Quaternary Ammonium Compounds Resistance Research Articles

Association analysis of antibiotic and disinfectant resistome in human and foodborne E. coli in Beijing, China

The widespread use of chemical disinfectants and antibiotics poses a major threat to food safety and human health. However, the mechanisms of co-transmission of antimicrobial resistance genes (ARGs) and biocides and metal resistance genes (BMRGs) of foodborne pathogens in the food chain is still unclear. This study isolated 343 E. coli strains from animal-derived foods in Beijing and incorporated online data of human-derived E. coli strains from NCBI. Our results demonstrated a relatively uniform distribution of strains from various regions in Beijing, indicating a lack of region-specific clustering. Additionally, predominant sequence types varied between food- and human-derived strains, suggesting a preference for different hosts and environments. Phenotypic association analysis showed that the chlorine disinfectants peroxides had a significant positive correlation with tetracyclines. Many more ARGs and BMRGs were enriched in human-associated E. coli compared with those in chicken- and pork-origin. The quaternary ammonium compounds (QACs) resistance gene qacEΔ1 had a strong correlation with aminoglycoside resistance gene aadA5, folate pathway antagonist resistance gene dfrA17, sul1 and macrolide resistance gene mph(A). The correlation results indicated a significant association between the copper resistance gene cluster pco and the silver resistance gene cluster sil. Coexistence of many resistance genes was observed within the qacEΔ1 gene structure, with qacEΔ1-sul1 being the most common combination. Our findings demonstrated that the epidemiological spread of resistance is affected by a combination of heavy metals, disinfectants and antibiotic use, suggesting that the prevention and control strategies of antimicrobial resistance need to be multifaceted and comprehensive.

Salinity-mediated enhancement of quaternary ammonium compounds resistance and removal in endophytic bacteria LSE01

The widespread usage of quaternary ammonium compounds (QACs) as disinfectants during the COVID-19 pandemic poses significant environmental risks, such as toxicity to organisms and the emergence of superbugs. In this study, different inorganic salts (NaCl, KCl, CaCl2, MgCl2) were used to induce endophytes LSE01 isolated from hyperaccumulating plants. After five generations of cultivation under 80 g/L NaCl, the minimum inhibitory concentration (MIC) of LSE01 to QACs increased by about 3-fold, while its degradation extent increased from 8% to 84% for C12BDMA-Cl and 5%–89% for C14BDMA-Cl. Transmission electron microscopy (TEM) and three-dimensional fluorescence spectra indicated that the cells induced by high concentration of salt caused plasmolysis and secreted more bound extracellular polymeric substances (B-EPS); these changes are likely to be an important reason for the observed increased resistance and enhanced degradation extent of LSE01 to QACs. Our findings suggest that salt-induction could be an effective way to enhance the resistance and removal of toxic organic pollutants by functional microorganisms.

Shifting from Ammonium to Phosphonium Salts: A Promising Strategy to Develop Next-Generation Weapons against Biofilms.

Since they are difficult and sometimes impossible to treat, infections sustained by multidrug-resistant (MDR) pathogens, emerging especially in nosocomial environments, are an increasing global public health concern, translating into high mortality and healthcare costs. In addition to having acquired intrinsic abilities to resist available antibiotic treatments, MDR bacteria can transmit genetic material encoding for resistance to non-mutated bacteria, thus strongly decreasing the number of available effective antibiotics. Moreover, several pathogens develop resistance by forming biofilms (BFs), a safe and antibiotic-resistant home for microorganisms. BFs are made of well-organized bacterial communities, encased and protected in a self-produced extracellular polymeric matrix, which impedes antibiotics' ability to reach bacteria, thus causing them to lose efficacy. By adhering to living or abiotic surfaces in healthcare settings, especially in intensive care units where immunocompromised older patients with several comorbidities are hospitalized BFs cause the onset of difficult-to-eradicate infections. In this context, recent studies have demonstrated that quaternary ammonium compounds (QACs), acting as membrane disruptors and initially with a low tendency to develop resistance, have demonstrated anti-BF potentialities. However, a paucity of innovation in this space has driven the emergence of QAC resistance. More recently, quaternary phosphonium salts (QPSs), including tri-phenyl alkyl phosphonium derivatives, achievable by easy one-step reactions and well known as intermediates of the Wittig reaction, have shown promising anti-BF effects in vitro. Here, after an overview of pathogen resistance, BFs, and QACs, we have reviewed the QPSs developed and assayed to this end, so far. Finally, the synthetic strategies used to prepare QPSs have also been provided and discussed to spur the synthesis of novel compounds of this class. We think that the extension of the knowledge about these materials by this review could be a successful approach to finding effective weapons for treating chronic infections and device-associated diseases sustained by BF-producing MDR bacteria.

Recombination-mediated dissemination of Methicillin-resistant S. aureus clonal complex 1 in the Egyptian health care settings

BackgroundMethicillin-resistant Staphylococcus aureus (MRSA) is a rapidly evolving pathogen that is frequently associated with outbreaks and sustained epidemics. This study investigated the population structure, resistome, virulome, and the correlation between antimicrobial resistance determinants with phenotypic resistance profiles of 36 representative hospital-acquired MRSA isolates recovered from hospital settings in Egypt.ResultsThe community-acquired MRSA lineage, clonal complex 1 (CC1) was the most frequently detected clone, followed by three other globally disseminated clones, CC121, CC8, and CC22. Most isolates carried SCCmec type V and more than half of isolates demonstrated multi-drug resistant phenotypes. Resistance to linezolid, a last resort antibiotic for treating multidrug resistant MRSA, was observed in 11.11% of the isolates belonging to different genetic backgrounds. Virulome analysis indicated that most isolates harboured a large pool of virulence factors and toxins. Genes encoding aureolysin, gamma hemolysins, and serine proteases were the most frequently detected virulence encoding genes. CC1 was observed to have a high pool of AMR resistance determinants including cfr, qacA, and qacB genes, which are involved in linezolid and quaternary ammonium compounds resistance, as well as high content of virulence-related genes, including both of the PVL toxin genes. Molecular clock analysis revealed that CC1 had the greatest frequency of recombination (compared to mutation) among the four major clones, supporting the role of horizontal gene transfer in modulating AMR and hypervirulence in this clone.ConclusionsThis pilot study provided evidence on the dissemination success of CA-MRSA clone CC1 among Egyptian hospitals. Co-detection of multiple AMR and virulence genes in this lineage pose a broad public health risk, with implications for successful treatment. The results of this study, together with other surveillance studies in Egypt, should be used to develop strategies for controlling MRSA infections in Egyptian health-care settings.

Genotyping of Acinetobacter baumannii isolates from a tertiary care hospital in Cochin, South India

Acinetobacter baumannii poses a significant challenge in healthcare settings across the globe, with isolates exhibiting carbapenem resistance at unprecedented rates. Here, we characterized a collection of A. baumannii isolates (n=64) recovered during the period September 2020 – November 2021 at a teaching hospital in Cochin, South India. The species identity of the isolates was confirmed with bla OXA-51-like PCR. The major carbapenemase determinants identified were bla OXA-23-like (45, 70.3 %) and bla NDM-1 (31, 48.4 %); co-occurrence of these genes was also observed in 27 (42.2 %) isolates. Other resistance genes identified included bla PER (34, 53.1 %), aac(6')-Ib-cr (42, 65.6 %), qnrS (25, 39.1 %), sul1 (32, 50 %), sul2 (33, 51.6 %), strA/strB (36, 56.3 %), aphA1-Iab (35, 54.7 %) and tetB (32, 50 %). Mapping PCR revealed the insertion element, ISAbaI upstream of bla OXA-23-like in all isolates possessing this gene. Concerning disinfectant resistance, all isolates carried the quaternary ammonium compound (QAC) resistance gene, qacEΔ1. Minimal inhibitory concentration (MIC) of benzalkonium chloride was high among the isolates and ranged from 8 to 128 µg ml−1. However, low MICs were observed for chlorhexidine and triclosan, with the majority (54, 80.6 %) of isolates showing an MIC of 2 µg ml−1 for chlorhexidine and all isolates exhibiting MICs of ≤0.125 µg ml−1 for triclosan. Further, all isolates were strong biofilm-producers, as assessed by the crystal violet-based microtitre plate assay. The ApaI-pulsed-field gel electrophoresis (PFGE) revealed the multi-clonal nature of the isolates, with 16 clusters and 16 unique pulsotypes identified at a cut-off of 80 %. In short, this study provides useful data on the molecular features of A. baumannii from this region, which could be helpful to assess the local epidemiology of this pathogen and also to devise infection control strategies.

Sequencing-based analysis of the microbiomes of Spanish food processing facilities reveals environment-specific variation in the dominant taxa and antibiotic resistance genes

In the last years, advances in high throughput sequencing technologies have opened the possibility to broaden environmental monitoring activities in facilities processing food, offering expanded opportunities for characterizing in an untargeted manner the microbiome and resistome of foods and food processing environments (FPE) with huge potential benefits in food safety management systems. Here the microbiome and resistome of FPE from slaughterhouses (n = 3), dairy (n = 12) and meat (n = 10) processing plants were assessed through whole metagenome sequencing of 2 composite samples for each facility, comprising 10 FPE swabs taken from food contact surfaces and 10 FPE samples from non-food contact surfaces, respectively. FPE from slaughterhouses had more diverse microbiomes and resistomes, while FPE from dairy processing plants showed the highest β-dispersion, consistent with a more heterogeneous microbiome and resistome composition. The predominant bacterial genera depended on the industry type, with Pseudomonas and Psychrobacter being highly dominant in surfaces from slaughterhouses and meat industries, while different lactic acid bacteria predominated in dairy industries. The most abundant antimicrobial resistance genes (ARG) found were associated with resistance to aminoglycosides, tetracyclines and quaternary ammonium compounds (QAC). ARGs relating to resistance to aminoglycosides and tetracyclines were significantly more prevalent in slaughterhouses than in food processing plants, while QAC resistance genes were particularly abundant in some food contact surfaces from dairy and meat processing plants, suggesting that daily sanitation under suboptimal conditions may be selecting for persistent microbiota tolerant to these biocides in some facilities. The taxonomic mapping of ARG pointed to specific bacterial genera, such as Escherichia, Bacillus, or Staphylococcus, as carriers of the most relevant resistance determinants. About 63% of all ARG reads were assigned to contigs classified as plasmid-associated, indicating that the resistome of FPE may be strongly shaped through the spread of mobile genetic elements. Overall, the relevance of FPE as reservoirs of ARG was confirmed and it was demonstrated that next generation sequencing technologies allowing a deep characterisation of sources and routes of spread of microorganisms and antimicrobial resistance determinants in food industry settings hold promise to be integrated in monitoring and food safety management programmes.

Shotgun-metagenomics reveals a highly diverse and communal microbial network present in the drains of three beef-processing plants.

Multi-species biofilms pose a problem in various environments, especially food-processing environments. The diversity of microorganisms in these biofilms plays a critical role in their integrity and protection against external biotic and abiotic factors. Compared to single-species biofilms, mixed-species biofilms are more resistant to various stresses, including antimicrobials like sanitizers. Therefore, understanding the microbiome composition and diversity in biofilms and their metabolic potential is a priority when developing intervention techniques to combat foodborne pathogens in food processing environments. This study aimed to describe and compare the microbiome profile of 75 drain biofilm samples obtained from five different locations (Hotscale, Hotbox, Cooler, Processing, & Grind room) of three beef-processing plants (Plant A, B & C) taken over two timepoints 2017-18 (T1) and 2021 (T2) by shotgun sequencing. Core microbiome analysis found Pseudomonas, Psychrobacter, and Acinetobacter to be the top three prevalent genera among the plants and locations. Alpha diversity analysis demonstrated a high diversity of microbiome present in all the plants and locations across the time points. Functional analysis showed the high metabolic potential of the microbial community with abundance of genes in metabolism, cell-adhesion, motility, and quorum sensing. Moreover, Quaternary Ammonium Compound (QAC) resistance genes were also observed, this is significant as QAC sanitizers are commonly used in many food processing facilities. Multi-functional genes such as transposases, polymerases, permeases, flagellar proteins, and Mobile Genetic Elements (MGEs) were found suggesting these are dynamic microbial communities that work together to protect themselves against environmental stresses through multiple defense mechanisms. This study provides a framework for understanding the collective microbial network spanning a beef processing system. The results can be used to develop intervention strategies to best control these highly communicative microbial networks.

Resistance profiles and genotyping of extended-spectrum beta-lactamase (ESBL) -producing and non-ESBL-producing E. coli and Klebsiella from retail market fishes.

Studies on antimicrobial resistance (AMR) profiles and epidemiological affirmation for AMR transmission are limited in fisheries and aquaculture settings. Since 2015, based on Global Action Plan on AMR by World Health Organization (WHO) and World Organization for Animal Health (OIE), several initiatives have been under taken to enhance the knowledge, skills and capacity to establish AMR trends through surveillance and strengthening of epidemiological evidence. The focus of this study was to determine the prevalence of antimicrobial resistance (AMR), its resistance profiles and molecular characterization with respect to phylogroups, antimicrobial resistance genes (ARGs), virulence genes (VGs), quaternary ammonium compounds resistance (QAC) genes and plasmid typing in retail market fishes. Pulse field gel electrophoresis (PFGE) to understand the genetic lineage of the two most important Enterobacteriaceae members, E. coli and Klebsiella sp. was performed. 94 fish samples were collected from three different sites viz., Silagrant (S1), Garchuk (S2) and North Guwahati Town Committee (NGTC) Region (S3) in Guwahati, Assam. Out of the 113 microbial isolates from the fish samples, 45 (39.82%) were E. coli; 23 (20.35%) belonged to Klebsiella genus. Among E. coli, 48.88% (n=22) of the isolates were alerted by the BD Phoenix M50 instrument as ESBL, 15.55% (n=7) as PCP and 35.55% (n=16) as non-ESBL. E. coli (39.82%) was the most prevalent pathogen among the Enterobacteriaceae members screened and showed resistance to ampicillin (69%) followed by cefazoline (64%), cefotaxime (49%) and piperacillin (49%). In the present study, 66.66% of E. coli and 30.43% of Klebsiella sp. were categorized as multi drug resistance (MDR) bacteria. CTX-M-gp-1, with CTX-M-15 variant (47%), was the most widely circulating beta-lactamase gene, while other ESBL genes blaTEM (7%), blaSHV (2%) and blaOXA-1-like (2%) were also identified in E. coli. Out of the 23 isolates of Klebsiella, 14(60.86%) were ampicillin (AM)-resistant (11(47.82%) K. oxytoca, 3(13.04%) K. aerogenes), whereas 8(34.78%) isolates of K. oxytoca showed intermediate resistance to AM. All Klebsiella isolates were susceptible to AN, SCP, MEM and TZP, although two K. aerogenes were resistant to imipenem. DHA and LAT genes were detected, respectively, in 7(16%) and 1(2%) of the E. coli strains while a single K. oxytoca (4.34%) isolate carried MOX, DHA and blaCMY-2 genes. The fluoroquinolone resistance genes identified in E. coli included qnrB (71%), qnrS (84%), oqxB (73%) and aac(6)-Ib-cr (27%); however, in Klebsiella, these genes, respectively, had a prevalence of 87%, 26%, 74% and 9%. The E. coli isolates belonged to phylogroup A(47%), B1(33%) and D(14%). All of the 22(100%) ESBL E. coli had chromosome-mediated disinfectant resistance genes viz., ydgE, ydgF, sugE(c), mdfA while 82% of ESBL E. coli had emrE. Among the non-ESBL E. coli isolates, 87% of them showed the presence of ydgE, ydgF and sugE(c) genes, while 78% of the isolates had mdfA and 39% had emrE genes respectively. 59% of the ESBL and 26% of the non-ESBL E. coli had showed the presence of qacEΔ1. The sugE(p) was present in 27% of the ESBL-producing E. coli and in 9% of non-ESBL isolates. Out of the 3 ESBL-producing Klebsiella isolates, 2(66.66%) K. oxytoca isolates were found harboring plasmid-mediated qacEΔ1 gene while one (33.33%) K. oxytoca isolate had sugE(p) gene. IncFI was the most prevalent plasmid type detected in the isolates studied, with A/C (18%), P (14%), X, Y (9% each) and I1-Iγ (14%, 4%). 50% (n=11) of the ESBL and 17% (n=4) of the non-ESBL E. coli isolates harboured IncFIB and 45% (n=10) ESBL and one (4.34%) non-ESBL E. coli isolates harboured IncFIA. Dominance of E. coli over other Enterobacterales and diverse phylogenetic profiles of E. coli and Klebsiella sp. suggests the possibility of contamination and this may be due to compromised hygienic practices along the supply chain and contamination of aquatic ecosystem. Continuous surveillance in domestic markets must be a priority in addressing antimicrobial resistance in fishery settings and to identify any unwarranted epidemic clones of E. coli and Klebsiella that can challenge public health sector.

Efficacy of Quaternary Ammonium Compounds for Control of Individual and Mixed Cultures of Escherichia coli with High- and Low-Quaternary Ammonium Compounds Resistance.

Escherichia coli is a well-characterized micro-organism in scientific literature. Similarly, quaternary ammonium compounds (QACs) are historical sanitizers in food processing. However, the use of QACs has been questioned due to bacterial resistance in some studies. Therefore, this study aimed to compare effects of single and mixed cultures of E. coli strains of different serogroups with either high (six strains) or low (five strains) resistance to QACs. Twenty-five combinations of strains with either high (H)- or low (L)-QAC resistance were analyzed (H + H vs. L + L). After exposure to QAC, combinations with statistical differences (p < 0.05) compared with individuals were selected and an inactivation model determined using GInaFit®. Only one combination of two strains (C23 and C20) with low-QAC resistance (mixture T18) had greater resistance (p < 0.05) than the individual isolates. The combination T18 and individual strain C23 presented a Weibull model, whereas the other isolated strain (C20) presented a biphasic inactivation model with a shoulder. Whole genome sequencing determined that unlike C20, C23 carried yehW, which may have led to Weibull inactivation. Possibly, very rapid interaction of C20 with the QAC favored increased survival of C23 and overall persistence of the T18 mixture. Consequently, our results indicate that individual E. coli with low-QAC resistance can synergistically interfere with QAC inactivation.

QAC Resistance Genes in ESBL-Producing E. coli Isolated from Patients with Lower Respiratory Tract Infections in the Central Slovenia Region-A 21-Year Survey.

Biocidal products prevent the spread of pathogenic microorganisms, including extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC), which is one of the most alarming health problems worldwide. Quaternary ammonium compounds (QACs) are surface-active agents that interact with the cytoplasmic membrane and are widely used in hospitals and food processing environments. A collection of 577 ESBL-EC, isolated from lower respiratory tract (LRT) samples, was screened for QAC resistance genes oqxA; oqxB; qacEΔ1; qacE; qacF/H/I; qacG; sugE (p); emrE; mdfA; sugE (c); ydgE; ydgF; and for class 1, 2, and 3 integrons. The prevalence of chromosome-encoded genes ranged from 77 to 100%, while the prevalence of QAC resistance genes encoded on mobile genetic elements (MGEs) was relatively low (0-0.9%), with the exception of qacEΔ1 (54.6%). PCR screening detected the presence of class 1 integrons in 36.3% (n = 210) of isolates, which were positively correlated with qacEΔ1. More correlations between QAC resistance genes, integrons, sequence type group ST131, and β-lactamase genes were presented. The results of our study confirm the presence of QAC resistance genes and also class 1 integrons commonly found in multidrug-resistant clinical isolates and highlight the potential role of QAC resistance genes in the selection of ESBL-producing E. coli in hospitals.

Functional Characterization of a Novel SMR-Type Efflux Pump RanQ, Mediating Quaternary Ammonium Compound Resistance in Riemerella anatipestifer.

Riemerella anatipestifer (R. anatipestifer) is a multidrug-resistant bacterium and an important pathogen responsible for major economic losses in the duck industry. Our previous study revealed that the efflux pump is an important resistance mechanism of R. anatipestifer. Bioinformatics analysis indicated that the GE296_RS02355 gene (denoted here as RanQ), a putative small multidrug resistance (SMR)-type efflux pump, is highly conserved in R. anatipestifer strains and important for the multidrug resistance. In the present study, we characterized the GE296_RS02355 gene in R. anatipestifer strain LZ-01. First, the deletion strain RA-LZ01ΔGE296_RS02355 and complemented strain RA-LZ01cΔGE296_RS02355 were constructed. When compared with that of the wild-type (WT) strain RA-LZ01, the mutant strain ΔRanQ showed no significant influence on bacterial growth, virulence, invasion and adhesion, morphology biofilm formation ability, and glucose metabolism. In addition, the ΔRanQ mutant strain did not alter the drug resistance phenotype of the WT strain RA-LZ01 and displayed enhanced sensitivity toward structurally related quaternary ammonium compounds, such as benzalkonium chloride and methyl viologen, which show high efflux specificity and selectivity. This study may help elucidate the unprecedented biological functions of the SMR-type efflux pump in R. anatipestifer. Thus, if this determinant is horizontally transferred, it could cause the spread of quaternary ammonium compound resistance among bacterial species.

Migration, Transformation, and Toxicity of Quaternary Ammonium Antimicrobial Agents in the Environment

Quaternary ammonium compounds (QACs) are one type of widely used cationic biocide, and their usage amount is growing rapidly due to the flu and COVID-19 pandemic. Many QACs were released into the environment in or after the course of their use, and thus they were widely detected in water, sediment, soil, and other environmental media. QACs have stronger surface activity and non-specific biotoxicity, which poses a potential threat to the ecosystem. In this study, the environmental fate and potential toxicity of QACs were documented in terms of their migration and transformation process, biological toxicity effects, and the main mechanisms of bacterial resistance to QACs. Aerobic biodegradation was the main natural way of eliminating QACs in the environment, and the reaction was mainly initiated by the hydroxylation of C atoms at different positions of QACs and finally mineralized to CO2and H2O through decarboxylation, demethylation, and β-oxidation reaction. Toxicological studies showed that QACs at environmental concentrations could not pose acute toxicity to the selected biotas but threatened the growth and reproduction of aquatic organisms like Daphnia magna. Their toxicity effects depended on their molecular structure, the tested species, and the exposed durations. Additionally, our team first investigated the toxicity effects and mechanisms of QACs toward Microcystis aeruginosa, which showed that QACs depressed the algae growth through the denaturation of photosynthetic organelles, suppression of electron transport, and then induction of cell membrane damage. In the environment, the concentrations of QACs were always lower than their bactericidal concentrations, and their degradation could induce the formation of a concentration gradient, which facilitated microbes resistant to QACs. The known resistance mechanisms of bacteria to QACs mainly included the change in cell membrane structure and composition, formation of biofilm, overexpression of the efflux pump gene, and acquisition of resistance genes. Due to the similar targets and mechanisms, QACs could also induce the occurrence of antibiotic resistance, mainly through co-resistance and cross-resistance. Based on the existing data, future research should emphasize the toxicity effect and the potential QACs resistance mechanism of microorganisms in real environmental conditions.

Quaternary Phosphonium Compound Unveiled as a Potent Disinfectant against Highly Resistant Acinetobacter baumannii Clinical Isolates.

Acinetobacter baumannii is classified as a highest threat pathogen, urgently necessitating novel antimicrobials that evade resistance to combat its spread. Quaternary ammonium compounds (QACs) have afforded a valuable first line of defense against antimicrobial resistant pathogens as broad-spectrum amphiphilic disinfectant molecules. However, a paucity of innovation in this space has driven the emergence of QAC resistance. Through this work, we sought to identify next-generation disinfectant molecules with efficacy against highly resistant A. baumannii clinical isolates. We selected 12 best-in-class molecules from our previous investigations of quaternary ammonium and quaternary phosphonium compounds (QPCs) to test against a panel of 35 resistant A. baumannii clinical isolates. The results highlighted the efficacy of our next-generation compounds over leading commercial QACs, with our best-in-class QAC (2Pyr-11,11) and QPC (P6P-10,10) displaying improved activities with a few exceptions. Furthermore, we elucidated a correlation between colistin resistance and QAC resistance, wherein the only pan-resistant isolate of the panel, also harboring colistin resistance, exhibited resistance to all tested QACs. Notably, P6P-10,10 maintained efficacy against this strain with an IC90 of 3 μM. In addition, P6P-10,10 displayed minimum biofilm eradication concentrations as low as 32 μM against extensively drug resistant clinical isolates. Lastly, examining the development of disinfectant resistance and cross-resistance, we generated QAC-resistant A. baumannii mutants and observed the development of QAC cross-resistance. In contrast, neither disinfectant resistance nor cross-resistance was observed in A. baumannii under P6P-10,10 treatment. Taken together, the results of this work illustrate the need for novel disinfectant compounds to treat resistant pathogens, such as A. baumannii, and underscore the promise of QPCs, such as P6P-10,10, as viable next-generation disinfectant molecules.

Short-term stress of quaternary ammonium compounds on intracellular and extracellular resistance genes in denitrification systems

The wastewater containing high quaternary ammonium compounds (QACs) concentrations from hospitals and other sources may cause the transient shock loads on the wastewater treatment plants (WWTPs). Denitrification is the key part of nitrogen removal from wastewater, however, the effects of QACs on the denitrification and the spread patterns of intracellular and extracellular resistance genes were still unknown. The short-term stress of three frequently detected QACs on denitrification were systematically investigated in this study. The inhibitory effect on nitrate removal had the following rules: BAC C12 > ATMAC C12 > DADMAC C12. The inhibition of QACs on denitrification performance was correlated with the reduction of Thauera abundance, and a higher QACs concentration made stronger inhibitory effect. Moreover, high concentrations of QACs could cause a significant decrease in reactive oxygen species, inhibit cell activity and extracellular polymeric substances secretion. Furthermore, QACs stimulated an overall increase of intracellular and extracellular QACs resistance genes in denitrification systems, both in sludge and in water. And co-selection among resistance genes occurred under shock loads of QACs, especially a high BAC C12 concentration caused a substantial raise in abundances of resistance genes in water. It was worth noting that resistance genes in sludge and water were mainly contributed by different bacteria. Rhizobium and Pseudomonas may be the potential hosts contributed to resistance genes in sludge while Thauera mainly contributed to that in water.

Metagenomic Analysis of Urban Wastewater Treatment Plant Effluents in Tokyo.

PurposeUrban wastewater treatment plant (WWTP) effluents, even with proper treatment, may cause antimicrobial resistance (AMR) burden, with a high frequency of acquired antimicrobial resistance genes (ARGs). The dissemination of ARGs into the environment increases the risk of infectious diseases; however, there is little direct evidence regarding their epidemiological effects. This study aimed to assess effluents from urban WWTPs around the Tama River and Tokyo Bay using metagenomic analysis of (AMR) genes (ARGs) and heavy-metal resistance genes.MethodsMetagenomic DNA-seq analysis of water samples and resistome analysis were performed.ResultsThe most prevalent ARG was the sulfonamide resistance gene, sul1, followed by the quaternary ammonium compound resistance gene, qacE, suggesting that basic gene sets (sul1 and ∆qacE) in the class 1 integrons are the predominant ARGs. The aminoglycoside resistance genes, aadA and aph, and macrolide resistance genes, msr(E) and mph(E), were the predominant ARGs against each antimicrobial. blaOXA and blaGES were frequently detected, whereas the blaCTX-M cluster was faintly detected. Non-metric multidimensional scaling plot analysis and canonical correspondence analysis results suggested that marked differences in ARGs could be involved in the seasonal differences; qnrS2, aac(6’)-Ib, and mef(C) increased markedly in summer, whereas msr(E) was more frequently detected in winter. Heavy-metal (Hg and Cu) resistance genes (HMRGs) were significantly detected in effluents from all WWTPs.ConclusionWe characterized a baseline level of the environmental ARG/HMRG profile in the overall community, suggesting that environmental AMR surveillance, particularly in urban WWTPs, is a valuable first step in monitoring the AMR dissemination of bacteria from predominantly healthy individuals carrying notable ARG/Bs.

The fate and behavior mechanism of antibiotic resistance genes and microbial communities in flocs, aerobic granular and biofilm sludge under chloroxylenol pressure

Chloroxylenol (PCMX), an antibacterial agent, has been widely detected in water environment and has toxic effects on biology and ecology. During 270 d, the influence of PCMX on the performance of three nitrification systems was investigated, including floc-based sequencing batch reactor (FSBR), aerobic granule-based SBR (AGSBR) and biofilm SBR (BSBR). The nitrification capability of three systems was inhibited by PCMX, but recovered after domestication, and PCMX made three systems realize partial nitrification for 10, 100 and 35 days, respectively. The extracellular polymeric substances of three systems increased first and then decreased with the increment of PCMX. The granular structure of AGSBR may be conducive to the enrichment of antibiotic resistance genes (ARGs), and almost all ARGs of BSBR were reduced during the addition of 5.0 mg/L PCMX. The microbial community results showed that Rhodococcus as potential degrading bacteria was continuously enriched in three systems. Piscinibacter was regarded as the potential antibiotic resistant bacteria, which was positively associated with multiple ARGs in three systems. Additionally, quaternary ammonium compounds resistance genes had a variety of positive correlations with bacteria in three systems. This study provided a new perspective for the usage and treatment of PCMX.

Fates of quaternary ammonium compound resistance genes and the corresponding resistant strain in partial nitrification/anammox system under pressure of hexadecyl trimethyl ammonium chloride

Hexadecyl trimethyl ammonium chloride (ATMAC-C16) is a kind of quaternary ammonium compound (QACs) which is extensively consumed as disinfectants, antimicrobials and surfactants. Here, the partial nitrification/anammox (PN/A) system was exposed to different levels of ATMAC-C16 (0–10 mg/L) and the main objective was to reveal the long-term microbiological responses of PN/A system to ATMAC-C16, importantly, explore the tolerance of PN/A to ATMAC-C16 and the key resistant strain. Nitrogen removal efficiency was influenced by environmental and extreme levels of ATMAC-C16 through mainly affecting the anammox (hzsB) gene. Two types of anammox, Candidatus Jettenia and Candidatus Kuenenia, were enriched under the pressure of ATMAC-C16, which allowed PN/A system to maintain good nitrogen removal performance. ATMAC-C16 might cause the hormesis of entire microbial population in PN/A system, leading to the enhancement of cell viability. ATMAC-C16 decreased the relative abundances of most antibiotics resistance genes (ARGs) but significantly enriched QACs resistance genes (QRGs). The tolerance of PN/A system to ATMAC-C16 might be strengthened by inducing the efflux pumps encoding genes (qacH-01/02). Microbial hosts dynamic and co-selection mechanism among ARGs and QRGs resulted in the opposite trends of qacEdeltal-01/02 and qacH-01/02. Pseudoxanthomonas mexicana was identified as the ATMAC-C16 resistant strain, and its resistance to 10 mg/L ATMAC-C16 might not only obtain by capturing the qacH gene, but also benefit from its own efflux pump system. Therefore, from the perspective of the transmission of resistance genes, especially for QRGs, the spread risk of QRGs and ATMAC-C16 resistant strain in PN/A technique should be taken seriously.

Molecular detection of quaternary ammonium compound resistance genes of Staphylococcus aureus from udder surface and mastitis milk of bovines

Quaternary ammonium compounds (QAC) are widely used biocides in the field of medical and veterinary practice. Resistance to QACs is an emerging problem due to this widespread use. The present study was undertaken to detect the presence of genes that contribute to resistance to QACs in Staphylococcus aureus isolates obtained from udder surface and mastitis milk. A total 22 isolates of S. aureus were obtained from udder washings and milk of bovine mastitis cases. Among these, seventeen isolates were from udder wash samples and five isolates from clinical and subclinical cases of mastitis. Broth microdilution assay was performed to assess the minimum inhibitory concentration (MIC) of the isolates against Cetyltrimethylammonium bromide (CTAB), a QAC. Polymerase chain reaction (PCR) was done targeting qac A/B gene which codes for efflux pump which targets QACs. In the present study qac A/B gene was detected in seven out of 22 isolates of S. aureus. Three of these isolates were obtained from udder washings before milking and four, after milking. None of the S. aureus isolates from mastitis milk possessed the gene. The average minimum inhibitory concentration of qac A/B positive isolates against CTAB was 0.63 ± 0.55 μg/ml. The average MIC between qac A/B positive and negative isolates were statistically insignificant. The study points to the fact that multiple factors could be contributing to biocide resistance in S. aureus.

178. Endemic Carbapenem Resistance Driven By Clonal and Horizontal Spread of blaIMP-4 Across Diverse Enterobacterales: Jumping Genes, Promiscuous Plasmids and Killer Clones

BackgroundCarbapenem-resistant Enterobacterales (CRE) have become endemic and cause significant morbidity and mortality globally. The metallo-beta-lactamase gene blaIMP-4 is a key CRE resistance determinant in Australia and Asia but its genomic context remains unknown. We aimed to determine the genomic epidemiology of blaIMP-4 in clinical and environmental isolates from 2008 – 2020 at our institution.MethodsWe performed whole genome sequencing on 219 blaIMP-4-carrying isolates from 134 patients (219 short-read and 75 long-read). Multi-locus sequence types (MLSTs), resistance determinants and plasmid replicons were assessed. High-quality de novo hybrid assemblies were used to identify location of blaIMP-4 gene. We conducted phylogenetic analysis for key MLSTs and plasmids.Results Bla IMP-4 was noted on a class I integron also harboring aminoglycoside, sulfamethoxazole, chloramphenicol and quaternary ammonium compound resistance genes. This integron was able to migrate over time to 10 bacterial species (42 STs) and 6 different plasmid types (Figure 1 and Figure 2). From 2008-2020, blaIMP-4 was present on IncC plasmids in Serratia marcescens and Klebsiella pneumoniae. We noted small outbreaks of Pseudomonas aeruginosa ST111 with chromosomal integration of blaIMP-4 from 2008-2018 (16 isolates) and Enterobacter cloacae complex ST114 with blaIMP-4 on IncFIB(K)/IncFIA(HI1) plasmids from 2011-2020 (19 isolates). From 2016-2020, there was an explosion of diverse IncHI2 plasmids carrying blaIMP-4. This was driven by clonal expansion of E. cloacae complex ST93/ST190 (79 isolates), with spillover of IncHI2 plasmids to Klebsiella spp (13 isolates), Citrobacter spp (2 isolates), S. marcescens (1 isolate), Escherichia coli (4 isolates). In addition to blaIMP-4, these plasmids carried mcr-9.1, a colistin resistance gene, and resistance determinants to nearly all key classes of Gram-negative antimicrobials.Figure 1. Bacterial species harboring blaIMP-4 2008-2020 BlaIMP-4 was noted in diverse bacterial species over the study period. Serratia marcescens and Klebsiella pneumoniae were present throughout. Outbreaks of Enterobacter cloacae complex ST114, ST190 and ST93 and Pseudomonas aeruginosa ST111 were noted.Figure 2. Diverse plasmids associated with blaIMP-4 carriage determined by de novo hybrid assembly Presence of blaIMP-4 on diverse plasmids that varied through the study period was noted. Plasmids were charaterised by analysing de novo hybrid assembly data and co-location of blaIMP-4 and plasmid replicons on the same contigs.Conclusion Bla IMP-4 spread on a class I integron was responsible for endemic carbapenem resistance at our institution. This mobile genetic element was able to persist due to both clonal spread and entry into diverse plasmids. Concerningly, we noted a large outbreak driven by IncHI2 plasmids harboring colistin resistance genes with spread to multiple bacterial species.Disclosures All Authors: No reported disclosures

Genomic analysis of antibiotic-resistant Enterobacter spp. from wastewater sources in South Africa: The first report of the mobilisable colistin resistance mcr-10 gene in Africa

This study delineated the resistome, virulome, mobile genetic elements, clonal lineages, phylogenetic and epidemiological insights of antibiotic-resistant Enterobacter cloacae complex (ECC) isolates obtained from a wastewater treatment plant (WWTP) and its associated waters in KwaZulu Natal, South Africa. The ECC isolates obtained from the final effluent of a (WWTP) and the receiving river upstream and downstream of the WWTP were sequenced on an Illumina MiSeq machine. Genomic analysis revealed that E. asburiae and E. kobei both harboured the fosfomycin resistance gene ( fosA ) and E. kobei was positive for the colistin resistance gene mcr-10 . The genetic environment of the fosA gene was not associated with any MGE and was found on the chromosome in both E. asburiae and E. kobei. The mcr-10 cassette was found associated with a tyrosine integrase and the insertion sequence IS3 (tyrosine-type recombinase/integrase: mcr-10 :IS3) genetic context located on an E. kobei plasmid pSTW0522-51-1 (with a 100% homology). All three isolates carried different bla ACT genes that were not associated with any MGE but had the sugE gene (encodes the quaternary ammonium compound resistance protein) in their genetic environment suggesting co-resistance to biocides and antibiotics. E. kobei harboured the virulence gene terC which confers resistance to tellurium. The E. kobei isolate was phylogenetically related to clinical isolates whereas E. asburiae and E. hormaechei isolates were closely related to environmental isolates. The occurrence of an E. kobei isolate carrying a mobilisable colistin resistance mcr-10 gene in the river water upstream of a major WWTP is the first report of the mcr-10 gene in Africa and is a public health concern warranting monitoring to gain genomic insights into the full extent of ABR in river systems in South Africa and globally.