Diversity of Bacterial Clones and Plasmids of NDM-1 Producing Escherichia coli Clinical Isolates in Central Greece

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer

Plethora of Resistance Genes in Carbapenem-Resistant Gram-Negative Bacteria in Greece: No End to a Continuous Genetic Evolution

Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca. This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution

Clostridioides difficile as a Dynamic Vehicle for the Dissemination of Antimicrobial-Resistance Determinants: Review and In Silico Analysis

The present paper is divided into two parts. The first part focuses on the role of Clostridioides difficile in the accumulation of genes associated with antimicrobial resistance and then the transmission of them to other pathogenic bacteria occupying the same human intestinal niche. The second part describes an in silico analysis of the genomes of C. difficile available in GenBank, with regard to the presence of mobile genetic elements and antimicrobial resistance genes. The diversity of the C. difficile genome is discussed, and the current status of resistance of the organisms to various antimicrobial agents is reviewed. The role of transposons associated with antimicrobial resistance is appraised; the importance of plasmids associated with antimicrobial resistance is discussed, and the significance of bacteriophages as a potential shuttle for antimicrobial resistance genes is presented. In the in silico study, 1101 C. difficile genomes were found to harbor mobile genetic elements; Tn6009, Tn6105, CTn7 and Tn6192, Tn6194 and IS256 were the ones more frequently identified. The genes most commonly harbored therein were: ermB, blaCDD, vanT, vanR, vanG and vanS. Tn6194 was likely associated with resistance to erythromycin, Tn6192 and CTn7 with resistance to the β-lactams and vancomycin, IS256 with resistance to aminoglycoside and Tn6105 to vancomycin

Diversity of Bacterial Clones and Plasmids of NDM-1 Producing <i>Escherichia coli</i> Clinical Isolates in Central Greece

The objective of the present study was to genetically characterize ten NDM-1 producing Escherichia coli isolates, recovered from patients in a hospital in Central Greece during the period 2017 to 2021.The isolates were studied by whole genome sequencing to obtain multi-locus sequencing typing (MLST), identification of blaNDM1-environment, resistome and plasmid content. MLST analysis showed the presence of eight sequence types: ST46* (two isolates), ST46, ST744, ST998, ST410, ST224, ST4380, ST683 and ST12 (one isolate each). Apart of the presence of blaNDM-1, the isolates carried a combination of various to β-lactams encoding resistance genes: blaTEM-1B, blaCTX-15, blaOXA-1, blaVIM-1, blaSHV-5, blaOXA-16, blaOXA-10 and blaVEB-1. Additionally, plurality of resistance genes to aminoglycosides, macrolides, rifamycin, phenicols, sulfonamides and tetracycline was detected. The presence of multiple replicons was observed, with predominance of IncFII and IncFIB. Analysis of blaNDM-1 genetic environment of the isolates showed that seven had 100% identity with the pS-3002cz plasmid (Accession Number KJ 958927), two with the pB-3002cz plasmid (Accession Number KJ958926) and one with the pEc19397-131 plasmid (Accession Number MG878866). Τhis latter plasmid was derived by the fusion of two, previously identified, plasmids, pAMPD2 and pLK75 (Accession Numbers CP078058 and KJ440076, respectively). The diversity of clones and plasmids of NDM-1 producing E. coli isolated from patients in Greece indicates a continuous horizontal gene transfer

First Detection and Molecular Characterization of <i>Pseudomonas aeruginosa bla</i>_NDM-1 ST308 in Greece

The objective of the present study is to report the detection and the molecular characterization of nine blaNDM-1-positive Pseudomonas aeruginosa isolates, all of which belonged to the epidemic high-risk international clone ST308, and all were isolated from patients in a tertiary care hospital in Central Greece from May to July 2023.The isolates were characterized by whole genome sequencing to obtain multi-locus sequencing typing (MLST) and identify the blaNDM1-environment and resistome and virulence genes content. In silico MLST analysis showed that all isolates belonged to the high-risk ST308 international clone. All strains possessed 22 different genes, encoding resistance to various antimicrobial agents. Whole genome sequencing revealed that the blaNDM-1 was chromosomally located within the integrative and conjugative element ICETn43716385 and that it was part of one cassette along with two other resistance genes, floR and msrE. Two additional resistance cassettes were also found in the genome, which included the arrays of aph(6)-Id, aph(3″)-Ib, floR, sul2 and aadA10, qnrVC1, aac(3)-Id, dfrB5, aac(6′)-II. Additionally, the strains possessed various virulence genes, e.g., aprA, exoU, lasA, lasB, toxA, and estA. All of the isolates shared identical genomes, which showed 98% similarity with the P. aeruginosa ST308 genome (acc. no CP020703), previously reported from Singapore. To our knowledge, this is the first report of ST308 blaNDM-1-positive P. aeruginosa isolation in Europe, which indicates the transmission dynamics of this high-risk clone

Combination of rRT-PCR and Anti-Nucleocapsid/Anti-Spike Antibodies to Characterize Specimens with Very Low Viral SARs-CoV-2 Load: A Real-Life Experience

The objective of the present study was to evaluate the true positivity among people, whose results of initial testing of nasopharyngeal swabs (NPS) showed a very low viral load of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Seventy-seven people detected with low viral loads of SARs-CoV-2 in nasopharyngeal samples (Ct ≥ 35) were enrolled in the study. For this purpose, a second NPS was collected for rRT-PCR (real-time reverse transcription polymerase chain reaction) combined with a pair of serum samples for detection of anti-nucleocapsid (anti-N) and anti-spike (anti-S) antibodies. In 8 people, subsequent examinations indicated an increase in viral loads, thereafter, followed by an increase of anti-N and anti-S antibodies, findings compatible with an early stage of COVID-19 infection. In 9 people, who already had increased anti-N antibodies, subsequent examination showed a decrease or absence of viral load and an increase in antibodies, indicative of a late stage of COVID-19 infection. In 60 people, subsequent examination showed absence of infection (as indicated by absence of viral load and antibodies). We propose that the combination of a second NPS and one serum-specimen, both taken three days after the first NPS, helps significantly to avoid false-positive results

Detection of Zoonotic Gastrointestinal Pathogens in Dairy Sheep and Goats by Using FilmArray&reg; Multiplex-PCR Technology

The objectives of this study were (a) to detect gastrointestinal pathogens in faecal samples of sheep and goats using the FilmArray&reg; GI Panel and (b) to evaluate factors that were associated with their presence. Faecal samples from ewes or does in 70 sheep flocks and 24 goat herds in Greece were tested for the presence of 22 gastrointestinal pathogens by means of the BioFire&reg; FilmArray&reg; Gastrointestinal (GI) Panel. The most frequently detected pathogens were Shiga-like toxin-producing Escherichia coli stx1/stx2 (94.7% of farms), Giardia lamblia (59.6%), and Campylobacter spp. (50.0% of farms). Other pathogens detected were Cryptosporidium spp., Salmonella spp., enterotoxigenic E. coli lt/st, Yersinia enterocolitica, E. coli O157, Rotavirus A, Shigella/enteroinvasive E. coli, and Plesiomonasshigelloides. There was a difference in the prevalence of detection of pathogens between sheep and goat farms only for Salmonella spp.: 18.3% versus 0.0%, respectively. Mixed infections were detected in 76 farms (80.9% of farms), specifically 57 sheep flocks and 19 goat herds, with on average, 2.5 &plusmn; 0.1 pathogens detected per farm. The body condition score of ewes in farms, in which only one pathogen was detected in faecal samples, was significantly higher than that of ewes in farms, in which at least two pathogens were detected: 2.55 &plusmn; 0.11 versus 2.31 &plusmn; 0.04. In sheep flocks, the number of pathogens in faecal samples was significantly higher in farms with semi-extensive management. In goat herds, the number of pathogens in faecal samples was positively correlated with average precipitation and inversely correlated with temperature range in the respective locations

emm Types and clusters and macrolide resistance of pediatric group A streptococcal isolates in Central Greece during 2011-2017.

BACKGROUND:The surveillance of emm types and macrolide susceptibility of group A streptococcus (GAS) in various areas and time periods enhances the understanding of the epidemiology of GAS infections and may guide treatment strategies and the formulation of type-specific vaccines. Greece has emerged as a country with high macrolide use. However, studies suggest a gradual reduction in macrolide consumption after 2007. METHODS:During a 7-year period (2011-2017), 604 GAS isolates were recovered from consecutive children presenting with pharyngeal or nonpharyngeal infections in Central Greece; 517 viable isolates underwent molecular analysis, including emm typing. RESULTS:Isolates belonged to 20 different emm types (in decreasing order of prevalence: 1, 89, 4, 12, 28, 3, 75 and 6, accounting for 88.2% of total isolates). The emm types comprised 10 emm clusters (five most common clusters: E4, A-C3, E1, A-C4 and A-C5). The emm89 isolates were acapsular ('new clade'). Overall macrolide resistance rate was 15.4%, and cMLSB emerged as the predominant resistance phenotype (56.4%). The lowest annual resistance rates occurred in 2014 (13.1%), 2016 (5.5%) and 2017(8.0%) (P for trend = 0.002). Consumption of macrolide/lincosamide/streptogramin B declined by 22.6% during 2011-2017. Macrolide resistance and emm28 and emm77 types were associated (both P<0.001). The most frequently identified genetic lineages of macrolide-resistant GAS included emm28/ST52, emm77/ST63, emm12/ST36, emm89/ST101 and emm4/ST39. We estimated that 98.8% of the isolates belonged to emm types incorporated into a novel 30-valent M protein vaccine. CONCLUSIONS:In Central Greece during 2011-2017, the acapsular emm89 isolates comprised the second most prevalent type. Susceptibility testing and molecular analyses revealed decreasing GAS macrolide resistance rates, which may be attributed to the reduction in the consumption of macrolides and/or the reduced circulation of macrolide-resistant clones in recent years. Such data may provide valuable baseline information in targeting therapeutic intervention and the formulation of type-specific GAS vaccines

Recovery of Staphylococci from Teatcups in Milking Parlours in Goat Herds in Greece: Prevalence, Identification, Biofilm Formation, Patterns of Antibiotic Susceptibility, Predictors for Isolation

The objectives of this work are (a) to describe staphylococci on the teatcups of milking parlours in goat farms and identify predictors for the presence of staphylococcal isolates on the teatcups, (b) to evaluate relationships with total bacterial counts and somatic cell counts in bulk-tank milk, and (c) to establish patterns of susceptibility to antibiotics for the staphylococcal isolates and identify predictors for the recovery of resistant isolates. In a cross-sectional study of 66 goat farms across Greece, swab samples were collected from 303 teatcups (upper and lower part) for staphylococcal recovery, identification, and assessment of biofilm formation. Details regarding health management on the farms (including conditions in the milking parlour) and the socio-demographic characteristics of farmers were collected by means of a structured questionnaire. A total of 87 contaminated teatcups (28.7%) were found on 35 goat farms (53.0%). Staphylococci were more frequently recovered from the upper than the lower part of teatcups: 73 versus 43 teatcups, respectively. After identification, 67 staphylococcal isolates (i.e., excluding similar isolates) were recovered from the teatcups; Staphylococcus aureus, Staphylococcus capitis, and Staphylococcus equorum predominated. Of these isolates, 82.1% were biofilm-forming. In multivariable analysis, the annual incidence of clinical mastitis in the herd emerged as the only significant factor associated with the isolation of staphylococci from the teatcups. Of the 67 isolates, 23 (34.3%) were resistant to at least one antibiotic, and 14 (22.4%) were multi-resistant. Resistance was found most commonly against penicillin and ampicillin (22.4% of isolates), fosfomycin (17.9%), clindamycin (14.9%), erythromycin, and tetracycline (13.4%). In multivariable analysis, the annual incidence of clinical mastitis in the herd and the use of detergent for parlour cleaning emerged as significant factors associated with the isolation of staphylococci resistant to antibiotics