Flavescence Dorée Strain-Specific Impact on Phenolic Metabolism Dynamics in Grapevine (Vitis vinifera) throughout the Development of Phytoplasma Infection.

Flavescence dorée phytoplasma (FDp) is a phytopathogenic bacterium associated with Grapevine yellowS disease, which causes heavy damage to viticultural production. Epidemiological data revealed that some FDp strains appear to be more widespread and aggressive. However, there is no data on mechanisms underlying the variable pathogenicity among strains. In this research, we employed chromatographic and spectrophotometric techniques to assess how two strains of FDp influence the levels of grapevine phenolic compounds, which are frequently utilized as indicative markers of stress conditions. The results pointed to the upregulation of all branches of phenolic metabolism through the development of infection, correlating with the increase in antioxidative capacity. The more aggressive strain M54 induced stronger downregulation of phenolics' accumulation at the beginning and higher upregulation by the end of the season than the less aggressive M38 strain. These findings reveal potential targets of FDp effectors and provide the first functional demonstration of variable pathogenicity between FDp strains, suggesting the need for future comparative genomic analyses of FDp strains as an important factor in exploring the management possibilities of FDp.

Dissemination of Clinical Acinetobacter baumannii Isolate to Hospital Environment during the COVID-19 Pandemic.

The aim of this study was to find the source of Acinetobacter baumannii in the intensive care unit (ICU) after an outbreak during the coronavirus disease 2019 (COVID-19) pandemic, as there was no A. baumannii detected on usually screened susceptible surfaces. The screening of the ICU environment was done in April 2021 when eleven different samples were taken. One A. baumannii isolate was recovered from the air conditioner and was compared with four clinical A. baumannii isolates obtained from patients hospitalized in January 2021. Isolates were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), minimum inhibitory concentrations (MICs) were determined, and the multilocus sequence typing (MLST) was performed. The molecular identification of A. baumannii isolates as ST208, the presence of the same blaOXA-23 carbapenemase gene, and the same antibiotic susceptibility profile suggest that the isolate recovered from the air conditioner is the same as the isolates recovered from hospitalized patients. The environmental isolate was recovered three months later than the clinical isolates, emphasizing the ability of A. baumannii to survive on dry abiotic surfaces. The air conditioner in the clinical environment is an important but undoubtedly neglected source of A. baumannii outbreaks, hence, frequent disinfection of hospital air conditioners with appropriate disinfectants is mandatory to mitigate the circulation of A. baumannii between patients and the hospital environment.

Molecular characterisation of colistin and carbapenem-resistant clinical isolates of Acinetobacter baumannii from Southeast Europe.

OBJECTIVES: To characterise 11 colistin- and carbapenem-resistant Acinetobacter baumannii isolates recently emerging in hospital settings. METHODS: A. baumannii isolates were collected from hospitalised patients under colistin treatment in three countries of Southeast Europe: Turkey, Croatia, and Bosnia and Herzegovina. Isolates were identified using molecular methods. RESULTS: Isolates from Turkey and Croatia belong to the sequence types ST195 or ST281 of the clone lineage 2, while the single isolate from Bosnia and Herzegovina belongs to the ST231 of clone lineage 1. All isolates turned out to be highly resistant to colistin (MIC ≥ 16 mg/L) and have point mutations in pmrCAB operon genes. The colistin-resistant isolate from Bosnia and Herzegovina had a unique P170L point mutation in the pmrB gene and the R125H point mutation in the pmrC gene. The L20S mutation in the pmrA gene was detected only in isolates from Croatia and has never been reported before in isolates from this country. CONCLUSION: Colistin resistance in A. baumannii in hospitalised patients receiving colistin treatment is a result of chromosomal mutations. The pattern of point mutations in pmrCAB genes suggests a spread of specific colistin-resistant isolates within the hospital.

First report of potato virus S and potato virus Y in tomatoes from Croatia.

Potato virus Y (PVY, genus Potyvirus) is an economically important aphid-transmissible virus with a very wide host range reported in many tomato-growing areas (Rivarez et al. 2021). Potato virus S (PVS, genus Carlavirus) has a limited host range (Lin et al. 2014) and occurs in tomato (Predajna et al. 2017), mostly in mixed infections with other viruses. In 2021, greenhouse tomatoes from Vidovec (46° 17' 3.4'' N, 16° 15' 37.0'' E) in the northwestern and Sedlarica (45° 54' 23.0'' N, 17° 12' 0.5'' E) in the eastern regions of Croatia were surveyed for virus-like diseases. In total, 30 plants were sampled (12 from Vidovec and 18 from Sedlarica) showing symptoms of mild mottling, leaf rugosity and mild bronzing followed by leaf necroses later in the season. Nucleic acids were extracted from leaves by adapted CTAB procedure (Murray and Thompson 1980) and DNase treated. Four representative samples from Vidovec and four from Sedlarica were pooled for high throughput sequencing (HTS). After rRNA depletion (RiboMinus™ Plant Kit for RNA-Seq, Invitrogen) and polyA tailing, two location specific libraries (PCR-cDNA sequencing kit, Oxford Nanopore Technologies) were prepared for nanopore HTS on MinION Mk1C device. From Vidovec samples, 459,285 raw reads (mean length 354 nt) were obtained and 206,718 (mean length 446 nt) from Sedlarica and mapped (Minimap2, v.2.17) against Kraken2 viral genome sequences database (https://benlangmead.github.io/aws-indexes/k2). The number of reads mapped to PVS genome was 1004 from Vidovec (coverage depth 1.56) and those mapped to PVY genome were 781 (coverage depth 0.99) and 57 (coverage depth 1), from Vidovec and Sedlarica, respectively. The PVS complete consensus genome from Vidovec (ON468562, 8485 nt) had 99.09% nucleotide identity (BLASTn) to a potato isolate from the Netherlands (MF418030). The PVY consensus genome sequences from Vidovec (ON505007, 9698 nt) and Sedlarica (ON505008, 9698 nt) had respectively 98.37% and 98.48% identities to a tomato isolate from Slovakia (MW685827). Reverse transcription polymerase chain reaction (RT-PCR) was performed for all 30 samples and amplicons were Sanger sequenced, with primers PVS-7773F/PVS-3'endR for a 720 nt PVS genome portion spanning the 3'-part of the CP and a complete 11K gene (Lin et al. 2014) and PVY-2F/2R primers for a 510 nt portion of PVY CP gene (Aramburu et al. 2006). Only one tomato out of 12 ('Borana') from Vidovec harbored PVS in the mixed infection with PVY. Two additional tomatoes from Vidovec and two from Sedlarica were infected solely by PVY. Amplicon sequences of PVS (ON651427) and PVY (ON707000-4, ON734067-8) had 100% identity with the HTS assembled sequences. The PVS isolate from Croatia grouped with PVSO (ordinary) strain in phylogenetic analysis and the PVY isolates from both sites grouped with the PVY-NTN strain (Cox and Jones 2010). Although PVY is considered to be widespread in tomato (Nikolic et al. 2018; Rivarez et al. 2021), this is its first report from Croatia. PVS, newly reported from Croatia here, is probably not associated with the symptoms recorded because the same symptomatology was observed in the singly and mixed infected 'Borana' tomato plants. The occurrence of PVY in the geographically distant (100 km apart) Vidovec and Sedlarica, suggests that it is widespread in the continental Croatia where tomatoes are commercially grown in plastic greenhouses. Further analyses are needed to elucidate PVY and PVS epidemiology and impact on the local tomato production.

Characterization of Burkholderia cepacia complex from environment influenced by human waste.

The natural environment is a primary source of infections caused by members of Burkholderia cepacia complex (BCC), but the release of human waste may in return enrich the natural environment with clinically relevant BCC. Seven BCC isolates from environment influenced by human liquid or solid waste across Croatia, and one clinical isolate was characterised. B. multivorans recovered from the soil at illegal dumpsite belonged to sequence type (ST)19; B. ambifaria from the agricultural soil fertilized with swine or poultry manure to ST927 or new ST; B. cenocepacia from creek sediment, river water and wound swab to new STs. Antimicrobial susceptibility of isolates ranged from sensitive to multidrug-resistant. A variety of blaTEM genes was confirmed in isolates. Isolates expressed the virulence factors and survived in river water during 50 days. The BCC present natural environments influenced by the human waste are of clinical relevance and a potential source of sporadic infections.

Legacy of Plant Virology in Croatia-From Virus Identification to Molecular Epidemiology, Evolution, Genomics and Beyond.

This paper showcases the development of plant virology in Croatia at the University of Zagreb, Faculty of Science, from its beginning in the 1950s until today, more than 70 years later. The main achievements of the previous and current group members are highlighted according to various research topics and fields. Expectedly, some of those accomplishments remained within the field of plant virology, but others make part of a much-extended research spectrum exploring subviral pathogens, prokaryotic plant pathogens, fungi and their viruses, as well as their interactions within ecosystems. Thus, the legacy of plant virology in Croatia continues to contribute to the state of the art of microbiology far beyond virology. Research problems pertinent for directing the future research endeavors are also proposed in this review.

Comparison of clinical and sewage isolates of Acinetobacter baumannii from two long-term care facilities in Zagreb; mechanisms and routes of spread.

In the previous studies OXA-23-like and OXA-24-like ß-lactamase were reported among Acinetobacter baumannii in both hospitals and long-term care facilities (LTCF) in Croatia. The aim of this study was to analyze clinical and sewage A. baumannii isolates from two nursing homes in Zagreb, with regard to antibiotic susceptibility and resistance mechanisms, to determine the route of spread of carbapenem-resistant isolates. Nine clinical isolates were collected from February to May 2017 whereas in April 2017, ten A. baumannii isolates were collected from sewage of two nursing homes in Zagreb. Antibiotics susceptibility was determined by broth microdilution method. The presence of carbapenemase and extended spectrum ß-lactamases (ESBL) encoding genes was explored by PCR. Conjugation and transformation experiments were performed as previously described. Genotyping was performed by SG determination, PFGE and MLST. Seven clinical isolates were positive for blaOXA24-like whereas two clinical and environmental carbapenem-resistant isolates, respectively, were found to possess blaOXA-23-like genes. Attempts to transfer imipenem resistance were unsuccessful indicating chromosomal location of blaOXA-23 gene. All carbapenem-resistant isolates belonged to SG- 1 (IC-2) whereas the rest of the isolates susceptible to carbapenems were allocated to SG- 2 (IC-1). PFGE analysis revealed low degree of genetic variability within both IC- I and IC- II. MLST corroborated that two environmental OXA-23 isolates belong to the ST-195. This study showed dissemination of OXA-23 producing A. baumannii from the nursing home into the urban sewage. Disinfection of nursing home sewage should be recommended in order to prevent the spread of resistance genes into the community sewage.

Carbapenem-Resistant Acinetobacter baumannii Recovered from Swine Manure.

Recently, concerns have been raised about the possibility of Acinetobacter baumannii transmission between animals and humans. So far, A. baumannii has been reported in animals with which people can come into contact. The presence of this pathogen in animal manure presents an equally important public health risk. In this study, we report the finding of two A. baumannii isolates in swine manure from a Croatian pig farm. Both isolates shared features with the widespread human clinical isolates: affiliation to the international clonal lineage 2 (ST-195), carbapenem, and extensive drug resistance and the plasmid-located acquired blaOXA-23 gene. These two A. baumannii isolates survived anaerobic conditions, competition with other microorganisms, and elevated concentrations of heavy metals in the stagnant swine manure for at least 2 weeks. These findings call for bacteriological analysis and disinfection of liquid swine manure before its application as a fertilizer in traditional extensive agriculture.

The genome of 'Candidatus Phytoplasma solani' strain SA-1 is highly dynamic and prone to adopting foreign sequences.

Bacteria of the genus 'Candidatus Phytoplasma' are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as 'Ca. P. solani', have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of 'Ca. P. solani' strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The 'Ca. P. solani' SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.

Extensively and multi drug-resistant Acinetobacter baumannii recovered from technosol at a dump site in Croatia.

In a karst pit above City of Rijeka in Croatia the hazardous industrial waste was continuously disposed from 1955 to 1990, and later it was periodically used as an illegal dump site. The surface part of a technosol at the edge of dump was analysed mineralogically, geochemically and bacteriologically. From the technosol rich in petroleum hydrocarbons and heavy metals three isolates of Acinetobacter baumannii were recovered. Isolates from technosol shared many features that are previously described for clinically isolates: the affiliation to IC1 and 2, multi-drug resistant (MDR) or extensively drug-resistant (XDR) antibiotic resistance profile, carbapenem resistance mediated by blaOXA72 and blaOXA23 genes, and the expression of virulence factors. In in vitro conditions, isolates were able to survive in contact with technosol during 58days of monitoring. The most probable source of A. baumannii in technosol was the illegally disposed hospital waste. Proper management and disposal of human solid waste is mandatory to prevent the spread of clinically important A. baumannii in nature.

Transmission and survival of carbapenem-resistant Acinetobacter baumannii outside hospital setting

Acinetobacter baumannii origin and itsepidemiology is under a great concern worldwide since this microorganism has become a leading nosocomial pathogen of the 21 th century among the "ESKAPE" group of microorganisms. The aim of the study was to monitor and explore the epidemiology of this important hospital pathogen in the second largest clinical university hospital in Croatia. The presence of A. baumannii in hospital wastewater, as a route for possible transmission outside of the hospital setting, as well as its survival in environmental conditions including seawater, was investigated. During the examination period ten both carbapenem and multidrug-resistant isolates of A. baumannii were recovered from hospital wastewater and compared to the clinical isolates originating from the same monitoring period. Multiplex PCR confirmed that four wastewater isolates harboured bla OXA-23-like ,while five wastewater isolatesharboured bla OXA-40-like genes sharing 100% sequence identity with bla OXA-72 sequence described in the same hospital in 2009, confirming the presence of an endemic cluster. Survival of A. baumannii in natural seawater was examined during 50 days of monitoring and to the best of our knowledge, was performed for the first time

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Pan Drug-Resistant Environmental Isolate of Acinetobacter baumannii from Croatia.

Acinetobacter baumannii is an emerging nosocomial pathogen with also emerging resistance to different antibiotics. Multidrug and pan drug-resistant clinical isolates were reported worldwide. Here we report the first evidence of pan drug-resistant environmental isolate of A. baumannii. The isolate was recovered from the effluent of secondary treated municipal wastewater of the City of Zagreb, Croatia. The isolate was resistant to penicillins/ß-lactamase inhibitors, carbapenems, fluoroquinolones, aminoglycosides, folate pathway inhibitors, and polymyxins, except intermediately susceptible to minocycline and tigecycline. Intrinsic chromosomally located blaOXA-51-like gene and acquired plasmid-located blaOXA-23-like gene were related to clinical isolates. Pan drug-resistant A. baumannii can occur in natural environments outside of the hospital. Secondary treated municipal wastewater represents a potential epidemiological reservoir of pan drug-resistant A. baumannii and carbapenem resistance gene.

Transmission and survival of carbapenem-resistant Acinetobacter baumannii outside hospital setting.

Acinetobacter baumannii origin and its epidemiology is under a great concern worldwide since this microorganism has become a leading nosocomial pathogen of the 21th century among the "ESKAPE" group of microorganisms. The aim of the study was to monitor and explore the epidemiology of this important hospital pathogen in the second largest clinical university hospital in Croatia. The presence of A. baumannii in hospital wastewater, as a route for possible transmission outside of the hospital setting, as well as its survival in environmental conditions including seawater, was investigated. During the examination period, ten both carbapenem and multidrug-resistant isolates of A. baumannii were recovered from hospital wastewater and compared to the clinical isolates originating from the same monitoring period. Multiplex PCR confirmed that four wastewater isolates harboured blaOXA-23-like, while five wastewater isolates harboured blaOXA-40-like genes sharing 100% sequence identity with blaOXA-72 sequence described in the same hospital in 2009, confirming the presence of an endemic cluster. Survival of A. baumannii in natural seawater was examined during 50 days of monitoring and to the best of our knowledge, was performed for the first time.

Emergence of Oxacillinases in Environmental Carbapenem-Resistant Acinetobacter baumannii Associated with Clinical Isolates.

Six carbapenem-resistant isolates of Acinetobacter baumannii were recovered from untreated and treated municipal wastewater of the capital city of Zagreb, Croatia. Molecular identification of environmental isolates of A. baumannii was performed by amplification, sequencing, and phylogenetic analyses of rpoB gene. The presence of blaOXA genes encoding OXA-type carbapenemases (OXA-51-like, OXA-23, and OXA-40-like) was confirmed by multiplex PCR and sequencing. Phylogenetic analyses corroborated the affiliation of detected blaOXA genes to three different clusters and showed association of environmental OXAs with those described from clinical isolates. This result suggests that isolates recovered from municipal wastewater are most probably of clinical origin. Furthermore, the presence of OXA-40-like (OXA-72) in an environmental A. baumannii isolate is reported for the first time. Persistence of A. baumannii harboring the clinically important OXAs in the wastewater treatment process poses a potentially significant source for horizontal gene transfer and implications for wider spread of antibiotic resistance genes.

Single-strand conformation polymorphism analysis for differentiating phytoplasma strains.

Single-strand conformation polymorphism (SSCP) analysis is a sensitive and rapid technique for detecting DNA polymorphisms and mutations in PCR-amplified fragments. Due to its technical simplicity, it is widely used as a screening tool in various investigations, ranging from clinical diagnosis of human hereditary diseases to the characterization of microbial communities. This method can also be used successfully on phytoplasmas as a tool for the detection of molecular variability in conserved housekeeping genes such as 16S rRNA and tuf, as well as in more variable genes, revealing the presence of polymorphisms undetected by routine RFLP analyses. The reliability of SSCP has been confirmed by multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles. However, it is not broadly applied in phytoplasma research yet. The technique provides an inexpensive, convenient, and sensitive method for determining sequence variation and to differentiate phytoplasma strains, and is particularly suitable for epidemiological studies or as a fast screening, typing tool when dealing with a large number of field samples.

Phytoplasma PMU1 exists as linear chromosomal and circular extrachromosomal elements and has enhanced expression in insect vectors compared with plant hosts.

Phytoplasmas replicate intracellularly in plants and insects and are dependent on both hosts for dissemination in nature. Phytoplasmas have small genomes lacking genes for major metabolic pathways. Nevertheless, their genomes harbour multicopy gene clusters that were named potential mobile units (PMUs). PMU1 is the largest most complete repeat among the PMUs in the genome of Aster Yellows phytoplasma strain Witches' Broom (AY-WB). PMU1 is c. 20 kb in size and contains 21 genes encoding DNA replication and predicted membrane-targeted proteins. Here we show that AY-WB has a chromosomal linear PMU1 (L-PMU1) and an extrachromosomal circular PMU1 (C-PMU1). The C-PMU1 copy number was consistently higher by in average approximately fivefold in insects compared with plants and PMU1 gene expression levels were also considerably higher in insects indicating that C-PMU1 synthesis and expression are regulated. We found that the majority of AY-WB virulence genes lie on chromosomal PMU regions that have similar gene content and organization as PMU1 providing evidence that PMUs contribute to phytoplasma host adaptation and have integrated into the AY-WB chromosome.

The use of SSCP analysis in the assessment of phytoplasma gene variability.

Single-strand conformation polymorphism (SSCP) analysis is a broadly used technique for detecting mutations. The aim of this work was to assess the applicability of SSCP as a new tool for the detection of the molecular variability of uncultivable mollicutes - phytoplasmas. Three phytoplasma regions were investigated: 16S rDNA, tuf gene, and dnaB gene. Fragments amplified by PCR were subjected to SSCP under conditions optimized for each fragment length. In all of the analyzed regions, SSCP revealed the presence of polymorphism undetected by routine RFLP analyses. Reliability of the method was confirmed by the multiple alignments and phylogenetic analyses of representative sequences showing different SSCP profiles.

Effect of indole-3-butyric acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro.

Phytoplasmas are noncultivable bacteria usually maintained in Catharanthus roseus shoots grown in vitro on MS medium with benzylaminopurine. The aim of our research was to examine the influence of indole-3-butyric acid (IBA) on C. roseus shoots infected with three different phytoplasma strains. Supplement of IBA in the medium supported plant growth, photosynthesis and remission of symptoms in all phytoplasma-infected shoots, but had no effect on the presence of EY-C and SA-I phytoplasma strains in tested tissue. However, HYDB phytoplasma was undetectable in approximately half of the tested shoots grown on the medium with IBA. After 1 year of IBA treatment, HYDB-infected periwinkle shoots were retransferred to the medium supplemented with benzylaminopurine. Some of the shoots showing remission of symptoms during the IBA treatment permanently escaped the infection and remained negative when tested for phytoplasma presence. This is the first report on the differential influence of plant growth regulators on phytoplasma-infected C. roseus shoots.