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Objectives: Despite the critical importance of colistin as a last-resort antibiotic, limited studies have investigated colistin resistance in human infections in Cambodia. This study aimed to investigate the colistin resistance and its molecular determinants among Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing (CP) Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolated in Cambodia between 2016 and 2020., Methods: E. coli (n = 223) and K. pneumoniae (n = 39) were tested for colistin minimum inhibitory concentration (MIC) by broth microdilution. Resistant isolates were subjected to polymerase chain reaction (PCR) for detection of mobile colistin resistance genes (mcr) and chromosomal mutations in the two-component system (TCS)., Results: Eighteen isolates (10 K. pneumoniae and 8 E. coli) revealed colistin resistance with a rate of 5.9% in E. coli and 34.8% in K. pneumoniae among ESBL isolates, and 1% in E. coli and 12.5% in K. pneumoniae among CP isolates. The resistance was associated with mcr variants (13/18 isolates, mcr-1, mcr-3, and mcr-8.2) and TCS mutations within E. coli and K. pneumoniae, with the first detection of mcr-8.2 in Cambodia, the discovery of new mutations potentially associated to colistin resistance in the TCS of E. coli (PhoP I47V, PhoQ N352K, PmrB G19R, and PmrD G85R) and the co-occurrence of mcr genes and colistin resistance conferring TCS mutations in 11 of 18 isolates., Conclusions: The findings highlight the presence of colistin resistance in ESBL- and CP- Enterobacteriaceae involved in human infections in Cambodia as well as chromosomal mutations in TCS and the emergence of mcr-8.2 in E. coli and K. pneumoniae. It underscores the need for continuous surveillance, antimicrobial stewardship, and control measures to mitigate the spread of colistin resistance., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Gene bioaugmentation is a bioremediation strategy that enhances biodegradative potential via dissemination of degradative genes from introduced microorganisms to indigenous microorganisms. Bioremediation experiments using 2,4-dichlorophenoxyacetic acid (2,4-D)-contaminated soil slurry and strains of Pseudomonas putida or Escherichia coli harboring a self-transmissible 2,4-D degradative plasmid pJP4 were conducted in microcosms to assess possible effects of gene bioaugmentation on the overall microbial community structure and ecological functions (carbon source utilization and nitrogen transformation potentials). Although exogenous bacteria decreased rapidly, 2,4-D degradation was stimulated in bioaugmented microcosms, possibly because of the occurrence of transconjugants by the transfer of pJP4. Terminal restriction fragment length polymorphism analysis revealed that, although the bacterial community structure was disturbed immediately after introducing exogenous bacteria to the inoculated microcosms, it gradually approached that of the uninoculated microcosms. Biolog assay, nitrate reduction assay, and monitoring of the amoA gene of ammonia-oxidizing bacteria and nirK and nirS genes of denitrifying bacteria showed no irretrievable depressive effects of gene bioaugmentation on the carbon source utilization and nitrogen transformation potentials. These results may suggest that gene bioaugmentation with P. putida and E. coli strains harboring pJP4 is effective for the degradation of 2,4-D in soil without large impacts on the indigenous microbial community.

To add the knowledge on the plasmid-mediated bioaugmentation in the activated sludge process, conjugative transfer of a self-transmissible, broad host range, mercury resistance, and partial 2,4-dichlorophenoxyactic acid (2,4-D)-degrading plasmid pJP4 from Escherichia coli HB101 or Pseudomonas putida KT2440 to activated sludge bacteria were examined by filter mating. Activated sludge samples were collected from an actual wastewater treatment plant and laboratory reactors operated under different sludge retention times (SRTs) of 5, 10, and 15 days. Transfer of pJP4 was observed in all combinations of donor and activated sludge (recipient) with the transfer frequency from 6.5 x 10⁻⁸ to 2.5 x 10⁻⁴ transconjugants per recipient, except from E. coli HB101 to the activated sludge sample of SRT 5 days. Although plasmid pJP4 was transferred mainly to dominant bacteria belonging to Pseudomonas, the host range of pJP4 in the activated sludge varied depending on the donor strain. Phenotypically various transconjugants were isolated from activated sludge sampled from the reactors with longer SRT operations. All transconjugants exhibited high mercury resistance equivalent to the donor strains; some were capable of degrading 2,4-D. These results suggest that the behavior of plasmids can be controlled partially through selection of appropriate donor strains and operational conditions of the activated sludge process., (Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

In situ transfer of a self-transmissible, antibiotic-multiresistant plasmid RP4 from a laboratory Escherichia coli strain C600 to indigenous activated sludge bacteria was investigated using filter mating. The transfer frequency of RP4 from the donor E. coli to the bacteria that was sampled from two wastewater treatment plants was 5.1x10(-2) to 7.5x10(-1) and 4.6x10(-3) to 1.3x10(-2)/potential recipient. The isolated transconjugants showed resistance to Ap, Km, and Tc and the presence of a plasmid with a similar size to RP4. The traG gene on RP4 was also detected from all transconjugants. Reverse-transfer experiments from the transconjugants to E. coli HB101 indicated that RP4 maintained self-transmissibility in the transconjugants. The transconjugant strains were dominant bacteria in activated sludge including Pseudomonas fluorescens, P. putida, and Ochrobactrum anthropi and minor populations of enteric bacterial strains including Citrobacter freundii, E. coli, Enterobacter cloacae, E. asburiae, and Klebsiella pneumoniae ssp. pneumoniae. The transconjugant strains K. pneumoniae ssp. pneumonia, E. cloacae, and E. asburiae had several naturally occurring plasmids. These results suggest that in situ transfer of plasmids and the exchange of antibiotic-resistant genes can occur between released and indigenous bacteria in activated sludge.

We investigated the possibility of conjugative plasmid transfer to the predominant bacteria in activated sludge and the factors influencing the transfer frequency in the activated sludge process. We performed conjugative transfers of a self-transmissible, broad-host-range plasmid RP4 from Escherichia coli C600 to activated sludge bacteria by broth mating. Most of the activated sludge bacteria tested could acquire plasmid RP4, although the transfer frequencies varied from 8.8 x 10(-7) to 1.3 x 10(-2) transconjugants per recipient. The transfer frequencies in several strains were similar to, or higher than, that in intraspecific transfer to E. coli HB101. Matings under various environmental conditions showed that factors relevant to physiological activity, such as temperature and nutrient conditions, seemed to affect the transfer frequency. In addition, conjugative transfer was detected even in filtered raw and treated wastewaters. Thus, the predominant activated sludge bacteria seem to have sufficient potential as recipients in conjugative plasmid transfer under the conditions likely to occur in the activated sludge process. Transfer frequency was reduced by agitation in the presence of suspended solid. This may suggest that conjugative plasmid transfer is physically inhibited in aeration tanks.

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25μg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria. [ABSTRACT FROM AUTHOR]