To date, carbapenem-resistant Enterobacteriaceae have been found predominantly in clinical settings worldwide. Raoultella belongs to the Enterobacteriaceae family which can cause hospital-acquired infections, and carbapenem-resistant Raoultella spp. (CRR) is sporadically reported in the environment. We investigated the distribution and underlying resistance mechanisms of CRR in a wastewater treatment plant (WWTP) from eastern China between January 2018 and February 2019. A total of 17 CRR were isolated from 324 environmental samples, including Raoultella ornithinolytica (n = 15) and Raoultella planticola (n = 2). The detection of CRR was more frequent in the water inlet compared to anaerobic tank, aerobic tank, sludge thickener, activated sludge, mud cake storage area, and water outlet, and CRR was detected in mud cake stacking area. All CRR were resistant to imipenem, meropenem, ampicillin, piperacillin-tazobactam, cefotaxime, ceftazidime, trimethoprim-sulfamethoxazole and fosfomycin. Four different carbapenemase genes were identified, including blaKPC₋₂ (n = 13), blaNDM₋₁ (n = 8), blaNDM₋₅ (n = 1), blaIMP₋₄ (n = 1). Interestingly, isolated R. ornithinolytica from the WWTP were closely related to those reported from human samples in China. Plasmid analysis indicated that IncFII(Yp), IncP6, and IncU mediated blaKPC₋₂ spread, IncX3 and IncN2 mediated blaNDM spread in the environment. The core structure of the Tn3-ISKpn27-blaKPC₋₂-ISKpn6, ISAba125-blaNDM-bleMBL-trpF-dsbD were identified. The study provides evidence that Raoultella spp. may spread alarming carbapenem resistance in the environment and, therefore, the continuous surveillance for carbapenem resistance in the WWTP should be conducted, especially sludge.

Coastal waters are confluences receiving large amounts of point and non-point sources of pollution. An attempt was made to explore microbial community interactions in response to carbon, nitrogen and metal pollution. Additionally, experiments were designed to analyze the influence of these factors on horizontal gene transfer (HGT). Shift in bacterial diversity dynamics by arsenic stress and nutrient addition in coastal waters was explored by metagenomics of microcosm setups. Phylogenetic analysis revealed equal distribution of Gammaproteobacteria (29%) and Betaproteobacteria (28%) in control microcosm. This proportional diversity from control switched to unique distribution of Gammaproteobacteria (44.5%)> Flavobacteria (17.7%)> Bacteriodia (11.92%)> Betaproteobacteria (11.52%) in microcosm supplemented with carbon, nitrogen and metal (C + N + M). Among metal-stressed systems, alpha diversity analysis indicated highest diversity of genera in C + N + M followed by N + M > C+M> metal alone. Arsenic and ampicillin sensitive E. coli XL1 blue and environmental strains (Vibrio tubiashii W85 and E. coli W101) were tested for efficiency of uptake of plasmid (P) pUCminusMCS (arsBᴿampᴿ) under varying stress conditions. Transformation experiments revealed that combined effect of carbon, nitrogen and metal on horizontal gene transfer (HGT) was significantly higher (p < 0.01) than individual factors. The effect of carbon on HGT was proved to be superior to nitrogen under metal stressed conditions. Presence of arsenic in experimental setups (P + M, P + N + M and P + C + M) enhanced the HGT compared to non-metal counterparts supplemented with carbon or nitrogen. Arsenic resistant bacterial isolates (n = 200) were tested for the ability to utilize various carbon and nitrogen substrates and distinct positive correlation (p < 0.001) was found between arsenic resistance and utilization of urea and nitrate. However, evident positive correlation was not found between carbon sources and arsenic resistance. Our findings suggest that carbon and nitrogen pollution in aquatic habitats under arsenic stress determine the microbial community dynamics and critically influence uptake of genetic material from the surrounding environment.

The effect of land application of sewage sludge on soil microbial communities and the possible spread of antibiotic- and metal-resistant strains and resistance determinants were evaluated during a 720-day field experiment. Enzyme activities, the number of oligotrophic bacteria, the total number of bacteria (qPCR), functional diversity (BIOLOG) and genetic diversity (DGGE) were established. Antibiotic and metal resistance genes (ARGs, MRGs) were assessed, and the number of cultivable antibiotic- (ampicillin, tetracycline) and heavy metal- (Cd, Zn, Cu, Ni) resistant bacteria were monitored during the experiment. The application of 10 t ha⁻¹ of sewage sludge to soil did not increase the organic matter content and caused only a temporary increase in the number of bacteria, as well as in the functional and structural biodiversity. In contrast to expectations, a general adverse effect on the tested microbial parameters was observed in the fertilized soil. The field experiment revealed a significant reduction in the activities of alkaline and acid phosphatases, urease and nitrification potential. Although sewage sludge was identified as the source of several ARGs and MRGs, these genes were not detected in the fertilized soil. The obtained results indicate that the effect of fertilization based on the recommended dose of sewage sludge was not achieved.

The environment is one of the main reservoirs of antibiotic resistance genes (ARGs) but multidrug resistant (MDR) environmental isolates are barely characterised. As suggested by the name, Pedobacter species have been predominantly isolated from soils, but are also recovered from water (including drinking water), chilled food, fish, compost, sludge, glaciers and other extreme environments. The susceptibility phenotype of Pedobacter lusitanus NL19 (isolated from a deactivated uranium mine), its closely related species and the genus type strain were investigated. All strains are MDR bacteria, resistant to β-lactams, colistin, aminoglycosides and ciprofloxacin. Therefore, Pedobacter spp. are likely intrinsically resistant to β-lactams (including ertapenem) and to other three classes of antibiotics. 6%–8% of their total protein-encoding genes encode a diverse collection of putative ARGs, including β-lactamases. These enzymes are highly abundant in all the other Pedobacter strains with sequenced genomes, especially class C, class B3 and class A. LUS-1 and PLN-1 were further characterised in E. coli. LUS-1 is a class A β-lactamase and it conferred an increase in the MIC of cefotaxime, albeit very low. PLN-1 is a class B3 β-lactamase with carbapenemase activity, conferring resistance to ertapenem and a 66x and 16x increase in the MIC of imipenem and meropenem, respectively. PLN-1 also hydrolyses ampicillin, 1st and 3rd generation cephalosporins, and at a lower extent cephamycins and 4th generation cephalosporins. Therefore, Pedobacter spp. encode a large and diverse arsenal of resistance mechanisms that make them environmental superbugs.

Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales have been classified as critical priority pathogens by the World Health Organization (WHO). ESBL are universally distributed and, in 2006, were firstly reported on a wild animal. Understanding the relative contributions of wild animals to ESBL circulation in the environment is urgently needed. In this work, we have conducted a nationwide study in Portugal to investigate the occurrence of bacteria carrying clinically significant antimicrobial resistance genes (ARG), using widely distributed wild ungulates as model species. A total of 151 antimicrobial resistant-Enterobacterales isolates were detected from 181 wild ungulates: 50% (44/88) of isolates from wild boar (Sus scrofa), 40.3% (25/62) from red deer (Cervus elaphus), 41.4% (12/29) from fallow deer (Dama dama) and 100% (2/2) from mouflon (Ovis aries subsp. musimon). Selected isolates showed a diversified resistance profile, with particularly high values corresponding to ampicillin (71.5%) and tetracycline (63.6%). Enterobacterales strains carried blaTEM, tetA, tetB, sul2, sul1 or dfrA1 ARG genes. They also carried blaCTX₋M-type genes, which are prevalent in human infections, namely CTX-M-14, CTX-M-15 and CTX-M-98. Strikingly, this is the first report of CTX-M-98 in wildlife. Almost 40% (n = 59) of Enterobacterales were multi-drug resistant. The diversity of plasmids carried by ESBL isolates was remarkable, including IncF, K and P. This study highlights the potential role of wild ungulates as environmental reservoirs of CTX-M ESBL-producing E. coli and in the spill-over of AMR bacteria and their determinants. Our findings suggest that wild ungulates are useful as strategic sentinel species of AMR in terrestrial environments, especially in response to potential sources of anthropogenic pollution, providing early warning of potential risks to human, animal and environmental health.

The increase of CO₂ concentration in the atmosphere, water and soil environment can lead to the changes in microbial activities. However, the transformation of antibiotic resistance genes has not been investigated in the presence of higher levels of CO₂. This study demonstrated that CO₂ facilitated the transformation of pUC19 plasmid, carrying ampicillin resistance genes, into Escherichia coli. Mechanism studies revealed that the type Ⅱ secretion system, type Ⅳ pilus and some other secretion systems were enhanced by CO₂, leading to DNA capture by pilus, larger cell pore sizes and more cell membrane channels. CO₂ also increased reactive oxygen species production, leading to SOS response and cell membrane damage. Besides, changes in intracellular Fe²⁺ and Mg²⁺ concentrations induced by CO₂ caused greater damage to the cell membrane and enhanced secretion systems, respectively. Overall, increased CO₂ provided more cell membrane channels for plasmid uptake and led to higher transformation efficiencies. The potential risk of a natural factor on the transformation of ARGs was first studied in this study, which helps us understand the fate of ARGs in ecosystems. As the carbon emission will continue to grow and enhance the enrichment of CO₂ in water and soil, the findings revealed a more severe public health issue under the background of carbon emission and CO₂ leakage.

Zerovalent iron powder (ZVI or Fe⁰) and nanoparticulate ZVI (nZVI or nFe⁰) are proposed as cost-effective materials for the removal of aqueous antibiotics. Results showed complete removal of Amoxicillin (AMX) and Ampicillin (AMP) upon contact with Fe⁰ and nFe⁰. Antibiotics removal was attributed to three different mechanisms: (i) a rapid rupture of the β-lactam ring (reduction), (ii) an adsorption of AMX and AMP onto iron corrosion products and (iii) sequestration of AMX and AMP in the matrix of precipitating iron hydroxides (co-precipitation with iron corrosion products). Kinetic studies demonstrated that AMP and AMX (20 mg L⁻¹) undergo first-order decay with half-lives of about 60.3 ± 3.1 and 43.5 ± 2.1 min respectively after contact with ZVI under oxic conditions. In contrast, reactions under anoxic conditions demonstrated better degradation with t1/2 of about 11.5 ± 0.6 and 11.2 ± 0.6 min for AMP and AMX respectively. NaCl additions accelerated Fe⁰ consumption, shortening the service life of Fe⁰ treatment systems. 21Fe⁰ is efficient for the aqueous removal of the β-lactam antibiotics and chlorides enhanced the removal rate by sustaining the process of iron corrosion.

This study investigated the effects of adding biochar (BC) on the fate of ciprofloxacin (CIP) and its related antibiotic tolerance (AT) in activated sludge. Three activated sludge reactors were established with different types of BC, derived from apple, pear, and mulberry tree, respectively, and one reactor with no BC. All reactors were exposed to an environmentally relevant level of CIP that acted as a definitive selective pressure significantly promoting AT to four representative antibiotics (CIP, ampicillin, tetracycline, and polymyxin B) by up to two orders of magnitude. While CIP removal was negligible in the reactor without BC, the BC-dosed reactors effectively removed CIP (70–95% removals) through primarily adsorption by BC and biodegradation/biosorption by biomass. The AT in the BC-added reactors was suppressed by 10–99%, compared to that without BC. The BC addition played a key role in sequestering CIP, thereby decreasing the selective pressure that enabled the proactive prevention of AT increase. 16S rRNA gene sequencing analysis showed that the BC addition alleviated the CIP-mediated toxicity to community diversity and organisms related to phosphorous removal. Machine learning modeling with random forest and support vector models using AS microbiome data collectively pinpointed Achromobacter selected by CIP and strongly associated with the AT increase in activated sludge. The identification of Achromobacter as an important AT bacteria revealed by the machine learning modeling with multiple models was also validated with a linear Pearson's correlation analysis. Overall, our study highlighted Achromobacter as a potential useful sentinel for monitoring AT occurring in the environment and suggested BC as a promising additive in wastewater treatment to improve micropollutant removal, mitigate potential AT propagation, and maintain community diversity against toxic antibiotic loadings.

This study investigated antibiotic resistance profiles and tetracycline resistance genes in Enterobacteriaceae family isolates from the Pearl rivers. The Enterobacteriaceae isolates were tested for susceptibility to seven antibiotics ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, sulphamethoxazole/trimethoprim, tetracycline and trimethoprim. In Liuxi reservoir, with an exception to ampicillin resistant strains (11%) no other antibiotic resistance bacterial strains were detected. However, multiple drug resistance in bacterial isolates from the other sites of Pearl rivers was observed which is possibly due to sewage discharge and input from other anthropogenic sources along the rivers. Four tetracycline resistance genes tet A, tet B, tet C and tet D were detected in the isolates from the rivers. The genes tet A and tet B were widely detected with the detection frequencies of 43% and 40% respectively. Ciprofloxacin and levofloxacin resistant enteric bacteria were also isolated from the pig and duck manures which suggest a wider distribution of human specific drugs in the environment. This investigation provided a baseline data on antibiotic resistance profiles and tetracycline resistance genes in the Pearl rivers delta.

Vibrio parahaemolyticus can cause severe gastroenteritis, septicaemia and even death in humans. Continuous monitoring of V. parahaemolyticus contamination in aquatic products is imperative for ensuring food safety. In this study, we isolated and characterized 561 V. parahaemolyticus strains recovered from 23 species of commonly consumed shellfish, crustaceans, and fish collected in July and August of 2017 in Shanghai, China. The bacterium was not isolated from two fish species Carassius auratus and Parabramis pekinensis. The results revealed a very low occurrence of pathogenic V. parahaemolyticus carrying the toxin genes trh (0.2%) and tdh (0.0%). However, high percentages of resistance to the antimicrobial agents ampicillin (93.0%), rifampin (82.9%), streptomycin (75.4%) and kanamycin (50.1%) were found. A high incidence of tolerance to the heavy metals Hg²⁺ (74.7%) and Zn²⁺ (56.2%) was also observed in the isolates. ERIC-PCR-based fingerprinting of MDR isolates (77.5%) revealed 428 ERIC-genotypes, demonstrating remarkable genetic variation among the isolates. The results of this study support the urgent need for food safety risk assessment of aquatic products.