the fight against and prevention of the diseases caused by these pathogens. In this review, molecular typing methods for Mycobacterium tuberculosis and nontuberculous mycobacteria elaborated over the last 2 decades are summarized. The relevance of these methods to the epidemiological investigation, diagnosis, evolution, and control of mycobacterial diseases is discussed.

Rapid Assays for Specific Detection of Fungi of Scopulariopsis and Microascus Genera and Scopulariopsis brevicaulis Species.

PURPOSE: Fungi of Scopulariopsis and Microascus genera cause a wide range of infections, with S. brevicaulis being the most prevalent aetiological agent of mould onychomycosis. Proper identification of these pathogens requires sporulating culture, which considerably delays the diagnosis. So far, sequencing of rDNA regions of clinical isolates has produced ambiguous results due to the lack of reference sequences in publicly available databases. Thus, there is a clear need for the development of new molecular methods that would provide simple, rapid and highly specific identification of Scopulariopsis and Microascus species. The objective of this study was to develop simple and fast assays based on PCR and real-time PCR for specific detection of fungi from Scopulariopsis and Microascus genera, and separately, S. brevicaulis species. METHODS: On the basis of alignment of ß-tubulin gene sequences, Microascus/Scopulariopsis-specific primers were designed and S. brevicaulis-specific primers were reevaluated. DNA from cultured fungal isolates, extracted in a two-step procedure, was used in Microascus/Scopulariopsis-specific and S. brevicaulis-specific PCR and real-time PCR followed by electrophoresis or melting temperature analysis, respectively. RESULTS: The specificity of the assays was confirmed, as positive results were obtained only for Scopulariopsis spp. and Microascus spp. isolates tested in Microascus/Scopulariopsis-specific assay, and only for S. brevicaulis and S. koningii (syn. S. brevicaulis) isolates in a S. brevicaulis-specific assay, respectively, and no positive results were obtained neither for other moulds, dermatophytes, yeast-like fungi, nor for human DNA. CONCLUSIONS: The developed assays enable fast and unambiguous identification of Microascus spp. and Scopulariopsis spp. pathogens.

In vitro activities of a wide panel of antifungal drugs against various Scopulariopsis and Microascus species.

The in vitro activities of 11 antifungal drugs against 68 Scopulariopsis and Microascus strains were investigated. Amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, voriconazole, and ciclopirox showed no or poor antifungal effect. The best activities were exhibited by terbinafine and caspofungin, where the MIC and MEC (minimal effective concentration) ranges were 0.0313 to >16 µg/ml and 0.125 to 16 µg/ml, respectively. The MIC and MEC modes were both 1 µg/ml for terbinafine and caspofungin; the MIC50 and MEC50 were 1 µg/ml for both drugs, whereas the MIC90 and MEC90 were 4 µg/ml and 16 µg/ml, respectively.

First Probable Case of Subcutaneous Infection Due to Truncatella angustata: a New Fungal Pathogen of Humans?

Truncatella angustata is a coelomycetous fungus, typically associated with vascular plants as either an endophyte or a pathogen. This organism has not previously been implicated in human disease. This report describes a case of T. angustata subcutaneous infection in an immunocompetent patient. A conclusive diagnosis was achieved through partial sequencing of ribosomal DNA (rDNA) cluster. The patient was successfully treated with voriconazole followed by itraconazole.

Mutation profiling for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: Progress in the detection of drug-resistant TB has been underpinned by the development and implementation of new, reliable and rapid diagnostic tools. These rely mostly on the detection of specific mutations conferring resistance to anti-TB drugs. The aim of this study was to search for mutations associated with isoniazid resistance among Mycobacterium tuberculosis clinical isolates. METHODS: A collection of 150 M. tuberculosis strains, including 50 MDR, 50 isoniazid-monoresistant and 50 pan-susceptible strains, was used. For all the strains, seven structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and two regulatory regions (mabA-inhA promoter and oxyR-ahpC intergenic region) were PCR amplified and sequenced in their entirety. RESULTS: Sixty-six distinct mutations were detected at all nine loci investigated, accounting for 109 (72.7%) of the strains tested. The number of strains with any mutation among the MDR, isoniazid-monoresistant and pan-susceptible groups was 49 (98%), 37 (74%) and 23 (46%), respectively. Mutations in the katG gene predominated, with 29 different types distributed among 46 (92%) MDR, 31 (62%) isoniazid-monoresistant and 2 (4%) pan-susceptible strains. Twenty-nine and 19 mutations were found exclusively in MDR and isoniazid-monoresistant strains, respectively. CONCLUSIONS: This study revealed 17 mutations, previously unreported, that might be of potential use as new surrogate markers of isoniazid resistance. Their diagnostic accuracy needs to be confirmed on larger strain samples and from different geographical settings. For isoniazid resistance detection, molecular approaches should still be a complement to rather than a replacement for conventional drug susceptibility testing. This is supported by the lack of mutations in any of the nine genetic loci investigated in 18 isoniazid-resistant strains from this study.

Lmo0171, a novel internalin-like protein, determines cell morphology of Listeria monocytogenes and its ability to invade human cell lines.

Internalins comprise a class of Listeria monocytogenes proteins responsible for activation of signalling pathways leading to phagocytic uptake of the bacterium by the host cell. In this paper, a possible role of Lmo0171-a new member of the internalin family was investigated. Disruption of the lmo0171 gene resulted in important cell morphology alterations along with a decrease in the ability to invade three eukaryotic cell lines, that is Int407, Hep-2 and HeLa and diminished adhesion efficiency to int407, thereby suggesting bifunctionality of the newly characterised Lmo0171 internalin.

Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells.

BACKGROUND: Listeriolysin O (LLO) is the main virulence factor of Listeria monocytogenes and facilitates the intracellular survival of the pathogen. Some of its characteristics endorse the growing popularity of LLO for use in biotechnology, particularly in the development of novel vaccines. Here, we evaluate the use of LLO to eradicate leukaemia cells. RESULTS: A purified LLO preparation was obtained by affinity chromatography. The LLO preparation procedure was optimized and purified LLO was tested for optimal conditions of storage including temperature, application of proteinase inhibitors and serum components. We demonstrated the possibility of regulating LLO activity by adjusting cell membrane cholesterol content. The LLO preparation had haemolytic activity and had a cytotoxic effect on the human T-leukaemia Jurkat cell line as well as mouse and human peripheral blood mononuclear cells. CONCLUSIONS: LLO has a very potent cytotoxic activity towards human leukocytes. Importantly, the cytotoxic activity was easily regulated in vitro and could be restricted to areas containing malignant cells, raising the possibility of future clinical application of LLO for leukaemia treatment.

Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates.

OBJECTIVES: To determine the prevalence of isoniazid resistance-conferring mutations among multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis from Poland. METHODS: Nine genetic loci, including structural genes (katG, inhA, ahpC, kasA, ndh, nat and mshA) and regulatory regions (i.e. the mabA-inhA promoter and oxyR-ahpC intergenic region) of 50 MDR M. tuberculosis isolates collected throughout Poland were PCR-amplified in their entirety and screened for mutations by direct sequencing methodology. RESULTS: Forty-six (92%) MDR M. tuberculosis isolates had mutations in the katG gene, and the katG Ser315Thr substitution predominated (72%). Eight (16%) isolates (six with a mutated katG allele) had mutations in the inhA promoter region and two such isolates also had single inhA structural gene mutations. Mutations in the oxyR-ahpC locus were found in five (10%) isolates, of which all but one had at least one additional mutation in katG. Mutations in the remaining genetic loci (kasA, ndh, nat and mshA) were detected in 12 (24%), 4 (8%), 5 (10%) and 17 (34%) MDR isolates, respectively. All non-synonymous mutants for these genes harboured mutations in katG. One isolate had no mutations in any of the analysed loci. CONCLUSIONS: This study accentuates the usefulness of katG and inhA promoter mutations as predictive markers of isoniazid resistance. Testing only for katG 315 and inhA -15 mutations would detect isoniazid resistance in 84% of the MDR M. tuberculosis sample. This percentage would increase to 96% if the sequence analysis was extended to the entire katG gene. Analysis of the remaining genetic loci did not contribute greatly to the identification of isoniazid resistance.