The spreading of extended-spectrum β-lactamases (ESBL)-producing thermotolerant coliforms (TC) in the water environment is a threat to human health but little is known about ESBL-producing TCs in the Yangtze River. We received 319 ESBL-producing stains obtained from the Chongqing basin and we investigated antibiotic susceptibility, bla gene types and the presence of integrons and gene cassettes. 16.8% of TC isolates were ESBL-producing bacteria and blaTEM+CTx-M was the predominant ESBL type. 65.2% of isolates contained class 1 integrons, but only 3 carried intI 2. Gene cassettes were amplified and sequenced. aadA, drfA, cmlA, sat1, aar3 and two ORF cassettes were found. In conclusion, Yangtze River is heavily polluted by ESBL-producing TC bacteria and the combined bla gene type could enhance antibiotic resistance. Class 1 integrons were widespread in ESBL-producing isolates and play an important role in multi-drug resistance. Characterization of gene cassettes could reveal the dissemination of antibiotic resistance genes.

Reuse of sewage sludge is a general trend and land application is an essential way to reuse sludge. The outbreak of coronavirus disease has raised concerns about human pathogens and their serious threat to public health. The risk of pathogenic bacterial contamination from land application of municipal sludge has not been well assessed. The purpose of this study was to investigate the presence of pathogenic bacteria in municipal sewage sludge and to examine the survival potential of certain multidrug-resistant enteroaggregative Escherichia coli (EAEC) strain isolated from sewage sludge during heat treatment. The sewage sludge produced in the two wastewater treatment plants contained pathogenic bacteria such as pathogenic E. coli, Shigella flexneri, and Citrobacter freundii. The environmental strain of EAEC isolated from the sludge was resistant to eight types of antibiotics. It could also enter the dormant state after 4.5 h of treatment at 55 °C and regrow at 37 °C, while maintaining its antibiotic resistance. Our results indicate that the dormancy of EAEC might be why it is heat-resistant and could not be killed completely during the sludge heat treatment process. Owing to the regrowth of the dormant pathogenic bacteria, it is risky to apply the sludge to land even if the sludge is heat-treated, and there is also a risk of spreading antibiotic resistance.

The environment of a large-scale vegetable production area can be exposed to antibiotic residues and antibiotic-resistant bacteria (ARB) via animal manure and irrigation with contaminated water, which can facilitate the dissemination of ARB. However, the occurrence of ARB in plantation areas and their dissemination in this environment remain largely unexplored. In total, 382 samples including those from vegetable (n = 106), soil (n = 87), well water (n = 24), river water (n = 20), river sediments (n = 20), farmer feces (n = 58) and farmer hands (n = 67) were collected in 2019 from a large-scale cultivation area in Shandong, China. Selective agar plates were used to screen for carbapenem-resistant Enterobacteriaceae (CRE) and whole-genome sequencing and Southern blotting were used to characterise isolates and mobile genetic elements carrying carbapenem resistance determinants. A total of nine NDM-5-producing isolates of Escherichia coli, Klebsiella pneumoniae, and Citrobacter spp. were identified from environmental sources and human feces, all of which were multidrug-resistant. Single nucleotide polymorphism analysis suggested clonal transmission of carbapenem-resistant Citrobacter sedlakii within greenhouse soils in the area. Eight of the isolates carried closely related or identical IncX3 plasmids carrying blaNDM₋₅, which were shown to be conjugative via filter mating experiments, indicating the highly transmissible nature of this genetic element. Isolates of E. coli and Citrobacter freundii were detected in the feces of local farm workers and contained similar IncX3 plasmids with blaNDM₋₅ environmental isolates, suggesting a potential risk of CRE transfer from the work environment to the farm workers. Thus, further research is required to investigate the potential health risks associated with environmental exposure to CRE in vegetable cultivation areas.

Carbapenems are used as last-resort drugs to treat infections caused by multidrug-resistant bacteria. Despite the increasing number of reports of carbapenem-resistant Enterobacteriaceae (CRE), there is still limited information on their distribution or prevalence in the environment. Our aim was to assess the occurrence of CRE in the Lis river (Portugal) and to characterize the genetic platforms linked to carbapenemase genes. We collected six water samples from sites near a wastewater treatment plant (n = 4 samples) and livestock farms (n = 2). Twenty-four CRE were characterized by BOX element-polymerase chain reaction (BOX-PCR), and thirteen representative isolates were analysed by Pulsed-Field Gel Electrophoresis (PFGE) and by sequencing the 16S rRNA gene. Antimicrobial susceptibility testing, PCR screening for carbapenemase-encoding genes, conjugation experiments and plasmid analysis were performed. Four isolates were chosen for whole-genome sequencing. All water samples contained CRE (4.0 CFU/mL on average). Representative isolates were multidrug-resistant (resistant to ciprofloxacin, trimethoprim-sulfamethoxazole and to all β-lactams tested) and were identified as K. pneumoniae, Enterobacter and Citrobacter. Isolates carried plasmids and harboured carbapenemase-encoding genes: blaKPC₋₃ in K. pneumoniae (n = 9), blaNDM₋₁ in Enterobacter (n = 3) and blaGES₋₅ in Citrobacter (n = 1). Conjugation experiments were successful in two Klebsiella isolates. Enterobacter PFGE profiles grouped in one cluster while Klebsiella were divided in three clusters and a singleton. Whole-genome sequencing analysis revealed blaGES₋₅ within a novel class 3 integron (In3-16) located on an IncQ/pQ7-like plasmid in Citrobacter freundii CR16. blaKPC₋₃ was present on IncFIA-FII pBK30683-like plasmids, which were subsequently confirmed in all K. pneumoniae (n = 9). Furthermore, blaKPC₋₃ was part of a genomic island in K. pneumoniae CR12. In E. roggenkampii CR11, blaNDM₋₁ was on an IncA/C₂ plasmid. The carbapenemase-encoding plasmids harboured other resistance determinants and mobile genetic elements. Our results demonstrate that Lis river is contaminated with CRE, highlighting the need for monitoring antibiotic resistance in aquatic environments, especially to last-resort drugs.

The present study reports for the first time, a novel disinfection method that combines ultrasonication with a natural biocide terpinolene to inhibit tough and opportunistic antimicrobial-resistant (AMR) microorganisms isolated from hospital wastewater treatment plant (HWWTP). The enhancement of the disinfection process was evaluated for the effect of ultrasonication power, operating temperature, and inoculum size. A hybrid methodology combining terpinolene with traditional physico-chemical method of acoustic cavitation delivered efficient disinfection of the secondary effluent of field scale HWWTP, amended with a higher inoculum size of multi-drug-resistant coliform bacteria Enterobactor sp., Citrobacter freundii, and Klebsiella pneumonia. A bacterial load of 6.4 log CFU/mL was completely eliminated in 25 min. The present study also reports that due to the hybrid process, a very small concentration of 0.312 mM (0.25 × Minimum Inhibitory Concentration or MBC) of terpinolene was enough to completely disinfect the multi-drug-resistant coliforms. The leakage of intracellular nucleic acids during the disinfection process suggested disruption of cell membrane as the primary mechanism of disinfection followed by disruption of cellular metabolic function measured by respiratory chain dehydrogenase activity. Moreover, this study is the first to prove that terpinolene remained stable even after the cavitation process, thus revealing possibilities of recycling of the natural compound for wastewater disinfection. The results of the present research suggest that using terpinolene as a bio-additive can efficiently eliminate hazardous multi-drug-resistant bacteria and drastically reduce operational time and cost thus rendering it suitable to replace conventional wastewater disinfection.

One of the bibliographic reference entry of this article had the author name and family name switched by mistake.

The aim of the present study was to isolate several bacterial consortia from a soil sample and to establish if they could colonize zirconium-tin alloy, such as Zircaloy-4. Two bacterial consortia containing aerobic heterotrophic bacteria and anaerobic sulfate-reducing bacteria were isolated from a soil sample. The aerobic heterotrophic bacteria exhibited a higher capability to utilize different sole carbon sources, as compared with anaerobic sulfate-reducing bacteria. Based on a morphological, biochemical, and molecular analysis, bacterial isolates were identified as Pseudomonas putida IBBHA₁, Pseudomonas aeruginosa IBBHA₂, Achromobacter spanius IBBHA₃, Citrobacter freundii IBBSR₁, Citrobacter youngae IBBSR₂, and Citrobacter braakii IBBSR₃. Isolated bacterial consortia which possess distinct DNA fingerprints were able to form biofilms and colonize the surface of zirconium-tin alloy coupons, although the colonization of coupons by the aerobic heterotrophic bacteria or anaerobic sulfate-reducing bacteria alone was lower compared with that observed when the coupon was immersed in a mixture of both bacterial consortia. Coupons immersed in these bacterial consortia revealed changes in the surface characteristics, which can facilitate or accelerate zirconium-tin alloy corrosion. The accumulation of corrosion products on coupons surface was less significant when the coupons were immersed solely in aerobic heterotrophic bacteria or anaerobic sulfate-reducing bacteria, compared with that observed when the coupon was immersed in a mixture of both bacterial consortia.

Environmental pollution by pentachlorophenol (PCP) is a critical concern worldwide, and microbial bioremediation could constitute an ecologically friendly solution. The main objectives of this study were at first to clarify the factors, affecting the ability and efficiency of PCP biodegradation by the bacterium isolate P6, and secondly to optimize the condition of using P6 for PCP bioremediation. The PCP mineralizing bacterium was isolated from the contaminated forest soil of Tunisia, and it was identified as Citrobacter freundii (C. freundii), by using conventional and molecular characteristics. The HPLC and spectroscopic analysis were used to investigate the PCP degradation and the biomass formation by this isolate P6. The main results showed that P6 was able to degrade or to transform more than 98 % of 640 mg/l PCP afterwards 168 h in mineral salt medium (MSM). As well, the optimal aerobic growth conditions of P6 in MSM include essentially the range of pH (4 ≤ pH ≤ 9) and of temperature (25 °C < temperature < 30 °C). The addition of glucose as extra carbon sources has an effect to enhance the PCP biodegradation. On the other side, this isolate of C. freundii is capable to remove or transform around 95.33 % of PCP added in the sterilized soil suspension supplemented with PCP and adjusted to a final concentration of around 400 mg/l during 2 weeks of incubation at 25 °C. This last result argues in favor of the use of this strain P6 of C. freundii as a microbial tool of remediation of PCP-contaminated site.

The performance of a laboratory-scale biofilter packed with a mixture of compost and woodchip on formaldehyde removal from polluted air streams was investigated. The reactor was inoculated with aerobic sludge as a source of bacteria, obtained from a municipal wastewater treatment plant. A nutrient solution was daily added to the reactor media. An airflow containing different concentrations of formaldehyde (from 20 ± 2 to 276 ± 5 mg m⁻³) was introduced into the reactor. In inlet formaldehyde concentration, an average removal efficiency and elimination capacity of 91 % and 0.36 g m⁻³ h⁻¹were attained, respectively, at180 s empty bed residence time (EBRT). After acclimatization of the system for increased formaldehyde concentrations of up to 276 ± 5 mg m⁻³and for EBRT of 180 s, those values were stabilized at around 72 % and 3.98g⁻³ h⁻¹, respectively. The experimental results showed that the system was effective for a high loading rate of formaldehyde with an acceptable EBRT. Compared to the application of compost alone as a media, a mixture of compost and woodchip (50/50 v/v%) enhanced the performance of the biofilter. The most predominant microorganism involved in the biodegradation of formaldehyde was a species of citrobacter called Citrobacter freundii, an aerobic gram-negative bacillus. Pressure drop of the reactor over the entire operations was about 1 mmH₂O m⁻¹.

Alcohol ethoxylates (AE) are a major component of the surfactant stream discharged into surface water. The “central fission” of AE with the formation of poly(ethylene glycols) (PEG) is considered to be the dominant biodegradation pathway. However, information as to which bacterial strains are able to perform this reaction is very limited. The aim of this work was to establish whether such an ability is unique or common, and which bacterial strains are able to split AE used as a sole source of organic carbon. Four bacterial strains were isolated from river water and were identified on the basis of phylogenetic trees as Enterobacter strain Z2, Enterobacter strain Z3, Citrobacter freundii strain Z4, and Stenotrophomonas strain Z5. Sterilized river water and “artificial sewage” were used for augmentation of the isolated bacteria. The test was performed in bottles filled with a mineral salt medium spiked with surfactant C₁₂E₁₀ (10 mg L⁻¹) and an inoculating suspension of the investigated bacterial strain. Sequential extraction of the tested samples by ethyl acetate and chloroform was used for separation of PEG from the water matrix. LC–MS was used for PEG determination on the basis of single-ion chromatograms. All four selected and investigated bacterial strains exhibit the ability to split fatty alcohol ethoxylates with the production of PEG, which is evidence that this property is a common one rather than specific to certain bacterial strains. However, this ability increases in the sequence: Stenotrophomonas strain Z5 < Enterobacter strain Z2 < Enterobacter strain Z3 = Citrobacter freundii strain Z4. Graphical Abstract Biodegradation by central fission of alcohol ethoxylates by bacterial strains isolated from river water