Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

OBJECTIVES: To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. METHODS: Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. RESULTS: In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. CONCLUSIONS: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

Multiplex lateral flow immunochromatographic assay is an effective method to detect carbapenemases without risk of OXA-48-like cross reactivity.

BACKGROUND: It is essential to detect carriers of carbapenemase-producing Enterobacterales in order to implement infection control measures. The objectives of this study was to evaluate the NG-Test® CARBA 5 (CARBA 5) assay for detection of five carbapenemases and to assess the cross reactivity of other OXA-type carbapenemases with the OXA-48-like specific antibodies. METHODS: A total of 197 Enterobacterales isolates were tested. To evaluate the cross reactivity, 73 carbapenem-resistant A. baumannii, harboring OXA-type variants, were tested. Polymerase chain reaction (PCR) served as gold standard for carbapenemase identification. RESULTS: Excellent agreement was found between PCR and CARBA 5, for all but one isolate. The single false positive result (a blaSME positive S. marcescens isolate) was incorrectly positive for blaOXA-48 by CARBA 5. No cross reactivity was observed. The sensitivity and specificity were 100.0% and 98.0%, respectively. CONCLUSIONS: The CARBA 5 assay is highly sensitive and specific and is recommended as a tool for the detection of the main carbapenemases of interest in clinical microbiology laboratories.

Fourier Transform Infrared Spectroscopy Is a New Option for Outbreak Investigation: a Retrospective Analysis of an Extended-Spectrum-Beta-Lactamase-Producing Klebsiella pneumoniae Outbreak in a Neonatal Intensive Care Unit.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

Quality of Irrigation Water Affects Soil Functionality and Bacterial Community Stability in Response to Heat Disturbance.

Anthropogenic activities alter the structure and function of a bacterial community. Furthermore, bacterial communities structured by the conditions the anthropogenic activities present may consequently reduce their stability in response to an unpredicted acute disturbance. The present mesocosm-scale study exposed soil bacterial communities to different irrigation water types, including freshwater, fertilized freshwater, treated wastewater, and artificial wastewater, and evaluated their response to a disturbance caused by heat. These effectors may be considered deterministic and stochastic forces common in agricultural operations of arid and semiarid regions. Bacterial communities under conditions of high mineral and organic carbon availability (artificial wastewater) differed from the native bacterial community and showed a proteobacterial dominance. These bacterial communities had a lower resistance to the heat treatment disturbance than soils under conditions of low resource availability (high-quality treated wastewater or freshwater). The latter soil bacterial communities showed a higher abundance of operational taxonomic units (OTUs) classified as Bacilli These results were elucidated by soil under conditions of high resource availability, which lost higher degrees of functional potential and had a greater bacterial community composition change. However, the functional resilience, after the disturbance ended, was higher under a condition of high resource availability despite the bacterial community composition shift and the decrease in species richness. The functional resilience was directly connected to the high growth rates of certain Bacteroidetes and proteobacterial groups. A high stability was found in samples that supported the coexistence of both resistant OTUs and fast-growing OTUs.IMPORTANCE This report presents the results of a study employing a hypothesis-based experimental approach to reveal the forces involved in determining the stability of a soil bacterial community to disturbance. The resultant postdisturbance bacterial community composition dynamics and functionality were analyzed. The paper demonstrates the relatedness of community structure and stability under cultivation conditions prevalent in an arid area under irrigation with water of different qualities. The use of common agricultural practices to demonstrate these features has not been described before. The combination of a fundamental theoretical issue in ecology with common and concerning disturbances caused by agricultural practice makes this study unique. Furthermore, the results of the present study have applicable importance regarding soil conservation, as it enables a better characterization and monitoring of stressed soil bacterial communities and possible intervention to reduce the stress. It will also be of valued interest in coming years, as fresh water scarcity and the use of alternative water sources are expected to rise globally.

Resilience of soil bacterial community to irrigation with water of different qualities under Mediterranean climate.

Limited freshwater (FW) availability due to climate change and increasing global populations is driving agriculture in arid and semi-arid regions to recycle vast quantities of water for irrigation. However, irrigation with treated wastewater (TWW), which contains dissolved organic matter, salts and microorganisms might alter soil microbial populations, and thus affect soil fertility. We characterized the effects of irrigation with TWW and FW on soil bacterial community composition for three consecutive years. Orchard samples were collected at the end of each of the rainy (winter) and irrigation (summer) season. Community composition, determined by 16S ribosomal DNA amplicon pyrosequencing, was highly similar in soil samples obtained at the end of three consecutive rainy seasons, regardless of irrigation season water quality. However, whereas composition in soil shifted slightly during irrigation seasons by FW irrigation, it was greatly influenced by TWW irrigation. During the irrigation season, a decrease in the relative abundance of Actinobacteria was observed; along with an increase in the relative abundance Gammaproteobacteria within TWW-irrigated soils. The return to the 'baseline state' during the rainy season demonstrates that the soil community is not resistant to anthropogenic impact imposed by irrigation water quality, yet is resilient in long term.

Carbapenem-resistant Acinetobacter baumannii carrier detection: a simple and efficient protocol.

Timely detection of carbapenem-resistant Acinetobacter baumannii (CRAB) carriers is essential to direct infection control measures. In this work, we aimed to develop a practical protocol to detect CRAB from screening samples. To choose a selective medium that detects CRAB with high sensitivity and specificity, 111 A. baumannii clinical isolates were inoculated on three types of agar: mSuperCARBA (SC), CHROMagar Acinetobacter (CaA), and modified CHROMagar Acinetobacter (mCaA) containing 4.5 mg/mL meropenem. SC was non-selective, CaA was the most sensitive (100%), but only moderately specific (72%), and mCaA was highly specific (97%) and sensitive (98%). Confirmation of the carbapenem-resistant phenotype using PCR-based detection of blaOXA-23, blaOXA-24, and blaOXA-58 genes was specific but not sensitive, detecting only 58% of CRAB isolates. Identification of A. baumannii using either gyrB or blaOXA-51 PCR was excellent. Next, we used the same methodology in routine screening for CRAB carriage. mCaA had the best yield, with high sensitivity but moderate specificity to differentiate between CRAB and other carbapenem-resistant organisms. Skin sampling using sponges and 6 hour enrichment was highly sensitive (98%), while other body sites had poor sensitivity (27%- 41%). Shorter incubation had slightly lower yield, and longer incubation did not improve the detection. Performing PCR for blaOXA-51 and gyrB on colonies growing on modified mCaA differentiated between CRAB and other species with high accuracy (98% and 99%, respectively). Based on our results, we present a procedure for easy and reliable detection of CRAB carriage using skin sampling, short enrichment, selection on mCaA, and PCR-based identification. IMPORTANCE: Carbapenem-resistant Acinetobacter baumannii (CRAB) is a substantial cause of nosocomial infections, classified among the most significant multidrug-resistant pathogens by the World Health Organization and by the US Centers for Disease Control. Limiting the spread of CRAB is an important goal of infection control, but laboratory methods for identification of CRAB carriers are not standardized. In this work, we compared different selective agar plates, tested the efficiency of A. baumannii identification by PCR for species-specific genes, and used PCR-based detection of common resistance genes to confirm the carbapenem-resistant phenotype. During a prospective study, we also determined the optimal sample enrichment time. Based on our results, we propose a simple and efficient protocol for the detection of CRAB carriage using skin sampling, short enrichment, selection on appropriate agar plates, and PCR-based identification, resulting in a turn-around time of 24 hours.

Impact of short-term acidification on nitrification and nitrifying bacterial community dynamics in soilless cultivation media.

Soilless medium-based horticulture systems are highly prevalent due to their capacity to optimize growth of high-cash crops. However, these systems are highly dynamic and more sensitive to physiochemical and pH perturbations than traditional soil-based systems, especially during nitrification associated with ammonia-based fertilization. The objective of this study was to assess the impact of nitrification-generated acidification on ammonia oxidation rates and nitrifying bacterial community dynamics in soilless growth media. To achieve this goal, perlite soilless growth medium from a commercial bell pepper greenhouse was incubated with ammonium in bench-scale microcosm experiments. Initial quantitative real-time PCR analysis indicated that betaproteobacterial ammonia oxidizers were significantly more abundant than ammonia-oxidizing archaea, and therefore, research focused on this group. Ammonia oxidation rates were highest between 0 and 9 days, when pH values dropped from 7.4 to 4.9. Pyrosequencing of betaproteobacterial ammonia-oxidizing amoA gene fragments indicated that r-strategist-like Nitrosomonas was the dominant ammonia-oxidizing bacterial genus during this period, seemingly due to the high ammonium concentration and optimal growth conditions in the soilless media. Reduction of pH to levels below 4.8 resulted in a significant decrease in both ammonia oxidation rates and the diversity of ammonia-oxidizing bacteria, with increased relative abundance of the r-strategist-like Nitrosospira. Nitrite oxidizers (Nitrospira and Nitrobacter) were on the whole more abundant and less sensitive to acidification than ammonia oxidizers. This study demonstrates that nitrification and nitrifying bacterial community dynamics in high-N-load intensive soilless growth media may be significantly different from those in in-terra agricultural systems.

OXA-900, a Novel OXA Sub-Family Carbapenemase Identified in Citrobacter freundii, Evades Detection by Commercial Molecular Diagnostics Tests.

Using whole-genome sequencing and cloning of the target gene, we identified blaOXA-900 carbapenemase, a novel blaOXA belonging to a distant and distinct sub-family of blaOXA-48-like. The plasmid-mediated gene was identified in a C. freundii isolate with elevated carbapenem MICs that evaded detection by commercial DNA-based methods. The novel gene, an OXA-48 family carbapenem-hydrolyzing class D ß-lactamase, OXA-900, likely originates from marine environmental Shewanella. Since this plasmid-mediated gene has entered a member of the Enterobacterales and evades detection by commonly used tests, it may gain wide dissemination among Enterobacterales.

A multi-institutional outbreak of New Delhi metallo-ß-lactamase-producing Escherichia coli with subsequent acquisition of the Klebsiella pneumoniae carbapenemase gene.

We characterized 57 isolates from a 2-phase clonal outbreak of New Delhi metallo-ß-lactamase-producing Eschericha coli, involving 9 Israeli hospitals; all but 1 isolate belonged to sequence-type (ST) 410. Most isolates in the second phase harbored blaKPC-2 in addition to blaNDM-5. Genetic sequencing revealed most dual-carbapenemase-producing isolates to be monophyletically derived from a common ancestor.

Increased Capsule Thickness and Hypermotility Are Traits of Carbapenem-Resistant Acinetobacter baumannii ST3 Strains Causing Fulminant Infection.

BACKGROUND: Acinetobacter baumannii is a successful nosocomial pathogen, causing severe, life-threatening infections in hospitalized patients, including pneumonia and bloodstream infections. The spread of carbapenem-resistant Acinetobacter baumannii (CRAB) strains is a major health threat worldwide. The successful spread of CRAB is mostly due to its highly plastic genome. Although some virulence factors associated with CRAB have been uncovered, many mechanisms contributing to its success are not fully understood. METHODS: Here we describe strains of CRAB that were isolated from fulminant cases in 2 hospitals in Israel. These isolates show a rare hypermucoid (HM) phenotype and were investigated using phenotypic assays, comparative genomics, and an in vivo Galleria mellonella model. RESULTS: The 3 isolates belonged to the ST3 international clonal type and were closely related to each other, as shown by Fourier-transform infrared spectroscopy and phylogenetic analyses. These isolates possessed thickened capsules and a dense filamentous extracellular polysaccharides matrix as shown by transmission electron microscopy (TEM), and overexpressed the capsule polysaccharide synthesis pathway-related wzc gene. CONCLUSIONS: The HM isolates possessed a unique combination of virulence genes involved in iron metabolism, protein secretion, adherence, and membrane glycosylation. HM strains were more virulent than control strains in 2 G. mellonella infection models. In conclusion, our findings demonstrated several virulence factors, all present in 3 CRAB isolates with rare hypermucoid phenotypes.

The effect of prophylaxis with ertapenem versus cefuroxime/metronidazole on intestinal carriage of carbapenem-resistant or third-generation-cephalosporin-resistant Enterobacterales after colorectal surgery.

OBJECTIVES: Compared to cephalosporin-based prophylaxis, ertapenem prophylaxis lowers the risk of surgical site infection among carriers of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PEs) undergoing colorectal surgery. We aimed to determine whether ertapenem prophylaxis leads to increased postoperative colonization with carbapenem-resistant Enterobacterales (CREs) and third-generation-cephalosporin-resistant Enterobacterales (3GCR-Es). METHODS: This study was nested within a quality improvement study of prophylaxis for ESBL-PE carriers undergoing colorectal surgery. Patients were screened 4-6 days after surgery for carriage of ESBL-PEs or other 3GCR-Es and CREs. When CREs were detected, pre- and postsurgical clones were compared using Fourier-transform infrared (FT-IR) spectroscopy. RESULTS: The sample consisted of 56 patients who carried ESBL-PEs before surgery and received cefuroxime/metronidazole prophylaxis (Group 1), 66 who carried ESBL-PEs before surgery and received ertapenem (Group 2), and 103 ESBL-PE non-carriers who received cefuroxime/metronidazole prophylaxis (Group 3). CRE carriage was detected postoperatively in one patient (1.5%) in Group 2 versus eight patients (14.3%) in Group 1 (RD -12.8%; 95%CI -22.4% to -3.1%). For seven out of nine patients, preoperative ESBL-PE and postoperative CRE isolates were compared; in five of them, the pre- and postoperative clones were identical. Postoperative 3GCR-E carriage was detected in 37 patients (56.1%) in Group 2 versus 46 patients in Group 1 (82.1%) (aRD -20.7%, 95%CI -37.3% to -4.1%). CONCLUSIONS: Among ESBL-PE carriers undergoing colorectal surgery, detection of short-term postsurgical colonization by CREs and 3GCR-Es was significantly lower among patients who received ertapenem prophylaxis than those who received cephalosporin-metronidazole prophylaxis. Resistance development in a colonizing bacterial clone, rather than carbapenemase acquisition, was the major mechanism of carbapenem resistance.

Investigation of Outbreaks of Extended-Spectrum Beta-Lactamase-Producing Klebsiella Pneumoniae in Three Neonatal Intensive Care Units Using Whole Genome Sequencing.

Infections caused by extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are on a constant rise and are a noted cause of outbreaks in neonatal intensive care units (NICUs). We used whole genome sequencing (WGS) to investigate the epidemiology of consecutive and overlapping outbreaks caused by ESBL-KP in NICUs in three hospitals in close proximity. Clonality of 43 ESBL-KP isolates from 40 patients was determined by BOX-PCR. Short-read sequencing was performed on representative isolates from each clone. The dominant clones from each NICU were sequenced using long-read sequencing. Bioinformatics methods were used to define multilocus sequence type (MLST), analyze plasmid content, resistomes, and virulence factors. In each NICU, we found a unique dominant clone (ST985, ST37, and ST35), each belonging to a distinct sequence type (ST), as well as satellite clones. A satellite strain in NICU-2 (ST35) was the dominant strain in NICU-3, where it was isolated four weeks later, suggesting transmission. NICU-1- and NICU-2-dominant strains had blaCTX-M-15 carried on a similar transposable element (Tn3-ISEcp1) but at different locations: on a plasmid and on the chromosome, respectively. We concluded that the overlapping ESBL-KP outbreaks were a combination of clonal transmission within NICUs, possible transposable element transmission between NICUs, and repeated importation of ESBL-KP from the community.

Host Specificity and Spatial Distribution Preference of Three Pseudomonas Isolates.

Plant hosts recruit and maintain a distinct root-associated microbiota based on host and bacterium traits. However, past studies disregarded microbial strain-host specificity and spatial micro-heterogeneity of the root compartment. Using genetic manipulation, confocal laser scanning microscopy, real-time quantitative PCR, and genome sequencing we characterized the colonization patterns of three Pseudomonas spp. isolates native to wheat roots, on the micro-scale. Namely, isolates P. fluorescens NT0133, P. stutzeri NT124, and P. stutzeri NT128. All three isolates preferentially colonized wheat over cucumber roots that served as control for host specificity. Furthermore, not only had the isolates strong host specificity but each isolate had a distinct spatial distribution on the root, all within a few millimeters. Isolate P. stutzeri-NT0124 preferentially colonized root tips, whereas P. fluorescens-NT0133 showed a preference for zones distant from the tip. In contrast, isolate P. stutzeri-NT0128 had no preference for a specific niche on the root. While all isolates maintained genetic potential for motility and biofilm formation their phenotype varied significantly and corresponded to their niche preference. These results demonstrate the importance of spatial colonization patterns, governed by both niche and bacterial characteristics which will have great importance in future attempts to manipulate the plant microbiome by constructing synthetic microbial consortia.

Seasonal effect and anthropogenic impact on the composition of the active bacterial community in Mediterranean orchard soil.

Several anthropogenic interventions, common in agriculture, may influence active bacterial communities in soil without affecting their total composition. Therefore, the composition of an active bacterial community in soil may reflect its relation to biogeochemical processes. This issue was addressed during two consecutive years in olive-orchard soil, irrigated with treated wastewater (TWW) in a Mediterranean climate, by following the active (rRNA) and total (rRNA gene) bacterial community in the soil. Although TWW irrigation did not affect the composition of the total soil bacterial community, it had an effect on the active fraction of the community. These results, based on 16S rRNA data, indicate that the organic matter and minerals in TWW were not directly utilized for the rapid proliferation of specific taxonomic groups. Activity levels, manifested by variance in the relative abundance of the active and total communities of selected operational taxonomic units, revealed annual and seasonal fluctuations and fluctuations dependent on the type of irrigation. The potential activity (nitrification rates) and community composition of ammonia-oxidizing bacteria were affected by TWW irrigation, and this group of bacteria was therefore further explored. It was concluded that irrigation with TWW had little effect on "who is there", i.e. which bacteria were present, but influenced "who is active", with a distinct effect on bacteria associated with the biochemical cycling of nitrogen.

Biogeography rather than association with cyanobacteria structures symbiotic microbial communities in the marine sponge Petrosia ficiformis.

The sponge Petrosia ficiformis is ubiquitous in the Mediterranean Sea and Eastern Atlantic Ocean, hosting a diverse assemblage of bacteria, including, in illuminated sites, cyanobacteria. Two closely related sponge color morphs have been described, one inside caves and at their entrance (white/pink), and one on the rocky cliffs (violet). The presence of the different morphs and their ubiquity in the Mediterranean (from North-West to South-East) provides an opportunity to examine which factors mostly affect the associated microbial communities in this species: (i) presence of phototrophic symbionts or (ii) biogeography. 16S rRNA gene tag pyrosequencing data of the microbial communities revealed that Chloroflexi, Gammaproteobacteria, and Acidobacteria dominated the bacterial communities of all sponges analyzed. Chlorophyll a content, TEM observations and DNA sequence data confirmed the presence of the cyanobacterium Synechococcus feldmannii in violet and pink morphs of P. ficiformis and their absence in white color morphs. Rather than cyanobacterial symbionts (i.e., color morphs) accounting for variability in microbial symbiont communities, a biogeographic trend was observed between P. ficiformis collected in Israel and Italy. Analyses of partial 18S rRNA and mitochondrial cytochrome c oxidase subunit I (COX1) gene sequences revealed consistent genetic divergence between the violet and pink-white morphotypes of P. ficiformis. Overall, data indicated that microbial symbiont communities were more similar in genetically distinct P. ficiformis from the same location, than genetically similar P. ficiformis from distant locations.

Effects of metal oxide nanoparticles on soil properties.

In recent years the behavior and properties of nanoparticles released to the environment have been studied extensively to better assess the potential consequences of their broad use in commercial products. The fate, transport and mobility of nanoparticles in soil were shown to be strongly dependent on environmental conditions. However, little is known about the possible effects of nanoparticles on soil chemical, physical and biological properties. In this study, two types of metal oxide nanoparticles, CuO and Fe(3)O(4) were mixed into two types of soil and the effects of the nanoparticles on various soil properties were assessed. Metal oxide nanoparticles were shown previously to catalyze the oxidation of organic pollutants in aqueous suspensions, and they were therefore expected to induce changes in the organic material in the soil, especially upon addition of an oxidant. It was found that the nanoparticles did not change the total amount of organic materials in the soil or the total organic carbon in the soil extract; however, three-dimensional fluorescence spectroscopy demonstrated changes in humic substances. The nanoparticles also affected the soil bacterial community composition, based on denaturing gradient gel electrophoresis (DGGE) fingerprinting, but had little impact on the macroscopic properties of the soil.

Effect of metal oxide nanoparticles on microbial community structure and function in two different soil types.

Increased availability of nanoparticle-based products will, inevitably, expose the environment to these materials. Engineered nanoparticles (ENPs) may thus find their way into the soil environment via wastewater, dumpsters and other anthropogenic sources; metallic oxide nanoparticles comprise one group of ENPs that could potentially be hazardous for the environment. Because the soil bacterial community is a major service provider for the ecosystem and humankind, it is critical to study the effects of ENP exposure on soil bacteria. These effects were evaluated by measuring bacterial community activity, composition and size following exposure to copper oxide (CuO) and magnetite (Fe3O4) nanosized (<50 nm) particles. Two different soil types were examined: a sandy loam (Bet-Dagan) and a sandy clay loam (Yatir), under two ENP concentrations (1%, 0.1%). Results indicate that the bacterial community in Bet-Dagan soil was more susceptible to change due to exposure to these ENPs, relative to Yatir soil. More specifically, CuO had a strong effect on bacterial hydrolytic activity, oxidative potential, community composition and size in Bet-Dagan soil. Few effects were noted in the Yatir soil, although 1% CuO exposure did cause a significant decreased oxidative potential and changes to community composition. Fe3O4 changed the hydrolytic activity and bacterial community composition in Bet-Dagan soil but did not affect the Yatir soil bacterial community. Furthermore, in Bet-Dagan soil, abundance of bacteria annotated to OTUs from the Bacilli class decreased after addition of 0.1% CuO but increased with 1% CuO, while in Yatir soil their abundance was reduced with 1% CuO. Other important soil bacterial groups, including Rhizobiales and Sphingobacteriaceae, were negatively affected by CuO addition to soil. These results indicate that both ENPs are potentially harmful to soil environments. Furthermore, it is suggested that the clay fraction and organic matter in different soils interact with the ENPs and reduce their toxicity.