Phenotypic and Molecular Characterization of a Hospital Outbreak Clonal Lineage of Salmonella enterica Subspecies enterica serovar Mikawasima Containing blaTEM-1B and blaSHV-2 That Emerged on a Neonatal Ward, During the COVID-19 Pandemic.

Nontyphoid salmonella can cause severe infections in newborns and is therefore declared a pathogen of major health significance at this age. The aim of the study was molecular and antimicrobial characterization of ß-lactamase-producing Salmonella Mikawasima outbreak clone on a Neonatal ward, University Hospital of Split (UHS), Croatia during the COVID-19 pandemic. From April 2020, until April 2023, 75 nonrepetitive strains of Salmonella Mikawasima were isolated from stool specimens and tested for antimicrobial resistance. All 75 isolates were resistant to ampicillin and gentamicin, while 98% of isolates were resistant to amoxicillin/clavulanic acid. A high level of resistance was observed to third-generation cephalosporins (36% to ceftriaxone and 47% to ceftazidime). Extended-spectrum ß-lactamase production was phenotypically detected by double-disk synergy test in 40% of isolates. Moderate resistance to quinolones was detected; 7% of isolates were resistant to pefloxacin and ciprofloxacin. All isolates were susceptible to carbapenems, chloramphenicol, and co-trimoxazole. Fourteen representative isolates, from 2020, 2021, 2022, and 2023, were analyzed with PFGE and all of them belong to the same clone. Whole-genome sequencing (WGS) analysis of three outbreak-related strains (SM1 and SM2 from 2020 and SM3 from 2023) confirmed that these strains share the same serotype (Mikawasima), multilocus sequence typing profile (ST2030), resistance genes [blaTEM-1B, aac(6')-Iaa, aac(6')-Im, and aph(2'')-Ib)] and carry incompatibility group C (IncC) plasmid. Furthermore, the gene blaSHV-2 was detected in SM1 and SM2. In summary, WGS analysis of three representative strains clearly demonstrates the persistence of ß-lactamase-producing Salmonella Mikawasima in UHS during the 4-year period.

Large-Scale Biogeographical Shifts of Abundance of Antibiotic Resistance Genes and Marine Bacterial Communities as Their Carriers along a Trophic Gradient.

The role of marine environments in the global spread of antibiotic resistance still remains poorly understood, leaving gaps in the One Health-based research framework. Antibiotic resistance genes (ARGs) encoding resistance to five major antibiotic classes, including sulfonamides (sul1, sul2), tetracyclines (tetA, tetB), ß-lactams (blaCTX-M, blaTEMblaVIM), macrolides (ermB, mphA), aminoglycosides (aac3-2), and integrase gene (intl1) were quantified by RT-qPCR, and their distribution was investigated in relation to environmental parameters and the total bacterial community in bottom layer and surface waters of the central Adriatic (Mediterranean), over a 68 km line from the wastewater-impacted estuary to coastal and pristine open sea. Seasonal changes (higher in winter) were observed for antibiotic resistance frequency and the relative abundances of ARGs, which were generally higher in eutrophic coastal areas. In particular, intl1, followed by blaTEM and blaVIM, were strongly associated with anthropogenic influence and Gammaproteobacteria as their predominant carriers. Water column stratification and geographic location had a significant influence on ARGs distribution in the oligotrophic zone, where the bacterial community exhibited a seasonal shift from Gammaproteobacteria in winter to Marine group II in summer.

Molecular Characterization and Mutational Analysis of Clarithromycin- and Levofloxacin-Resistance Genes in Helicobacter pylori from Gastric Biopsies in Southern Croatia.

Point mutations in the 23S rRNA, gyrA, and gyrB genes can confer resistance to clarithromycin (CAM) and levofloxacin (LVX) by altering target sites or protein structure, thereby reducing the efficacy of standard antibiotics in the treatment of Helicobacter pylori infections. Considering the confirmed primary CAM and LVX resistance in H. pylori infected patients from southern Croatia, we performed a molecular genetic analysis of three target genes (23S rRNA, gyrA, and gyrB) by PCR and sequencing, together with computational molecular docking analysis. In the CAM-resistant isolates, the mutation sites in the 23S rRNA gene were A2142C, A2142G, and A2143G. In addition, the mutations D91G and D91N in GyrA and N481E and R484K in GyrB were associated with resistance to LVX. Molecular docking analyses revealed that mutant H. pylori strains with resistance-related mutations exhibited a lower susceptibility to CAM and LVX compared with wild-type strains due to significant differences in non-covalent interactions (e.g., hydrogen bonds, ionic interactions) leading to destabilized antibiotic-protein binding, ultimately resulting in antibiotic resistance. Dual resistance to CAM and LVX was found, indicating the successful evolution of H. pylori resistance to unrelated antimicrobials and thus an increased risk to human health.

Marine resistome of a temperate zone: Distribution, diversity, and driving factors across the trophic gradient.

Marine and ocean environments are the most widespread habitats in the world but are still the least studied from the aspect of antibiotic resistance. The indigenous and tetracycline (TET)- and sulfamethoxazole (SXT)-resistant planktonic bacterial communities were simultaneously investigated for the first time along a trophic gradient of a temperate zone, regarding their taxonomic and functional structures as well as biotic and abiotic factors affecting their dynamics as vehicles of antibiotic resistance genes (ARGs), thus impacting the ARGs distribution at seasonal and spatial scales. A total of 80 microbiomes, recovered seasonally from bottom layer and surface waters along a 68-km transect from wastewater-impacted estuary to coastal and pristine open sea in the central Adriatic (Mediterranean Sea), were analysed using 16S rRNA amplicon sequencing, PICRUSt2 bioinformatics and extensive biostatistics. Eighty-one bacterial phyla were identified, with majority (n = 49) in summer when communities were found to be more species enriched across the gradient. Microbial diversity was more site-specific and pronounced in surface microbiomes in winter. Nevertheless, both richness and community diversity decreased with distance from the coast. Although the microbiomes from human-influenced sites significantly differed from those in oligotrophic offshore area, Proteobacteria were still the most abundant phylum during both seasons at the surface and seabed along the gradient, and the major contributors to the marine resistome regarding native and TET- and SXT-resistant microbial communities. Resistome structure was more diverse in winter, whereas peptide, vancomycin and multidrug resistance modules predominated regardless of season, trophic status, or antibiotic. However, multidrug, beta-lactam resistance modules as well as macrolide, phenicol, aminoglycoside, and particularly imipenem resistance genes were much more frequent in winter, suggesting that the diversity of indigenous resistomes is highly dependent on seasonal variations of the water column, driven by thermohaline stratification and nutrients. Moreover, several pathogenic genera stood out as important carriers of multiple resistance traits in TET- and SXT-related resistomes in both seasons, particularly Acinetobacter, Vibrio, Bacillus and Pseudomonas, beside which Proteus, Serratia and Bacteroides prevailed in native resistomes. This study evidenced seasonal and spatial variations of the marine microbiome and resistome and their driving forces along the trophic gradient, providing a comprehensive insight into the diversity and distribution of antibiotic resistance in the marine ecosystem of a temperate zone.

Characterization of Third Generation Cephalosporin- and Carbapenem-Resistant Aeromonas Isolates from Municipal and Hospital Wastewater.

Antibiotic resistance (AR) remains one of the greatest threats to global health, and Aeromonas species have the potential to spread AR in the aquatic environment. The spread of resistance to antibiotics important to human health, such as third-generation cephalosporins (3GCs) and carbapenems, is of great concern. We isolated and identified 15 cefotaxime (3GC)- and 51 carbapenem-resistant Aeromonas spp. from untreated hospital and treated municipal wastewater in January 2020. The most common species were Aeromonas caviae (58%), A. hydrophila (17%), A. media (11%), and A. veronii (11%). Almost all isolates exhibited a multidrug-resistant phenotype and harboured a diverse plasmidome, with the plasmid replicons ColE, IncU, and IncR being the most frequently detected. The most prevalent carbapenemase gene was the plasmid-associated blaKPC-2 and, for the first time, the blaVIM-2, blaOXA-48, and blaIMP-13 genes were identified in Aeromonas spp. Among the 3GC-resistant isolates, the blaGES-5 and blaMOX genes were the most prevalent. Of the 10 isolates examined, three were capable of transferring carbapenem resistance to susceptible recipient E. coli. Our results suggest that conventionally treated municipal and untreated hospital wastewater is a reservoir for 3GC- and carbapenem-resistant, potentially harmful Aeromonas spp. that can be introduced into aquatic systems and pose a threat to both the environment and public health.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources.

Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we determined for the first time the antibiotic resistance and virulence genes, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside-resistance genes aac(6')-Ie-aph(2″)-Ia, and aph(2″)-Ib and aph(2″)-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence gene. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination. To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.

Metagenomic analysis of pioneer biofilm-forming marine bacteria with emphasis on Vibrio gigantis adhesion dynamics.

Marine biofilms occur frequently and spontaneously in seawater, on almost any submerged solid surface. At the early stages of colonization, it consists of bacteria and evolves into a more complex community. Using 16S rRNA amplicon sequencing and comparative metagenomics, the composition and predicted functional potential of one- to three-day old bacterial communities in surface biofilms were investigated and compared to that of seawater. This confirmed the autochthonous marine bacterium Vibrio gigantis as an early and very abundant biofilm colonizer, also functionally linked to the genes associated with cell motility, surface attachment, and communication via signaling molecules (quorum sensing), all crucial for biofilm formation. The dynamics of adhesion on a solid surface of V. gigantis alone was also monitored in controlled laboratory conditions, using a newly designed and easily implementable protocol. Resulting in a calculated percentage of bacteria-covered surface, a convincing tendency of spontaneous adhering was confirmed. From the multiple results, its quantified and reproducible adhesion dynamics will be used as a basis for future experiments involving surface modifications and coatings, with the goal of preventing adhesion.

Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment.

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43-90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes bla KPC-2 and bla OXA-48. The bla OXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the bla KPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as bla KPC-2 vesicles, providing first report of these bla KPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of bla KPC-2 and bla OXA-48 carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.

Microbiome and antibiotic resistance profiling in submarine effluent-receiving coastal waters in Croatia.

Wastewater treatment plant (WWTP) effluents are pointed as hotspots for the introduction of both commensal and pathogenic bacteria as well as their antibiotic resistance genes (ARGs) in receiving water bodies. For the first time, the effect of partially treated submarine effluents was explored at the bottom and surface of the water column to provide a comprehensive overview of the structure of the microbiome and associated AR, and to assess environmental factors leading to their alteration. Seawater samples were collected over a 5-month period from submarine outfalls in central Adriatic Sea, Croatia. 16S rRNA amplicon sequencing was used to establish taxonomic and resistome profiles of the bacterial communities. The community differences observed between the two discharge areas, especially in the abundance of Proteobacteria and Firmicutes, could be due to the origin of wastewaters treated in WWTPs and the limiting environmental conditions such as temperature and nutrients. PICRUSt2 analysis inferred the total content of ARGs in the studied microbiomes and showed the highest abundance of resistance genes encoding multidrug efflux pumps, such as MexAB-OprM, AcrEF-TolC and MdtEF-TolC, followed by the modified peptidoglycan precursors, transporter genes encoding tetracycline, macrolide and phenicol resistance, and the bla operon conferring ß-lactam resistance. A number of pathogenic genera introduced by effluents, including Acinetobacter, Arcobacter, Bacteroides, Escherichia-Shigella, Klebsiella, Pseudomonas, and Salmonella, were predicted to account for the majority of efflux pump-driven multidrug resistance, while Acinetobacter, Salmonella, Bacteroides and Pseudomonas were also shown to be the predominant carriers of non-efflux ARGs conferring resistance to most of nine antibiotic classes. Taken together, we evidenced the negative impact of submarine discharges of treated effluents via alteration of physico-chemical characteristics of the water column and enrichment of bacterial community with nonindigenous taxa carrying an arsenal of ARGs, which could contribute to the further propagation of the AR in the natural environment.

Bacteria tolerant to colistin in coastal marine environment: Detection, microbiome diversity and antibiotic resistance genes' repertoire.

The global spread of mobilized colistin resistance (mcr) genes in clinical and natural environments dangerously diminishes the effectiveness of this last-resort antibiotic, becoming an urgent health threat. We used a multidisciplinary approach to detect mcr-1 gene and colistin (CL)-resistant bacteria in seawater from two Croatian public beaches. Illumina-based sequencing of metagenomic 16S rRNA was used to assess the taxonomic, functional, and antibiotic resistance genes (ARGs) profiling of the bacterial community tolerant to CL regarding different culture-based isolation methodologies. Data revealed that the choice of methodology alters the diversity and abundance of taxa accounting for the CL-resistance phenotype. The mcr-1 gene was identified by cloning and sequencing in one sample, representing the first report of mcr-1 gene in Croatia. Culturing of CL-resistant strains revealed their resistance phenotypes and concurrent production of clinically significant ß-lactamases, such as CTX-M-15, CTX-M-3 and SHV-12. We also report the first identification of blaCTX-M-15 gene in Klebsiella huaxiensis and K. michiganensis, as well as the blaTEM-1+CTX-M-3 in Serratia fonticola. ARGs profiles derived from metagenomic data and predicted by PICRUSt2, showed the highest abundance of genes encoding for multidrug efflux pumps, followed by the transporter genes accounting for the tetracycline, macrolide and phenicol resistance. Our study evidenced the multidrug resistance features of CL-tolerant bacterial communities thriving in surface beach waters. We also showed that combined application of the metagenomic approaches and culture-based techniques enabled successful detection of mcr-1 gene, which could be underreported in natural environment.