The inappropriate use of antibiotics is widely recognized as the primary driver of bacterial antibiotic resistance. However, less attention has been given to the potential induction of multidrug-resistant bacteria through exposure to disinfectants. In this study, Klebsiella pneumonia, an opportunistic pathogen commonly associated with hospital and community-acquired infection, was experimentally exposed to NaClO at both minimum inhibitory concentration (MIC) and sub-MIC levels over a period of 60 days. The result demonstrated that NaClO exposure led to enhanced resistance of K. pneumonia to both NaClO itself and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin). Concurrently, the evolved resistant strains exhibited fitness costs, as evidenced by decreased growth rates. Whole population sequencing revealed that both concentrations of NaClO exposure caused genetic mutations in the genome of K. pneumonia. Some of these mutations were known to be associated with antibiotic resistance, while others had not previously been identified as such. In addition, 11 identified mutations were located in the virulence factors, demonstrating that NaClO exposure may also impact the pathogenicity of K. pneumoniae. Overall, this study highlights the potential for the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic to contribute to the emergence of antibiotic-resistant bacteria. ENVIRONMENTAL IMPLICATION: Considering the potential hazardous effects of disinfectant residues on environment, organisms and biodiversity, the sharp rise in use of disinfectants during COVID-19 pandemic has been considered highly likely to cause worldwide secondary disasters in ecosystems and human health. This study demonstrated that NaClO exposure enhanced the resistance of K. pneumonia to both NaClO and five antibiotics (erythromycin, polymyxin B, gentamicin, tetracycline, and ciprofloxacin), highlighting the widespread use of NaClO-containing disinfectants during the COVID-19 pandemic may increase the emergence of antibiotic-resistant bacteria in the environment.

Salmonella has presented increasingly alarming rates of antimicrobial resistance believed to be a result of a high prevalence of integrons. It is speculated that disinfectant-resistant isolates are due to the expression of qacEΔ1, an efflux pump located in the 3\' conserved sequence (3\'CS) of class 1 integrons. With this concern, we tested the antibiotic and disinfectant resistance of 581 Salmonella strains collected from different sources, and characterized their integron structures. Gene expression and induction experiments were also performed. Results showed that Salmonella have high resistance to antimicrobials, especially to sulfonamides (SAs, 78.83 %), tetracyclines (TCs, 75.04 %) and benzalkonium chloride (BC, 87.26 %). The multi-drug resistance (MDR) frequency reached up to 63.17 %, and the prevalence of intI1 was 45.78 %. Molecular characterization of class 1 integrons exhibited nine different gene cassette arrays, of these, dfrA12-orf-aadA2 (n = 75), EstX (n = 25) and aadA2 (n = 14) were the most frequent. Importantly, 74.06 % of intI1-positive isolates were carrying qacEΔ1-sul1 genes in the 3\'CS. This study also demonstrated that phenotypic resistance to both antibiotics and disinfectants was significantly correlated with the emergence of intI1 (p < 0.05). 91.37 % of qacEΔ1-sul1 positive Salmonella were found with disinfectant resistance. Additionally, expression of qacEΔ1 gene in Escherichia coli confirmed qacEΔ1 is predominantly involved in conferring disinfectant resistance. Disinfectant induction experiments further implicated qacEΔ1 in disinfectant resistance. RT-qPCR revealed a disinfectant-mediated increase in the relative expression of antibiotic-resistant genes (ARGs), aadA2 and dfrA12 on the integron, and efflux pump genes (mdtH and acrD) indicating that disinfectant could trigger co or cross-resistance. Therefore, our study confirmed that using disinfectant could provide selection pressure for strains with acquired resistance to antibiotics, providing new insights into the public health impact of Salmonella and guide continued efforts in antimicrobial stewardship and prevention of antibiotic resistance.

The emergence of disinfectant-resistant pathogens in water is a major threat to public health. However, whether human-consumed pharmaceuticals can induce bacterial resistance to disinfectants remains unclear. Herein, Escherichia coli was exposed to 12 antidepressants, and susceptibility of antidepressant-induced chloramphenicol (CHL)-resistant mutants to disinfectants was tested. Whole genome sequencing, global transcriptomic sequencing, and real-time quantitative polymerase chain reaction were used to elucidate the underlying mechanisms. We observed that duloxetine, fluoxetine, amitriptyline, and sertraline significantly increased the mutation frequency of E. coli against CHL by 15- to 2948-fold. The resultant mutants increased the average MIC50 of sodium hypochlorite, benzalkonium bromide, and triclosan roughly 2- to 8-fold. Consistently, marRAB and acrAB-tolC genes, together with ABC transporter genes (e.g., yddA, yadG, yojI, and mdlA), were triggered to increase the efflux of disinfectants out of the cell, while ompF was inhibited, reducing disinfectant penetration into the cell. Additionally, the occurrence of DNA mutations in marR and acrR in the mutants was observed, potentially resulting in increased synthesis of the AcrAB-TolC pump. This study indicates that pharmaceutical exposure may create disinfectant-resistant bacteria, which may then be released into water systems, providing novel insights into the potential source of water-borne disinfectant-resistant pathogens.

The widespread escalation of bacterial resistance threatens the safety of the food chain. To investigate the resistance characteristics of E. coli strains isolated from disinfected tableware against both disinfectants and antibiotics, 311 disinfected tableware samples, including 54 chopsticks, 32 dinner plates, 61 bowls, 11 cups, and three spoons were collected in Chengdu, Sichuan Province, China to screen for disinfectant- (benzalkonium chloride and cetylpyridinium chloride) and tigecycline-resistant isolates, which were then subjected to antimicrobial susceptibility testing and whole genome sequencing (WGS). The coliform-positive detection rate was 51.8% (161/311) and among 161 coliform-positive samples, eight E. coli strains were multidrug-resistant to benzalkonium chloride, cetylpyridinium chloride, ampicillin, and tigecycline. Notably, a recently described mobile colistin resistance gene mcr-10 present on the novel IncFIB-type plasmid of E. coli EC2641 screened was able to successfully transform the resistance. Global phylogenetic analysis revealed E. coli EC2641 clustered together with two clinically disinfectant- and colistin-multidrug-resistant E. coli strains from the US. This is the first report of mcr-10-bearing E. coli detected in disinfected tableware, suggesting that continuous monitoring of resistance genes in the catering industry is essential to understand and respond to the transmission of antibiotic resistance genes from the environment and food to humans and clinics.

Klebsiella pneumoniae infection and antimicrobial resistance among children are major concerns. The occurrence of hypervirulent K. pneumoniae (hvKp) infections is gradually increasing worldwide, and disinfectant resistance is also being reported. Carbapenem- and disinfectant-resistant hvKp infection has made clinical treatment and nosocomial infection control among children increasingly challenging. In this study, whole-genome sequencing was conducted among 34 Carba NP-positive carbapenem-resistant K. pneumoniae (CRKP) strains, and the distribution of antibiotic resistance genes, virulence genes and disinfectant resistance genes was determined. Eleven distinct STs were identified, and most of them were ST11 (58.8%). Among the carbapenem resistance genes, KPC-2 was predominant (61.8%), followed by NDM-1 (26.5%) and IPM-4 (11.8%), and no other carbapenemase genes were found. Twelve virulence genes were investigated. All 34 CRKP strains carried the following virulence genes: rcsA/B, entA, fimA/H and mrkA/D. The gene iucB was present in only 3 (8.9%) CRKP strains. The positive detection rates of the iroN and ybtA genes were 94.1% and 64.7%, respectively. None of the strains was found to carry the rmpA and iroB genes. Two disinfectant resistance genes were investigated in this study. Twenty-one (61.8%) strains carried both the qacE and cepA disinfectant resistance genes, 13 (38.2%) CRKP strains carried only the cepA gene, and no strains with only the qacE gene was detected. The correlations among virulence, drug resistance and disinfectant tolerance showed that the virulence and disinfectant resistance genes were distinct among several types of carbapenemase-producing CRKP strains.

Disinfectant resistance is evolving into a serious problem due to the long-term and extensive use of disinfectants, which brings great challenges to hospital infection control. As a notorious multidrug-resistant bacterium, carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the most common and difficult pathogens of nosocomial infection. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests of seven kinds of disinfectants (0.1% benzalkonium bromide, 4% aqueous chlorhexidine, 75% alcohol, entoiodine II, 2% glutaraldehyde, 2000 mg/L chlorine-containing disinfectants, and 3% hydrogen peroxide) were detected by the broth dilution method. Three efflux pump genes (oqxA, oqxB, and qacE∆1-sul1) were detected by PCR. The mean MIC value of aqueous chlorhexidine from the intensive care unit (ICU) (0.0034%) was significantly higher than that from non-ICUs (0.0019%) (p < 0.05). The positive rates of three efflux pump genes oqxA, oqxB and qacE∆1-sul1 were 60.9% (39/64), 17.2% (11/64) and 71.9% (46/64) in the detected CRKP isolates, respectively. This study discovered that CRKP strains demonstrated extensive resistance to clinical disinfectants and suggest that it is necessary to perform corresponding increases in the concentration of aqueous chlorhexidine and chlorine-containing disinfectants on the basis of current standards in the healthcare industry.

Phthalate esters (PAEs) are commonly released from plastic pipes in some water distribution systems. Here, we show that exposure to a low concentration (1-10 μg/L) of three PAEs (dimethyl phthalate (DMP), di-n-hexyl phthalate (DnHP), and di-(2-ethylhexyl) phthalate (DEHP)) promotes Pseudomonas biofilm formation and resistance to free chlorine. At PAE concentrations ranging from 1 to 5 μg/L, genes coding for quorum sensing, extracellular polymeric substances excretion, and oxidative stress resistance were upregulated by 2.7- to 16.8-fold, 2.1- to 18.9-fold, and 1.6- to 9.9-fold, respectively. Accordingly, more biofilm matrix was produced and the polysaccharide and eDNA contents increased by 30.3-82.3 and 10.3-39.3%, respectively, relative to the unexposed controls. Confocal laser scanning microscopy showed that PAE exposure stimulated biofilm densification (volumetric fraction increased from 27.1 to 38.0-50.6%), which would hinder disinfectant diffusion. Biofilm densification was verified by atomic force microscopy, which measured an increase of elastic modulus by 2.0- to 3.2-fold. PAE exposure also stimulated the antioxidative system, with cell-normalized superoxide dismutase, catalase, and glutathione activities increasing by 1.8- to 3.0-fold, 1.0- to 2.0-fold, and 1.2- to 1.6-fold, respectively. This likely protected cells against oxidative damage by chlorine. Overall, we demonstrate that biofilm exposure to environmentally relevant levels of PAEs can upregulate molecular processes and physiologic changes that promote biofilm densification and antioxidative system expression, which enhance biofilm resistance to disinfectants.

Metals are widely used in animal feed for their growth-stimulating and antimicrobial effects, yet their use may potentially promote the proliferation of antibiotic resistance through co-selection. We studied the prevalence and associations of metal, antibiotic, and disinfectant resistances of 300 Salmonella Typhimurium isolates from pig meat, pig manure, chicken meat, poultry manure, and human stool from Sichuan, China. Seventy four percent of the 300 Salmonella Typhimurium isolates were considered resistant to Cu, almost 50% to Zn and Cr, over 25% to Mn and Cd, and almost 10% to Co. Most of the isolates carried at least one heavy metal resistance gene (HMRG). The Cr-Zn-Cd-resistance gene czcD was carried by 254 isolates and the Cu-resistance genes pcoR and pcoC by 196 and 179 isolates, respectively. Most of the isolates were resistant to at least one antibiotic and almost 80% were multidrug-resistant. The prevalence of resistance to six antibiotics was higher among the pig meat and manure isolates than among other isolates, and that of streptomycin and ampicillin were highest among the pig meat isolates and that of ciprofloxacin and ofloxacin among the pig manure isolates. From 55 to 79% of the isolates were considered resistant to disinfectants triclosan, trichloroisocyanuric acid, or benzalkonium chloride. The metal resistances and HMRGs were associated with resistance to antibiotics and disinfectants. Especially, Cu-resistance genes were associated with resistance to several antibiotics and disinfectants. The transfer of the Cr-Zn-Cd-resistance gene czcD, Cu-resistance gene pcoC, and Co-Ni-resistance gene cnrA into Escherichia coli and the increased Cu-resistance of the transconjugants implied that the resistance genes were located on conjugative plasmids. Thus, the excessive use of metals and disinfectants as feed additives and in animal care may have the potential to promote antibiotic resistance through co-selection and maintain and promote antibiotic resistance even in the absence of antibiotics.