Pathogens Detected in Questing Ixodes ricinus Ticks in a Mountainous Area in Greece.

Ixodes ricinus ticks are vectors of a plethora of pathogens. The purpose of this study was to screen 398 I. ricinus ticks for a variety of pathogens. Following the pooling, homogenization, and extraction of total nucleic acids, a real-time PCR was applied for the detection of a panel of tick-borne pathogens, while additional conventional PCRs combined with Sanger sequencing were applied for the detection of viruses and typing of Rickettsia and Borrelia species. At least one pathogen was detected in 60 of the 80 (75%) tick pools. Rickettsia spp. predominated, as it was detected in 63.75% of the pools (51/80; MIR 12.81%), followed by Borrelia spp. (35 pools (45%); MIR 8.79%), while Anaplasma phagocytophilum was detected in 2 pools (2.5%, MIR 0.5%). The ticks of six Rickettsia-positive pools were tested individually (from stored half-ticks); all sequences were identical to those of R. monacensis. Similarly, the ticks of six Borrelia-positive pools were tested individually, and it was shown that four belonged to the genospecies Borrelia garinii and two to Borrelia valaisiana. Phleboviruses were detected in 3 pools (3.75%; MIR 0.75%), with sequences clustering in the Ixovirus genus, while nairoviruses were detected in 7 pools (8.75%; MIR 1.76%), with one sequence clustering in the Orthonairovirus genus, and six clustering in the Norwavirus genus. Although a small number of ticks from only one area in Greece were tested, a variety of pathogens together with recently identified viruses were detected, prompting further studies in ticks and surveillance studies in humans.

West Nile virus spread in Europe: Phylogeographic pattern analysis and key drivers.

BACKGROUND: West Nile virus (WNV) outbreaks in birds, humans, and livestock have occurred in multiple areas in Europe and have had a significant impact on animal and human health. The patterns of emergence and spread of WNV in Europe are very different from those in the US and understanding these are important for guiding preparedness activities. METHODS: We mapped the evolution and spread history of WNV in Europe by incorporating viral genome sequences and epidemiological data into phylodynamic models. Spatially explicit phylogeographic models were developed to explore the possible contribution of different drivers to viral dispersal direction and velocity. A "skygrid-GLM" approach was used to identify how changes in environments would predict viral genetic diversity variations over time. FINDINGS: Among the six lineages found in Europe, WNV-2a (a sub-lineage of WNV-2) has been predominant (accounting for 73% of all sequences obtained in Europe that have been shared in the public domain) and has spread to at least 14 countries. In the past two decades, WNV-2a has evolved into two major co-circulating clusters, both originating from Central Europe, but with distinct dynamic history and transmission patterns. WNV-2a spreads at a high dispersal velocity (88km/yr-215 km/yr) which is correlated to bird movements. Notably, amongst multiple drivers that could affect the spread of WNV, factors related to land use were found to strongly influence the spread of WNV. Specifically, the intensity of agricultural activities (defined by factors related to crops and livestock production, such as coverage of cropland, pasture, cultivated and managed vegetation, livestock density) were positively associated with both spread direction and velocity. In addition, WNV spread direction was associated with high coverage of wetlands and migratory bird flyways. CONCLUSION: Our results suggest that-in addition to ecological conditions favouring bird- and mosquito- presence-agricultural land use may be a significant driver of WNV emergence and spread. Our study also identified significant gaps in data and the need to strengthen virological surveillance in countries of Central Europe from where WNV outbreaks are likely seeded. Enhanced monitoring for early detection of further dispersal could be targeted to areas with high agricultural activities and habitats of migratory birds.

Neonatal Bloodstream Infection with Ceftazidime-Avibactam-Resistant blaKPC-2-Producing Klebsiella pneumoniae Carrying blaVEB-25.

BACKGROUND: Although ceftazidime/avibactam (CAZ/AVI) has become an important option for treating adults and children, no data or recommendations exist for neonates. We report a neonatal sepsis case due to CAZ/AVI-resistant blaKPC-2-harboring Klebsiella pneumoniae carrying blaVEB-25 and the use of a customized active surveillance program in conjunction with enhanced infection control measures. METHODS: The index case was an extremely premature neonate hospitalized for 110 days that had been previously treated with multiple antibiotics. Customized molecular surveillance was implemented at hospital level and enhanced infection control measures were taken for early recognition and prevention of outbreak. Detection and identification of blaVEB-25 was performed using next-generation sequencing. RESULTS: This was the first case of a bloodstream infection caused by KPC-producing K. pneumoniae that was resistant to CAZ/AVI without the presence of a metalo-ß-lactamase in the multiplex PCR platform in a neonate. All 36 additional patients tested (12 in the same NICU and 24 from other hospital departments) carried wild-type blaVEB-1 but they did not harbor blaVEB-25. CONCLUSION: The emergence of blaVEB-25 is signal for the horizontal transfer of plasmids at hospital facilities and it is of greatest concern for maintaining a sharp vigilance for the surveillance of novel resistance mechanisms. Molecular diagnostics can guide appropriate antimicrobial therapy and the early implementation of infection control measures against antimicrobial resistance.

The 2022 West Nile Virus Season in Greece; A Quite Intense Season.

Since 2010, the West Nile virus (WNV) has been established in Greece. We describe the epidemiology of diagnosed human WNV infections in Greece with a focus on the 2022 season. During the transmission period, clinicians were sending samples from suspected cases for testing. Active laboratory-based surveillance was performed with immediate notification of diagnosed cases. We collected clinical information and interviewed patients on a timely basis to identify their place of exposure. Besides serological and molecular diagnostic methods, next-generation sequencing was also performed. In 2022, 286 cases of WNV infection were diagnosed, including 278 symptomatic cases and 184 (64%) cases with neuroinvasive disease (WNND); 33 patients died. This was the third most intense season concerning the number of WNND cases, following 2018 and 2010. Most (96%) cases were recorded in two regions, in northern and central Greece. The virus strain was a variant of previous years, clustering into the Central European subclade of WNV lineage 2. The 2022 WNV season was quite intense in Greece. The prompt diagnosis and investigation of cases are considered pivotal for the timely response, while the availability of whole genome sequences enables studies on the molecular epidemiology of the disease.

Genetic Characterization of Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates in a Tertiary Hospital in Greece, 2018-2022.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious public health issue. The study aimed to identify the antimicrobial resistance and accessory genes, the clonal relatedness, and the evolutionary dynamics of selected CRKP isolates recovered in an adult and pediatric intensive care unit of a tertiary hospital in Greece. METHODS: Twenty-four CRKP isolates recovered during 2018-2022 were included in the study. Next-generation sequencing was performed using the Ion Torrent PGM Platform. The identification of the plasmid content, MLST, and antimicrobial resistance genes, as well as the comparison of multiple genome alignments and the identification of core genome single-nucleotide polymorphism sites, were performed using various bioinformatics software. RESULTS: The isolates belonged to eight sequence types: 11, 15, 30, 35, 39, 307, 323, and 512. A variety of carbapenemases (KPC, VIM, NDM, and OXA-48) and resistance genes were detected. CRKP strains shared visually common genomic regions with the reference strain (NTUH-K2044). ST15, ST323, ST39, and ST11 CRKP isolates presented on average 17, 6, 16, and 866 recombined SNPs, respectively. All isolates belonging to ST15, ST323, and ST39 were classified into distinct phylogenetic branches, while ST11 isolates were assigned to a two-subclade branch. For large CRKP sets, the phylogeny seems to change approximately every seven SNPs. CONCLUSIONS: The current study provides insight into the genetic characterization of CRKP isolates in the ICUs of a tertiary hospital. Our results indicate clonal dispersion of ST15, ST323, and ST39 and highly diverged ST11 isolates.

Overview of Mpox Outbreak in Greece in 2022-2023: Is It Over?

In May 2022, for the first time, multiple cases of mpox were reported in several non-endemic countries. The first ever case of the disease in Greece was confirmed on 8 June 2022, and a total of 88 cases were reported in the country until the end of April 2023. A multidisciplinary response team was established by the Greek National Public Health Organization (EODY) to monitor and manage the situation. EODY's emergency response focused on enhanced surveillance, laboratory testing, contact tracing, medical countermeasures, and the education of health care providers and the public. Even though management of cases was considered successful and the risk from the disease was downgraded, sporadic cases continue to occur. Here, we provide epidemiological and laboratory features of the reported cases to depict the course of the disease notification rate. Our results suggest that measures for raising awareness as well as vaccination of high-risk groups of the population should be continued.

Simultaneous Multiple-Stages Mpox Genital Lesions on the Same Site in a Traveler to Greece: A Case Report.

A 47-year-old Caucasian traveller from an mpox (formerly monkeypox and also best suited abbreviated MPX)-endemic country was referred for a skin rash, of recent onset, confined to the genital area. The rash consisted of erythematous umbilicated papules, vesicles and pustules with a characteristic white ring. The lesions were observed simultaneously at different stages of progression on the same anatomical site, a clinical presentation that is not encountered frequently. The patient was febrile, fatigued and had blood-tinged cough. The clinical suspicion of mpox was raised, and the initial real-time PCR identified a non-variola orthopox virus, which was confirmed at the National Reference Laboratory to belong to the West African clade.

West Nile Virus in Culex Mosquitoes in Central Macedonia, Greece, 2022.

In 2022, Greece was the second most seriously affected European country in terms of the West Nile virus (WNV), after Italy. Specifically, Central Macedonia was the region with the most reported human cases (81.5%). In the present study, 30,816 female Culex pipiens sensu lato mosquitoes were collected from May to September 2022 in the seven regional units of Central Macedonia; they were then grouped into 690 pools and tested for WNV, while next-generation sequencing was applied to the samples, which showed a cycle threshold of Ct < 30 in a real-time RT-PCR test. WNV was detected in 5.9% of pools, with significant differences in the detection rate among regional units and months. It is of interest that in the Thessaloniki regional unit, where most of the human cases were observed, the virus circulation started earlier, peaked earlier, and lasted longer than in the other regional units. All sequences clustered into the Central European subclade of WNV lineage 2, and the virus strain differed from the initial Greek strain of 2010 by 0.52% and 0.27% at the nucleotide and amino acid levels, respectively. Signature substitutions were present, such as S73P and T157A in the prM and E structural proteins, respectively. The screening of mosquitoes provides useful information for virus circulation in a region with a potential for early warning, while the availability of whole-genome sequences is essential for further studies, including virus evolution.

Development of Toehold Switches as a Novel Ribodiagnostic Method for West Nile Virus.

West Nile virus (WNV) is an emerging neurotropic RNA virus and a member of the genus Flavivirus. Naturally, the virus is maintained in an enzootic cycle involving mosquitoes as vectors and birds that are the principal amplifying virus hosts. In humans, the incubation period for WNV disease ranges from 3 to 14 days, with an estimated 80% of infected persons being asymptomatic, around 19% developing a mild febrile infection and less than 1% developing neuroinvasive disease. Laboratory diagnosis of WNV infection is generally accomplished by cross-reacting serological methods or highly sensitive yet expensive molecular approaches. Therefore, current diagnostic tools hinder widespread surveillance of WNV in birds and mosquitoes that serve as viral reservoirs for infecting secondary hosts, such as humans and equines. We have developed a synthetic biology-based method for sensitive and low-cost detection of WNV. This method relies on toehold riboswitches designed to detect WNV genomic RNA as transcriptional input and process it to GFP fluorescence as translational output. Our methodology offers a non-invasive tool with reduced operating cost and high diagnostic value that can be used for field surveillance of WNV in humans as well as in bird and mosquito populations.

Clinical Performance Evaluation of the NeuMoDx Flu A-B/RSV/SARS-CoV-2 Vantage Assay.

SARS-CoV-2 infections may present with various symptoms that are similar to those of other respiratory diseases. For this reason, the need for simultaneous detection of at least RSV and influenza viruses together with SARS-CoV-2 was evident from the early stages of the pandemic. In the present study, we evaluated the clinical performance of the NeuMoDx™ Flu A-B/RSV/SARS-CoV-2 Vantage Assay against the conventional low-plex PCR utilized to detect influenza A-B, RSV, and SARS-CoV-2. There were 115 known positive clinical samples and 35 negative controls obtained from asymptomatic health-care workers included in the study; 25 samples were positive for influenza viruses, 46 for RSV, and 44 for SARS-CoV-2. The sensitivity, specificity, positive predictive value, and negative predictive value of the evaluated method for influenza and SARS-CoV-2 were 100%. The Spearman correlation coefficient was 0.586 (p < 0.05) for influenza and 0.893 (p < 0.05) for SARS-CoV-2. The sensitivity of the aforementioned assay for RSV was 93.47%; the specificity and the positive predictive value were 100%, and the negative predictive value was 92.10%, while the Spearman correlation coefficient was not applicable for the RSV. Overall, the assay under evaluation was shown to be a reliable alternative for the simultaneous detection of influenza viruses, RSV and SARS-CoV-2.

Prevalence, Infectious Characteristics and Genetic Diversity of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus (MRSA) in Two Raw-Meat Processing Establishments in Northern Greece.

In the present study, we investigated the isolation frequency, the genetic diversity, and the infectious characteristics of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) from the incoming meat and the meat products, the environment, and the workers' nasal cavities, in two meat-processing establishments in northern Greece. The isolated S. aureus strains were examined for their resistance to antimicrobials, carriage of the mecA and mecC genes, carriage of genes encoding for the production of nine staphylococcal enterotoxins, carriage of the Panton-Valentine Leukocidin and Toxic Shock Syndrome genes, and the ability to form biofilm. The genetic diversity of the isolates was evaluated using Pulsed Field Gel Electrophoresis (PFGE) and spa typing. S. aureus was isolated from 13.8% of the 160 samples examined, while only one sample (0.6%) was contaminated by MRSA carrying the mecA gene. The evaluation of the antimicrobial susceptibility of the isolates revealed low antimicrobial resistance. The higher resistance frequencies were observed for penicillin (68.2%), amoxicillin/clavulanic acid (36.4%) and tetracycline (18.2%), while 31.8% of the isolates were sensitive to all antimicrobials examined. Multidrug resistance was observed in two isolates. None of the isolates carried the mecC or lukF-PV genes, and two isolates (9.1%) harbored the tst gene. Eight isolates (36.4%) carried the seb gene, one carried the sed gene, two (9.1%) carried both the sed and sei genes, and one isolate (4.5%) carried the seb, sed and sei genes. Twenty-one (95.5%) of the isolates showed moderate biofilm production ability, while only one (4.5%) was characterized as a strong biofilm producer. Genotyping of the isolates by PFGE indicates that S. aureus from different meat-processing establishments represent separate genetic populations. Ten different spa types were identified, while no common spa type isolates were detected within the two plants. Overall, our findings emphasize the need for the strict application of good hygienic practices at the plant level to control the spread of S. aureus and MRSA to the community through the end products.

Spa diversity and genetic characterization of t127 methicillin-resistant Staphylococcus aureus in a tertiary Greek hospital.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) causes severe community and hospital acquired infections. Identification of staphylococcal cassette chromosome mec (SCCmec), multilocus-sequence typing, and sequencing of S. aureus protein A (spa) gene are used for MRSA typing. The aim was to investigate the spa types of MRSA isolates in a tertiary hospital in Greece and analyse the whole genome sequences of two t127 MRSA isolates. Methods: Totally, 39 MRSA isolates collected from July 2019 to June 2020 in "Georgios Gennimatas" General Hospital of Thessaloniki, Greece, were included in the study. Identification and antimicrobial susceptibility testing were performed using VITEK II automated system, and spa typing was performed. A minimum spanning tree was used to display the spa type frequencies and the genetic distances among them. Two t127-MRSA isolates (IM-MRSA and PD-MRSA) were selected for WGS. Results: Six isolates (15.4%) were resistant to mupirocin, 18 (46.2%) to fusidic acid, three (7.7%) to vancomycin and two (5.1%) to teicoplanin. Twenty-two different spa types were detected, with t002, t003, and t422 being the most frequent (5/39, 12.8% each), followed by t1994 (4/39, 10.3%). The isolates presented high genetic diversity and, taking into account the time between hospital admission and sampling, intrahospital spread did not occur. Even the two t127 isolates were assigned to different sequence types, ST9-XII-t127 and ST1-IVa-t127. Plasmids and genes conferring antimicrobial resistance and virulence were also identified. Conclusions: Various spa types were identified and together with the information about the time between hospital admission and sampling supports polyclonal MRSA spread in the hospital excluding a nosocomial infection. WGS provides a more detailed analysis distinguishing even the isolates belonging to the same spa type.

Changes in molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in the intensive care units of a Greek hospital, 2018-2021.

The spread of multi-drug resistant (MDR) Gram-negative bacteria, including Klebsiella pneumoniae, constitutes a global threat. The most frequent mechanism of acquired carbapenem resistance is the production of carbapenemases, especially KPC, NDM, VIM, IMP and OXA-48. We analyzed the epidemiological trend of carbapenem resistance genes of carbapenem-resistant K. pneumoniae (CRKP) strains isolated from critically ill patients in a Greek tertiary hospital. The study included 150 CRKP isolates collected from 116 (77.4%) patients hospitalized in the adult ICU and 17 (11.3%) each in the pediatric and the two neonatal ICUs between March 2018 and March 2021. Identification and antimicrobial susceptibility testing were performed using VITEK-2. A multiplex lateral flow immunoassay was used for the detection of carbapenemases, while the detection of bla VIM, bla KPC, bla NDM, bla IMP and bla OXA-48-like genes was achieved by multiplex PCR. The bla NDM was mainly detected in adults (54/116, 46.9%), while in children the most often detected gene was bla KPC (24/34, 70.6%). The predominant carbapenem resistance gene during 2018-2019 was bla KPC alone or in combination with bla VIM, reaching 44.4% in 2019, while during 2020-2021 the detection of bla NDM prevailed significantly, reaching 45.5 and 60.7% for 2020 and 2021, respectively. A shift in the molecular epidemiology of CRKP was seen during 2018-2021, which is probably associated with the recent excessive empiric use of newer antimicrobials. Surveillance studies and proper and strict implementation of infection control measures are highly needed to decrease the spread of MDR bacteria, including CRKP.

Detection and molecular characterization of West Nile virus in Culex pipiens mosquitoes in Central Macedonia, Greece, 2019-2021.

Since 2010 when West Nile virus (WNV) emerged in Greece, it causes seasonal outbreaks of human infections almost every year. During May-October of 2019-2021 a total number of 51,504 Culex pipiens mosquitoes were trapped in all seven regional units of Central Macedonia in northern Greece. They were grouped into 1099 pools and tested for WNV. The virus was detected in 5% of the mosquito pools (1.5%, 3.6% and 9.6% pools in 2019, 2020, and 2021, respectively), with significant rate differences among the regional units and years. The highest maximum likelihood estimation for WNV infection rates calculated per 1000 mosquitoes for 2019 and 2020 were 1.89 and 3.84 in Serres, and 7.08 for 2021 in Pella regional unit. Sixteen whole genome sequences were taken by applying a recently described PCR-based next generation sequencing protocol. Phylogenetic analysis showed that the sequences belonged to the Central European clade of WNV lineage 2, and that a virus strain introduced in Greece in 2019 continued to circulate and spread further during 2020-2021. The data are useful for public health and mosquito control programs' operational scheduling, while the whole genome sequences are an added value for molecular epidemiology and evolutionary studies.

Early initiation of the respiratory syncytial virus season in 2021-2022, Greece.

Respiratory syncytial virus (RSV) is the most common viral pathogen causing respiratory disease in the pediatric population. An unexpected sudden upsurge of RSV infections among children was observed in September 2021 in Greece. Forty-one rhinopharyngeal samples from children under the age of 2 years with confirmed RSV bronchiolitis were tested to identify the genotype(s) of the RSV strain(s). The children were hospitalized during September-November 2021 in three tertiary hospitals in northern Greece. A one-step RT-PCR which amplifies a fragment of the second hypervariable region of the G protein gene was applied. PCR products were sequenced, and phylogenetic analysis was performed. Most (80.5%) RSV cases were typed as RSV-A, with RSV-B accounting for 19.5% of cases. RSV-A and RSV-B sequences clustered within the ON1 and BA genotypes, respectively. As the same genotypes were detected in cases observed during 2016-2018 in northern Greece, it was suggested that the early upsurge of infections was not related to the emergence of novel strain(s), but it was the result of the absence of immunity among children and their mothers due to the restriction measures taken during the COVID-19 pandemic in the previous RSV season. Awareness is needed to diagnose even the out-of-season RSV infections, while molecular epidemiology plays a key role in monitoring the efficacy of currently available therapeutics and for those under development.

Emergence of ST39 carbapenem-resistant Klebsiella pneumoniae producing VIM-1 and KPC-2.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKP) causes life-threatening hospital-acquired infections. KPC and VIM carbapenemase production is the main molecular mechanism for carbapenem resistance. The aim of the current study was the genetic characterization of four ST39 CRKP isolates simultaneously producing VIM-1 and KPC-2, obtained in a Greek tertiary hospital. METHODS: Identification and antimicrobial susceptibility testing were performed through VITEK 2. Multiplex PCR, multiplex lateral flow immunoassay, phenotypic tests and next generation sequencing were applied. The sequence reads were de novo assembled and annotated, while antimicrobial resistance genes and plasmids were identified using bioinformatics software. Genomic comparison and core genome single-nucleotide polymorphism-based phylogenetic analysis were also performed. RESULTS: Three isolates were pandrug-resistant, and one was extensively drug-resistant; they all carried blaVIM-1 and blaKPC-2 genes and were assigned to ST39. BlaVIM-1 was integrated in a class 1 integron. They all harboured many antimicrobial resistance genes and various plasmids. The mgrB gene of all isolates was disrupted by an insertion sequence (ISKpn14). Genome comparison and phylogenetic analysis revealed that the isolates were closely related. CONCLUSION: To our knowledge this is the first report on detection of CRKP ST39 isolates simultaneously producing VIM-1 and KPC-2 in addition to colistin resistance. The knowledge of the clonal relatedness of the isolates can lead to the implementation of strict infection control measures absolutely needed to eliminate their spread.

PCR-based next-generation West Nile virus sequencing protocols.

The epidemiology of West Nile virus (WNV) is unpredictable and changing. Availability of whole genome sequences enables the detailed molecular epidemiology studies and the evaluation and design of diagnostic tools. In the present study we provide two PCR-based protocols which can be applied directly on biological samples from hosts infected by WNV strains belonging to lineage 1 or lineage 2. It was shown that the protocols worked successfully even on samples with relatively low viral load.

Prospective evaluation of specimen pooling strategy for detection of SARS-CoV-2 using pools of five and six specimens.

The increased demand for SARS-CoV-2 molecular testing during the COVID-19 pandemic resulted in shortage of reagents and consumables. Pooling of specimens could be an alternative strategy to overcome these problems. Initial evaluation of the pooling strategy was performed using known positive specimens, previously tested individually, and their respective pools of plus four (5X), five (6X) and nine (10X) known negative specimens. Subsequently, 35 positive 5X and 35 positive 6X pools containing only one positive specimen per pool were analyzed prospectively regarding the difference in Ct values in pooled versus individual specimens. When the number of samples in the pool were five or six, the average deviation of Ct differences was < 1; therefore, this strategy was followed in the prospective study. Significant difference in Ct values was observed in positive specimens when tested individually and in 5X pools (p = 0.006), while the difference was not significant when positive specimens were tested individually and in 6X pools (p = 0.07). The difference in Ct values was not significant between the 5X and 6X pools. Testing in pools of five or six specimens is a reliable option for SARS-CoV-2 RNA detection when mass testing is needed.

West Nile fever upsurge in a Greek regional unit, 2020.

During the 2020 West Nile virus (WNV) transmission season, Greece was the most affected EU Member State. More than one third of human cases occurred in Serres regional unit in northern Greece, which is characterized by the presence of a major wetland (Kerkini lake and Strimon river). A total of 2809 Culex pipiens mosquitoes collected in Serres were grouped into 70 pools and tested for WNV. Ten (14.3%) pools were found positive, and all WNV sequences belonged to the Central European subclade of WNV lineage 2. The first human case occurred in a village nearby the lake, and all following cases occurred across the connected river and its tributaries. Similar distribution presented the sites where WNV-positive mosquitoes were detected. The number of Culex spp. mosquitoes per trap per night was higher in 2020 than in previous years (2017-2019). The spatial and temporal distribution of human cases and WNV-positive mosquitoes in 2020 in Serres regional unit suggest that the upsurge of the virus circulation was probably related with factors that affected the ecosystem of the wetland.

Spread of NDM-producing Klebsiella pneumoniae in a tertiary Greek hospital.

Bacterial carbapenem resistance, especially when mediated by transferable carbapenemases, is of important public health concern. An increased number of metallo-ß-lactamase (MBL)-producing Klebsiella pneumoniae strains isolated in a tertiary hospital in Thessaloniki, Greece, called for further genetic investigation.The study included 29 non-repetitive carbapenem resistant K. pneumoniae isolates phenotypically characterized as MBL-producers collected in a tertiary hospital in Greece. The isolates were screened for the detection of carbapenemase genes (K. pneumoniae carbapenemase (blaKPC), Verona-integron-encoded MBL-1 (blaVIM-1), imipenemase (blaIMP), oxacillinase-48 (blaOXA-48) and New Delhi MBL (blaNDM)). The genetic relationship of the isolates was determined by Random Amplified Polymorphic DNA (RAPD) analysis. The whole genome sequences (WGS) from two NDM-positive K. pneumoniae isolates were further characterized.The presence of New Delhi MBL (blaNDM) gene was confirmed in all K. pneumoniae isolates, while blaKPC and blaVIM-1 genes were co-detected in one and two isolates, respectively. The RAPD analysis showed that the isolates were clustered into two groups. The whole genome sequence analysis of two K. pneumoniae isolates revealed that they belonged to the sequence type 11, they carried the blaNDM-1 gene, and exhibited differences in the number and type of the plasmids and the resistant genes.All MBL-producing K. pneumoniae isolates of the study harbored a blaNDM gene, while WGS analysis revealed genetic diversity in resistance genes. Continuous surveillance is needed to detect the emergence of new clones in a hospital setting, while application of antimicrobial stewardship is the only way to reduce the spread of multi-resistant bacteria.