Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato.

BACKGROUND: Potato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY. RESULTS: The presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY. CONCLUSIONS: The new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Late blight resistance genes in potato breeding.

MAIN CONCLUSION: Using late blight resistance genes targeting conservative effectors of Phytophthora infestans and the constructing gene pyramids may lead to durable, broad-spectrum resistance, which could be accelerated through genetic engineering. Potato (Solanum tuberosum L.) is one of the most important food crops worldwide. In 2020, potato production was estimated to be more than 359 million tons according to the Food and Agriculture Organization (FAO). Potato is affected by many pathogens, among which Phytophthora infestans, causing late blight, is of the most economic importance. Crop protection against late blight requires intensive use of fungicides, which has an impact on the environment and humans. Therefore, new potato cultivars have been bred using resistance genes against P. infestans (Rpi genes) that originate from wild relatives of potato. Such programmes were initiated 100 years ago, but the process is complex and long. The development of genetic engineering techniques has enabled the direct transfer of resistance genes from potato wild species to cultivars and easier pyramiding of multiple Rpi genes, which potentially increases the durability and spectrum of potato resistance to rapidly evolving P. infestans strains. In this review, we summarize the current knowledge concerning Rpi genes. We also discuss the use of Rpi genes in breeding as well as their detection in existing potato cultivars. Last, we review new sources of Rpi genes and new methods used to identify them and discuss interactions between P. infestans and host.

Molecular characterization of Candida isolates from intensive care unit patients, Krakow, Poland / Caracterización molecular de aislamientos de Candida de pacientes en cuidados intensivos en Cracovia, Polonia

Background. Over the last decades, Candida species have emerged as important pathogens in immunocompromised patients. Nosocomial infections are mainly of endogenous origin. Nevertheless, some cases of exogenous candidiasis have also been reported. Aims. The aim of this study was to evaluate the genetic relatedness between Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida kefyr isolates recovered from intensive care unit (ICU) patients. Methods. A total of 132 Candida clinical isolates (62 C. albicans, 40 C. glabrata, 13 C. tropicalis, 11 C. krusei, 6 C. kefyr), obtained from specimens of endotracheal aspirate, urine and blood taken from patients of a tertiary hospital in Poland, were included in the study. Species identification was performed by PCR method and genetic relatedness was assessed by randomly amplified polymorphic DNA assay (RAPD) with five primers. Results. The RAPD analysis revealed high genetic diversity among the studied Candida isolates, indicating that most of the strains were from endogenous sources. Only two clonal strains of C. glabrata isolated from different patients were observed, suggesting a possible cross-transmission of these pathogens. Conclusions. Our study confirmed the high discriminatory power of the RAPD assay. This genotyping method can be applied to local epidemiological studies of Candida species (AU)

Antecedentes. En las últimas décadas, el hongo Candida se ha convertido en un patógeno importante para los pacientes con trastornos del sistema inmune. Las infecciones nosocomiales son fundamentalmente de origen endógeno; sin embargo, también se han documentado algunos casos de candidiasis exógena. Objetivos. El objetivo del estudio fue evaluar la relación genética entre las cepas de Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei y Candida kefyr aisladas de pacientes en cuidados intensivos. Métodos. Se estudiaron 132 aislamientos de Candida (62 C. albicans, 40 C. glabrata, 13 C. tropicalis, 11 C. krusei, 6 C. kefyr) obtenidos de muestras procedentes de aspirado endotraqueal, orina y sangre tomadas de pacientes de un hospital en Polonia. La identificación de las especies se realizó mediante PCR, y el estudio de la relación genética con el método de amplificación aleatoria de ADN polimórfico (RAPD) con cinco oligonucleótidos. Resultados. El análisis de la amplificación por RAPD mostró una alta diversidad genética entre los aislamientos objeto de estudio, lo que indica que la mayoría de ellos tenían un origen endógeno. Solo se observaron dos cepas clonales de C. glabrata procedentes de diferentes pacientes, lo que evidencia una posible transmisión cruzada de estos patógenos. Conclusiones. Nuestro estudio confirma el alto poder discriminatorio de la técnica RAPD, lo que validaría este método de genotipificación para el estudio de la epidemiología local de especies de Candida (AU)

EVALUATION OF IN VITRO TIGECYCLINE ACTIVITY AGAINST MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII CLINICAL ISOLATES FROM POLAND.

Acinetobacter baumannii is a major cause of nosocomial infections worldwide. Therapeutic options in management of this bacteria are limited. Tigecycline is considered as an alternative treatment of infections caused by multidrug-resistant A. baumannii strains, however this resistance has emerged recently. Another growing problem is a lack of international consensus between U.S. FDA and EUCAST recommendations, regarding tigecycline breakpoints for Acinetobacter spp., and frequently off-label use. The aim of the present study was to assess the in vitm susceptibility to tigecycline and other antimicrobials, routinely used in the treatment of infections, among 155 A. baumannii isolates, collected between 2008-2013 from a hospital in Poland. The most active agent against the tested MDR strains was colistin (99.3% susceptible isolates). Our study has shown a low efficiency of tigecycline, with 74.2% of non-susceptible strains (according to the U.S. FDA guidelines). Tigecycline MIC values ranged from 0.125 to 48 mg/L. The MIC50 and MIC90 were 3 and 8 mg/L, respectively, and 25.8% (40) of the isolates displayed MIC = 2 mg/L. The highest percentage of tigecycline-resistant strains were noted in 2010 (56.3%). Our study revealed remarkably high tigecycline non-susceptibili- ty rates among MDR A. baumannii isolates, therefore this antimicrobial should be administered with caution.

Molecular characterization of Candida isolates from intensive care unit patients, Krakow, Poland.

BACKGROUND: Over the last decades, Candida species have emerged as important pathogens in immunocompromised patients. Nosocomial infections are mainly of endogenous origin. Nevertheless, some cases of exogenous candidiasis have also been reported. AIMS: The aim of this study was to evaluate the genetic relatedness between Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida kefyr isolates recovered from intensive care unit (ICU) patients. METHODS: A total of 132 Candida clinical isolates (62 C. albicans, 40 C. glabrata, 13 C. tropicalis, 11 C. krusei, 6 C. kefyr), obtained from specimens of endotracheal aspirate, urine and blood taken from patients of a tertiary hospital in Poland, were included in the study. Species identification was performed by PCR method and genetic relatedness was assessed by randomly amplified polymorphic DNA assay (RAPD) with five primers. RESULTS: The RAPD analysis revealed high genetic diversity among the studied Candida isolates, indicating that most of the strains were from endogenous sources. Only two clonal strains of C. glabrata isolated from different patients were observed, suggesting a possible cross-transmission of these pathogens. CONCLUSIONS: Our study confirmed the high discriminatory power of the RAPD assay. This genotyping method can be applied to local epidemiological studies of Candida species.

Acinetobacter baumannii: biology and drug resistance - role of carbapenemases.

Acinetobacter baumannii is a Gram-negative, glucose-non-fermenting, oxidase-negative coccobacillus, most commonly associated with the hospital settings. The ability to survive in adverse environmental conditions as well as high level of natural and acquired antimicrobial resistance make A. baumannii one of the most important nosocomial pathogens. While carbapenems have long been considered as antimicrobials of last-resort, the rates of clinical A. baumannii strains resistant to these antibiotics are increasing worldwide. Carbapenem resistance among A. baumannii is conferred by coexisting mechanisms including: decrease in permeability of the outer membrane, efflux pumps, production of beta-lactamases, and modification of penicillin-binding proteins. The most prevalent mechanism of carbapenem resistance among A. baumannii is associated with carbapenem-hydro-lysing enzymes that belong to Ambler class D and B beta-lactamases. In addition, there have also been reports of resistance mediated by selected Ambler class A carbapenemases among A. baumannii strains. Resistance determinants in A. baumannii are located on chromosome and plasmids, while acquisition of new mechanisms can be mediated by insertion sequences, integrons, transposons, and plasmids. Clinical relevance of carbapen-em resistance among strains isolated from infected patients, carriers and hospital environment underlines the need for carbapenemase screening. Currently available methods vary in principle, accuracy and efficiency. The techniques that deserve particular attention belong to both easily accessible unsophisticated methods as well as advanced techniques based on mass spectrometry or molecular biology. While carbapenemases limit the therapeutic options in A. baumannii infections, studies concerning novel beta-lactamase inhibitors offer a new insight into effective therapy.

ACTIVITY OF THYME OIL (OLEUM THYMI) AGAINST MULTIDRUG-RESISTANT ACINETOBACTER BAUMANNII AND PSEUDOMONAS AERUGINOSA.

Almost as soon as antibiotics were introduced to treat infectious diseases, it could be observed that bacteria were able to develop resistance against them. Currently, multidrug-resistant strains are being isolated mainly in the hospital environment. These are primarily non-fermenting Gram-negative bacilli, which exhibit both natural and acquired resistance to multiple antibiotics and disinfectants rendering them difficult to eradicate. The development of new, effective and safe substances that prevent troublesome infections is greatly needed to provide alternative therapeutic options for patients. There is increasing interest in drugs of natural origin, including essential oils. It is of particular interest that, although active against many bacterial strains, they do not contribute to antibacterial resistance against their components. The aim of our study was to evaluate the in vino antibacterial activity of thyme oil against multidrug-resistant strains of A. baumannii and P. aeriginosa using the disc diffusion and macrodilution methods. The strains were isolated from patients hospitalized in the years 2013-2014. The in vitto antibacterial activity of thyme oil was assessed by the disc diffusion method and the inhibition zones for the oil at different concentrations, produced against A. baumannii, ranged from 7 to 44 mm. Low level of activity of thyme oil was observed against P. aeruginosa strains. The results of serial dilution tests confirmed the high activity of thyme oil against A. baumannii isolates, expressed as MIC values ranging from 0.25 to 2 µL/mL. These results suggest the need for further studies of antibacterial activity of essential oils, especially against multidrug-resistant bacterial isolates.

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland.

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.

Molecular epidemiology of Candida albicans and Candida glabrata strains isolated from intensive care unit patients in Poland

Over the last decades, Candida spp have been responsible for an increasing number of infections, especially in patients requiring intensive care. Knowledge of local epidemiology and analysis of the spread of these pathogens is important in understanding and controlling their transmission. The aim of this study was to evaluate the genetic diversity of 31 Candida albicans and 17 Candida glabrata isolates recovered from intensive care unit patients from the tertiary hospital in Krakow between 2011-2012. The strains were typed by random amplified polymorphic DNA (RAPD) polymerase chain reaction using five primers (CD16AS, HP1247, ERIC-2, OPE-3 and OPE-18). The results of the present investigation revealed a high degree of genetic diversity among the isolates. No clonal relationship was found among the C. albicans strains, whereas two C. glabrata isolates were identical. The source of Candida infection appeared to be mostly endogenous; however, the presence of two clonal C. glabrata strains suggested the possibility of cross-transmission of these pathogens. Our study confirmed the high discriminatory power of the RAPD technique in the molecular typing of Candida clinical isolates. This method may be applied to the evaluation of transmission routes of pathogenic fungi on a local level.

Co-occurrence of carbapenem and aminoglycoside resistance genes among multidrug-resistant clinical isolates of Acinetobacter baumannii from Cracow, Poland.

BACKGROUND: Acinetobacter baumannii is a significant hospital pathogen, possessing a considerable degree of antimicrobial resistance. A. baumannii resistance to carbapenems and aminoglycosides is mostly conferred by class D OXA carbapenemases and aminoglycoside-modifying enzymes, respectively. The aim of this study was to determine the prevalence of selected genes encoding OXA carbapenemases and aminoglycoside-modifying enzymes in multidrug-resistant strains of A. baumannii. MATERIAL AND METHODS: The study included 61 carbapenem-resistant and aminoglycoside-nonsusceptible A. baumannii isolates, collected between 2009 and 2011 in Cracow, Poland. Selected resistance genes, including: blaOXA-51-like, blaOXA-23-like, blaOXA-40-like, blaOXA-58-like, aac(6')-Ih, aac(3)-Ia, aac(3)-IIa, aac(6')-Ib, aph(3')-Ia and aph(3')-VI, were detected by PCR method. RESULTS: The blaOXA-51-like genes were detected in all isolates, while acquired carbapenemase encoding genes were found in 96.7% of tested strains. Presence of blaOXA-40-like and blaOXA-23-like genes was observed among 65.6% and 27.9% of isolates, respectively. Assayed aminoglycoside resistance genes were found to harbor 98.4% of isolates. Among tested strains, we observed the following percentages of resistance determinants: aac(3)-Ia - 78.7%, aph(3')-VI - 78.7% and aph(3')-Ia - 27.9%. Analysis of co-occurrence of carbapenem and aminoglycoside resistance genes revealed the highest percentage of strains possessing blaOXA-40-like, aac(3)-Ia, and aph(3')-VI genes (44.3%). CONCLUSIONS: The blaOXA-40-like and aac(3)-Ia/aph(3')-VI were the most prevalent genes encoding acquired OXA carbapenemases and aminoglycoside-modifying enzymes, respectively, among A. baumannii strains in Cracow, Poland. Genes conferring resistance to carbapenems and aminoglycosides coexisted in the clinical strains of A. baumannii. The phenomenon of A. baumannii resistance indicates the necessity of monitoring for the presence of the resistance genes.

[Occurrence of alert pathogens in hospital environment. Part I. ESBL-producing enterobacteriaceae strains]. / Wystepowanie patogenów alarmowych w srodowisku szpitalnym. Czesc I. Paleczki z rodziny Enterobacteriaceae wytwarzajace beta-laktamazy ESBL.

INTRODUCTION: Gram-negative bacteria belonging to the family Enterobacteriaceae cause severe and difficult to treat nosocomial infections. Strains of different species that produce extended-spectrum beta-lactamases (ESBL) were classified as alert pathogens. The purpose of this study was to analyze the occurrence and determination of antimicrobial susceptibility patterns of 134 ESBL-producing Enterobacteriaceae strains. METHODS: 96 (72%) isolates of Klebsiella pneumoniae and 38 (28%) isolates of Escherichia coli, were cultured from patients of Specialistic Hospital in Krakow, in the period from 2008 to 2010. Bacterial identification and antimicrobial susceptibility testing were performed by automated system Vitek 2 Compact (bioMerieux, Poland). Condition for inclusion in the study was the production by the strains of beta-lactamases with extended substrate spectrum (ESBL), which was confirmed using the automated method (Vitek 2 Compact system) and the disc-diffusion assay (DDST). Taken into consideration the first isolate from the patient. RESULTS: Bacilli of the species K. pneumoniae ESBL(+) were mainly isolated from respiratory tract samples (46%), urine (27%) and blood (12%). The dominant divisions in terms of frequency of isolation of these pathogens were anesthesiology and intensive care (42%), neurology and brain strokes (16%) and internal medicine (11%). Drugs with the highest efficiency against K. pneumoniae ESBL(+), in our in vitro studies, were: imipenem (100%), meropenem (100%), amikacin (90%) and tetracycline (75%). E. coli ESBL(+) isolates derived from patients of Anesthesiology and Intensive Care Unit (32%), Internal Medicine Unit (16%) and Division of Hematology (13%). Among all tested strains majority were obtained from respiratory tract samples, urine, swabs from wounds and blood (respectively 24%, 24%, 21% and 18%). Isolates of E. coli ESBL(+) demonstrated the greatest susceptibility in case of amikacin (92%) and piperacillin with tazobactam (76%), which suggests the highest activity of that antimicrobials against infections caused by examined strains. None of the analyzed bacilli were resistant to carbapenems. CONCLUSIONS: Our study highlights the importance of characteristics of distribution of ESBL-producing K. pneumoniae and E. coli strains among hospitalized patient. Good antibiotic policies based on antibiotic resistant patterns can decrease the risk of ESBL infection.

[Occurrence of alert pathogens in hospital environment Part II. Multidrug-resistant non-fermenting bacilli]. / Wystepowanie patogenów alarmowych w srodowisku szpitalnym. Czesc II. Wielolekooporne paleczki niefermentujace.

INTRODUCTION: Multidrug-resistant gram-negative non-fermenting bacilli are an important cause of nosocomial infection. Aim of this study was to analyze the prevalence and antimicrobial susceptibility of rods of the species Acinetobacter baumannii and Pseudomonas aeruginosa, belonging to multidrug-resistant alert pathogens. METHODS: 105 (70%) strains of A. baumannii and 46 (30%) strains of P. aeruginosa were isolated from 125 patients hospitalized in the Specialistic Hospital in Krakow, in the years 2008-2010. Taken into account first isolate from the patient. The condition for inclusion in the study was the resistance or reduced susceptibility to selected groups of antibiotics, such as beta-lactams, aminoglycosides and fluoroquinolones. Bacterial identification and antimicrobial susceptibility testing were performed by automated system Vitek 2 Compact (bioMerieux, Poland). All strains were tested with phenotypic method Etest MBL (AB Biodisk, Sweden) for the presence of resistance mechanism associated with the production of metallo-beta-lactamases. RESULTS: Bacilli of the species A. baumannii were isolated most frequently from patients from the Department of Anesthesiology and Intensive Care (52%) and Burn Therapy Unit (25%), with clinical materials collected from the respiratory tract (51%), the wound swabs (18%), urine (11%) and blood (11%). Production of metallo-beta-lactamases was found in 24 (22.9%) strains of A. baumannii. Drugs effective against multidrug-resistant isolates of A. baumannii were colistin and amikacin. Department of anesthesiology and intensive care (59%) and unit of internal medicine (11%) were the main source of multidrug-resistant strains of P. aeruginosa. Pathogens were mainly isolated from clinical specimens collected from the respiratory tract (61%), urine (15%) and wound swabs (13%). Seven (15.2%) strains of P. aeruginosa produced the metallo-beta-lactamases. With regard to colistin and piperacillin with tazobactam was noted the highest percentage of susceptible isolates. CONCLUSIONS: MDR bacteria belonging to alert pathogens are an important cause of many severe and difficult to treat infections which greatly increases the morbidity and mortality among hospitalized patients worldwide. Epidemiological studies and detection of local resistance patterns can provide useful information which can be used in the development of strategies to combat the rising tide of microbial antibiotic resistance.

Distribution of blaOXA genes among carbapenem-resistant Acinetobacter baumannii nosocomial strains in Poland.

Acinetobacter baumannii is an important nosocomial pathogen occurring particularly in intensive care (ICU) as well as burn therapy units (BTU). A. baumannii strains have emerged as resistant to almost all antimicrobial agents, including carbapenems. b-lactamase-mediated resistance is the most common mechanism for carbapenem resistance in this species. Carbapenem-hydrolysing class D b-lactamases - OXA are widespread among A. baumannii strains. It is suggested that ISAba1 plays an important role in drug resistance. The aims of the study were detection of OXA encoding genes and presence of ISAba1. The study included the total of 104 isolates of carbapenem-resistant A. baumannii, obtained from patients hospitalized in ICU and BTU of Specialized Hospital in Krakow. Multiplex PCR was applied for detection of selected OXA carbapenemases encoding genes. PCR analysis showed the presence of bla OXA-51-like gene and ISAba1 in all isolates. 46 strains carried bla OXA-51-like and bla OXA-23-like genes while 48 bla OXA-51-like and bla OXA-40-like genes. 3 isolates carried: bla OXA-51-like , bla OXA-23-like and bla OXA-40-like genes. 7 strains encoded an OXA-51-like carbapenemase but were negative for enzymes belonging to the other families tested. Comparative analysis of ICU and BTU isolates revealed the dominance of: bla OXA-51-like and bla OXA-40-like among ICU while bla OXA-51-like and bla OXA-23-like in BTU.

[The genetic description of Acinetobacter baumannii and Pseudomonas aeruginosa strains isolated from patients with hospital acquired pneumonia hospitalized in intensive care unit]. / Genetyczna charakterystyka szczepów Acinetobacter baumannii oraz Pseudomonas aeruginosa izolowanych od chorych z zapaleniem pluc hospitalizowanych w oddziale intensywnej terapii.

The aim of study was to recognize the epidemiology of hospital acquired pneumonia on the base of genotypes of Acinetobacter baumannii and Pseudomonas aeruginosa strains. Gram negative-non fermenting bacilli are the most frequent aetiologic agent of hospital acquired pneumonia among patients from Anesthesiology and Intensive Care Unit in the Rydygiers hospital in Cracow. In the following research RAPD-PCR method was applied and there 272, 208, ERIC- 2 and PAL- 2 primers were used. The investigation was conducted among 33 Acinetobacter baumannii and 31 Pseudomonas aeruginosa strains which were isolated from endotracheal aspirates (ETA). ETA were taken from 19 patients with microbiologically confirmed pneumonia. The result of our study shows that most A. baumannii strains came from hospital habitat. In the investigated group of A. baumannii strains, 31 belonged to 3 clonal groups and probably came from two bacteria subpopulations which were present in ICU from a long time. There were only two isolates which had got the posthospital origin. Moreover, the assay of genetic similarity between A. baumannii strains, isolated from individual patients, showed that only in two patients were observed strains with different genetic profiles. Isolates which came from 8 patients had got the same pattern of bands. There were many genetic differences between investigated P. aeruginosa strains. In the group of 31 isolates, which were investigated by using 208, 272, ERIC- 2 and PAL- 2 primers, recognized respectively 10, 10, 13 and 8 of genetic profiles. All isolates of P. aeruginosa probably came from a few subpopulations of hospital strain which has undergone evolutionary divergence phenomenon in time as a result of changing condition of hospital environment and application of antibiotics. The assay of genotype of P. aeruginosa strains which were repeatedly isolated from individual patients showed that only from two patients strains with 100% genetic homology were isolated.