Quaternary Ammonium Compounds Resistance Genes Research Articles

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.

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.

The responses of microbial metabolic activity, bacterial community and resistance genes under the coexistence of nanoplastics and quaternary ammonium compounds in the sewage environment

Nanoplastics (NPs) and quaternary ammonium compounds (QACs) are frequently detected in sewage. However, little is known about the risks of coexistence of NPs and QACs. In this study, the responses of microbial metabolic activity, bacterial community and resistance genes (RGs) to the exposure of polyethylene (PE), polylactic acid (PLA), silicon dioxide (SiO2) and dodecyl dimethyl benzyl ammonium chloride (DDBAC) were focused on 2nd and 30th day of incubation in sewer environment. Bacterial community contributed 25.01 % to shape RGs and mobile genetic elements (MGEs) after two days of incubation in sewage and plastisphere. After 30 days of incubation, the most important individual factor (35.82 %) was turned to microbial metabolic activity. The metabolic capacity of the microbial communities in plastisphere was stronger than that from SiO2 samples. Moreover, DDBAC inhibited the metabolic capacity of microorganisms in sewage samples, and increased the absolute abundances of 16S rRNA in plastisphere and sewage samples which might be similar to the hormesis effect. After 30 days of incubation, Aquabacterium was the predominant genus in plastisphere. As for SiO2 samples, Brevundimonas was the predominant genus. QACs RGs (qacEdelta1-01, qacEdelta1-02) and antibiotic RGs (ARGs) (aac(6′)-Ib, tetG-1) significantly enriched in plastisphere. There was also co-selection among qacEdelta1-01, qacEdelta1-02 and ARGs. In addition, VadinBC27 which enriched in plastisphere of PLA NPs was positively correlated with the potentially disease-causing genus Pseudomonas. It showed that after 30 days of incubation, plastisphere had an important effect on distribution and transfer of pathogenic bacteria and RGs. Plastisphere of PLA NPs also carried the risk of spreading disease.

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

Presence of Qac genes in clinical isolates of methicillin‐resistant and methicillin‐susceptible Staphylococcus pseudintermedius and their impact on chlorhexidine digluconate susceptibility

To evaluate the presence of quaternary ammonium compound (QAC) (resistance genes, qac A/B, smr, qacG, and qacJ, in clinical isolates of methicillin-susceptible Staphylococcus pseudintermedius (MSSP) and methicillin-resistant S pseudintermedius (MRSP) from dogs and the impact on in vitro chlorhexidine susceptibility. Experimental in vitro study. Seventy isolates from dogs colonized or infected with MRSP (n = 50) or MSSP (n = 20). Agar dilution was used to determine the minimum inhibitory concentration (MIC) of chlorhexidine digluconate. Real-time polymerase chain reaction was used to detect the presence of QAC resistance genes, qacA/B, smr, qacG, and qacJ genes. One or more qac genes were identified in 52 of 70 (74%) isolates. Overall, there was no association between chlorhexidine MIC and the presence of one or more qac genes (P = .85) or the presence of qacA/B (P = .31), smr (P = .72) or qacJ (P = .93) individually. There was an association between qacG and MIC (P = .012), with a median MIC of 1.5 μg/mL for isolates possessing this gene and 1 μg/mL for those not possessing it. Quaternary ammonium compound resistance genes were present in MRSP and MSSP isolates. With the exception of qacG, the presence of these genes was not associated with increased MIC. All isolates exhibited MIC 5000 to 80 000 times lower than the concentration recommended for use. Despite the presence of QAC genes, chlorhexidine digluconate should be effective against MRSP and MSSP if used correctly.

Coexistence of bla NDM-1 and rmtC on a Transferrable Plasmid of a Novel ST192 Klebsiella aerogenes Clinical Isolate.

IntroductionThe occurrence and development of antibiotic resistance are mainly caused by the spread of large plasmids carrying multiple antibiotic resistance genes. Recently, the association between 16S rRNA methyltransferase genes and β-lactamase genes carried by the same plasmid is of concern.MethodsThe Klebsiella aerogenes 1564 was isolated from the catheter tip of a patient in a tertiary hospital, Shanghai, China. The presence of the blaNDM-1 and rmtC genes were assessed by PCR. Complete sequence of plasmid p1564 was determined. The K. aerogenes 1564 was characterized by antimicrobial susceptibility testing, Carbapenemase phenotype confirmation testing, conjugation experiment, S1-PFGE and multilocus sequence typing (MLST).ResultsHerein, we found that a New Delhi Metallo-β-lactamase-1 gene (blaNDM-1) and a 16S rRNA methyltransferase gene (rmtC) coexisted on a transferrable plasmid of a carbapenem-resistant K. aerogenes clinical isolate. The K. aerogenes clinical isolate was found to belong to a novel sequence type 192 (ST192) determined by MLST. The sequencing results of the plasmid p1564 carrying blaNDM-1 gene and rmtC gene showed that the size and guanine-cytosine content of the plasmid were 136, 902 bp and 51.8%, with 164 putative ORFs and two multidrug resistance gene islands. In addition to blaNDM-1and rmtC, the plasmid contained bleomycin resistance gene (bleMBL), CMY-6β-lactamase gene (blaCMY-6), quaternary ammonium compound resistance gene (sugE), truncated quaternary ammonium compound resistance gene (qacEΔ1), aminoglycoside resistance gene (aacA4) and sulfonamide resistance gene (sul1). By comparison, p1564 has high homology with pHS36-NDM from Salmonella enterica subsp. enterica serovar Stanley reported in China, with similar size and both belonging to plasmid incompatibility group A/C.ConclusionThe present study demonstrated for the first time the co-existence of rmtC and blaNDM-1 in a novel ST192 K. aerogenes. The spread of plasmids harboring both blaNDM-1 and rmtC may occur among Enterobacteriaceae in China.

Genome Analysis and Multiplex PCR Method for the Molecular Detection of Coresistance to Cephalosporins and Fosfomycin in Salmonella enterica Serovar Heidelberg

Heidelberg is among the top three Salmonella enterica serovars associated with human foodborne illness in Canada. Traditional culture and antimicrobial susceptibility testing techniques can be time-consuming to identify Salmonella Heidelberg resistant to cephalosporins and fosfomycin. Rapid and accurate detection of such antibiotic-resistant Salmonella Heidelberg isolates is essential to adopt appropriate control measures. In this study, 15 Salmonella Heidelberg strains isolated from feces of Canadian broiler chickens were characterized by whole genome sequencing. Salmonella Heidelberg genomes had an average coverage of greater than 80-fold, an average of 4,761 protein-coding genes, and all belonged to multilocus sequence type ST15. Genome sequences were compared with genomes in the National Center for Biotechnology Information Pathogen Detection database ( www.ncbi.nlm.nih.gov/pathogens/ ), including human outbreak isolates. The Canadian broiler isolates clustered with chicken isolates from the United States and an equine clinical isolate from Ontario, Canada. In agreement with their antimicrobial resistance phenotypes, several chromosomally encoded specific antimicrobial resistance genes including fosA7 and multidrug resistance efflux pump determinants were detected. An AmpC-like β-lactamase gene, blaCMY-2, linked with a quaternary ammonium compound resistance gene, sugE, on a replicon type IncI1 plasmid was detected in all 15 broiler Salmonella Heidelberg isolates. Of the 205,031 published Salmonella genomes screened in silico, 4,954 (2.4%) contained blaCMY-2, 8,143 (4.0%) contained fosA7, and 919 (0.4%) contained both resistance genes. The combination of both resistance genes (fosA7 and blaCMY-2) was detected in 64% of the Heidelberg genomes and in a small proportion of various other serovars. A PCR method was developed to detect Salmonella Heidelberg in pure culture and chicken feces based on specific primers targeting genes conferring fosfomycin (fosA7) and third-generation cephalosporin (blaCMY-2) resistance as well as the Salmonella-specific invA gene and the universal 16S rRNA genes. The PCR assay was specific and sensitive for blaCMY-2 and fosA7 harboring Salmonella Heidelberg. However, some other Salmonella serovars containing these two resistance genes could also be detected by the developed PCR method.

The impact and mechanism of quaternary ammonium compounds on the transmission of antibiotic resistance genes.

The emergence of antibiotic resistance genes (ARGs) in microbes can be largely attributed to the abuse and misuse of antibiotics and biocides. Quaternary ammonium compounds (QACs) have been used worldwide as common disinfectants and detergents; however, their potential impact on the spread and diffusion of ARGs is still unknown. In this study, we detected the QAC resistance gene (qacEΔ1), the 1 integron gene (intI1), and 12 ARGs (sul1, sul2, cfr, cml, fexA, tetA, tetG, tetQ, tetX, ermB, blaTEM, and dfrA1) in 48 water samples from three watersheds by quantitative PCR (qPCR). We investigated the evolution of bacterial antibiotic resistance under QAC and antibiotic environmental pressures by long-term continuous culture. In addition, five QACs were selected to investigate the effect of QAC on the efficiency of conjugation transfer. The changes in bacterial cell membrane and production of reactive oxygen species (ROS) were detected by flow cytometry, revealing the mechanism by which QAC affects the spread of antibiotic resistance. Our results showed that the QAC resistance gene was ubiquitous in watersheds and it had significant correlation with intI1 and seven ARGs (r = 0.999, p < 0.01). QACs could increase the resistance of bacteria to multiple antibiotics. Furthermore, all five QACs promoted the conjugation transfer of the RP4 plasmid; the optimal concentration of QACs was about 10-1-10-2mg/L and their transfer efficiencies were between 1.33 × 10-6 and 8.87 × 10-5. QACs enhanced membrane permeability of bacterial cells and stimulated bacteria to produce ROS, which potentially promoted the transfer of plasmids between bacteria. In conclusion, this study demonstrated that QACs may facilitate the evolution and gene transfer of antibiotic resistance gene among microbiome.

Molecular study of resistance of Staphylococcus aureus to antiseptic quaternary ammonium compounds.

This study determined the prevalence of qac and smr genes in clinical Staphylococcus aureus isolates from hospital-acquired infections and their susceptibility to quaternary ammonium compounds (QACs) and antibiotics, and correlated the presence of antiseptic resistance genes with antibiotic resistance. Susceptibility of 150 non-duplicate clinical S. aureus isolates to antimicrobials and benzalkonium chloride (BAC) was determined by disk diffusion and MIC method, respectively. Resistant strains were analysed by multiplex PCR for the presence of qac and smr genes. Reduced susceptibility to BAC was detected in 30% of isolates (MIC cut-off >8mg/L). QAC resistance genes were detected in 13 isolates with reduced BAC susceptibility. The most frequently detected genes were qacA/B (10 isolates; 22.2%), followed by qacJ (10; 22.2%), smr (8; 17.8%), qacG (8; 17.8%) and qacH (3; 6.7%). There was a strong positive correlation between presence of QAC resistance genes and higher BAC MIC associated with qacA/B, qacJ and smr genes. There was a statistically significant prevalence of antiseptic resistance genes among isolates resistant to cefoxitin, ciprofloxacin, clindamycin, oxacillin, tetracycline and erythromycin. This study highlights the prevalence of qac and smr genes in clinical S. aureus isolates with resistance to QACs. There was an association between the presence of antiseptic resistance genes and resistance to different antibiotics, which may be attributed to the presence of both groups of genes on the same plasmid or to selection of resistant strains. More studies are needed on the clinical relevance of the presence of genes controlling resistance to antiseptics.

Genes significantly associated with lineage II food isolates of Listeria monocytogenes

BackgroundListeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments.ResultsNo SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates.ConclusionsWe identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.

Examination of Quaternary Ammonium Compound Resistance in Proteus mirabilis Isolated from Cooked Meat Products in China.

The aim of this study was to examine the presence of genes responsible for resistance to quaternary ammonium compounds (QACs) and the association of qac genes with class 1 integrons in Proteus mirabilis isolated from cooked meat products. A total of 52 P. mirabilis isolates (29.2%) were detected from 178 samples, and their minimum inhibitory concentrations (MICs) of benzalkonium chloride (BC) ranged from 4 to >32 μg/mL. The isolates with BC MICs of 24 μg/mL were observed most frequently. PCR assays indicated that mdfA, ydgE/ydgF, qacE, qacEΔ1, emrE, sugE(c), and sugE(p) were commonly present (32.7%–100%) in these isolates, but qacH was less prevalent (3.8%). Five groups of resistance gene cassettes were identified in 10 intI1-positive isolates. An unusual gene cassette array dfrA32-ereA-aadA2 was found in one foodborne isolate of P. mirabilis. Two isolates harbored qacH- and sul3- associated non-classic integrons: aadA2-cmlA1-aadA1-qacH-IS440-sul3 and a new arrangement dfrA32-ereA1-aadA2-cmlA1-aadA1-qacH-IS440-sul3, which is first reported in P. mirabilis. Non-classic class 1 integrons were located on conjugative plasmids of 100 kb in two tested isolates. Our data showed that the QAC resistance genes were commonly present among P. mirabilis isolates from cooked meats and qacH was associated with non-classic class 1 integrons. The creation of transconjugants demonstrated that qacH-associated non-classic class 1 integrons were located on conjugative plasmids and therefore could facilitate the co-dissemination of disinfectant and antimicrobial resistance genes among bacteria, an increasing area of concern.

Characterization of a blaNDM‑1‑harboring plasmid from a Salmonellaenterica clinical isolate in China.

The plasmid-mediated transmission of antibiotic resistance genes has been reported to be involved in the development of antibiotic resistance in bacteria, and poses a serious threat for the success of bacterial infection treatment and human health worldwide. The present study used a 454 GS-FLX pyrosequencing system to determine the ~140 kb nucleotide sequence of plasmid pHS36-NDM, which was identified in a Salmonella Stanley isolate from the stool sample of an 11-month-old girl at Lishui Central Hospital, China, and which contains a New Delhi metallo-β-lactamase-1 (NDM-1) carbapenem resistance gene (blaNDM-1). The 181 open reading frames encode proteins with functions including replication, stable inheritance, antibiotic resistance and mobile genetic elements. Both horizontal transfer and passage stability-related genes were identified in pHS36-NDM, including a conserved type 4 secretion system and stbA (stable plasmid inheritance protein A). Two multidrug resistance gene islands were identified: The ISEcp1-blaCMY transposition unit which contains a CMY-6 β-lactamase gene (blaCMY-6) and a quaternary ammonium compound resistance gene (sugE); and the intI1-ISCR27 accessory region, which contained a trimethoprim resistance gene (dfrA12), two aminoglycoside resistance genes (aadA2 and rmtC), a truncated quaternary ammonium compound resistance gene (qacE∆1), a sulfonamide resistance gene (sul1), the blaNDM-1 carbapenemase and a bleomycin resistance gene (bleMBL). pHS36-NDM shared high homology with other blaNDM-1-containing plasmids reported in Sweden, Italy and Japan. However, no previous international travel history was documented for the patient and her family, even to neighboring cities. Furthermore, pHS36-NDM is of a different incompatibility group to other published blaNDM-1-carrying plasmids reported in China, with low homology in the surrounding structure of blaNDM-1. The present study will facilitate the understanding of the underlying resistance and dispersal mechanism of pHS36-NDM, and will deepen our recognition of the ongoing spread of the blaNDM-1-containing plasmids.

Occurrence of Extended-Spectrum β-Lactamases, Plasmid-Mediated Quinolone Resistance, and Disinfectant Resistance Genes in Escherichia coli Isolated from Ready-To-Eat Meat Products.

There are growing concerns about the coselection of resistance against antibiotics and disinfectants in bacterial pathogens. The aim of this study was to characterize the antimicrobial susceptibility profiles, the prevalence of extended-spectrum β-lactamases (ESBLs), plasmid-mediated quinolone resistance genes (PMQRs), and quaternary ammonium compound resistance genes (QACs) in Escherichia coli isolated from ready-to-eat (RTE) meat products obtained in Guangzhou, China, and to determine whether these genes were colocalized in the isolates. A total of 64 E. coli isolates were obtained from 720 RTE meat samples. Multidrug resistance was observed in 70.3% of the isolates. A 100% of the isolates were resistant to benzalkonium chloride. Four types of β-lactamase genes were identified in the 16 ESBL-producing E. coli isolates: blaSHV (9.4%), blaTEM (7.8%), blaCTX-M-15 (1.6%), and blaCTX-M-9 (1.6%). PMQRs were present in nine isolates (14.1%), with aac(6')-Ib-cr and qnrD detected in eight (12.5%) and one isolate (1.6%), respectively. The QACs ydgE/ydgF were most commonly present (60.9%), while qacF, mdfA, sugE(p), emrE, qacG, sugE(c), and qacE were less prevalent (1.6%-18.8%). Coexistence of ESBLs and/or PMQRs with QACs was found in 21 isolates (32.8%). The aac(6')-Ib-cr and blaCTX-M-15 genes were found to be cotransferred with qacF in one isolate. The data obtained in this study indicate that ESBLs and/or PMQRs with QACs can not only be colocalized but can also be cotransferred in E. coli isolates from RTE meat products. The E. coli isolates with multiple antimicrobial resistance genes may transmit to humans through food chain and thus require further investigation and increased awareness.

Tolerance to quaternary ammonium compound disinfectants may enhance growth of Listeria monocytogenes in the food industry

The antibacterial effect of disinfectants is crucial for the control of Listeria monocytogenes in food processing environments. Tolerance of L. monocytogenes to sublethal levels of disinfectants based on quaternary ammonium compounds (QAC) is conferred by the resistance determinants qacH and bcrABC. The presence and distribution of these genes have been anticipated to have a role in the survival and growth of L. monocytogenes in food processing environments where QAC based disinfectants are in common use. In this study, a panel of 680 L. monocytogenes from nine Norwegian meat- and salmon processing plants were grouped into 36 MLVA profiles. The presence of qacH and bcrABC was determined in 101 isolates from the 26 most common MLVA profiles. Five MLVA profiles contained qacH and two contained bcrABC. Isolates with qacH and bcrABC showed increased tolerance to the QAC Benzalkonium chloride (BC), with minimal inhibitory concentrations (MICs) of 5–12, 10–13 and <5ppm for strains with qacH (two allele variants observed), bcrABC, and neither gene, respectively. Isolates with qacH or bcrABC were not more tolerant to BC in bactericidal tests in suspension or in biofilms compared with isolates lacking the genes. Water residue samples collected from surfaces in meat processing plants after QAC disinfection had bactericidal effect against L. monocytogenes when the sample BC levels were high (>100ppm). A sample with lower BC concentrations (14ppm of chain length C-12 and 2.7ppm of chain length C-14) inhibited growth of L. monocytogenes not containing bcrABC or qacH, compared to strains with these genes.The study has shown that L. monocytogenes harbouring the QAC resistance genes qacH and bcrABC are prevalent in the food industry and that residuals of QAC may be present in concentrations after sanitation in the industry that result in a growth advantage for bacteria with such resistance genes.

Virulence factors and antimicrobial resistance in Escherichia coli strains isolated from hen egg shells

Eggs may contain extraintestinal pathogenic (ExPEC) and diarrheogenic (DEC) Escherichia coli which in addition may carry antibiotic resistance. The wide use of biocides and disinfectants in the food industry may induce biocide tolerance in bacteria. The aim of the present study was to evaluate biocide tolerance and antibiotic resistance in E. coli from hen egg shells. A total of 27 isolates obtained from a screening of 180 eggs were studied. Seven isolates carried both eae and bfpA genes of typical enteropathogenic E. coli (EPEC) strains, while 14 isolates only carried eae associated with atypical EPEC strains. Shiga toxin genes stx and stx2 were detected in four isolates. Heat-stable and heat-labile enterotoxin genes as well as aggR were also detected. Several isolates had minimum inhibitory concentrations (MICs) that were higher than the wild-type for the biocide hexadecylpyridinium chloride (HDP, 18.52%) or the commercial disinfectant P3 oxonia (OX, 14.81%). Antibiotic resistance was detected for ampicillin (37.03%), streptomycin (37.03%), tetracycline (37.03%), chloramphenicol (11.11%), nalidixic acid (18.51%) and trimethoprim-sulfamethoxazole (14.81%). Eight isolates (29.63%) were biocide tolerant and antibiotic resistant. Efflux pump genes detected included acrB (96.29%), mdfA (85.18%) and oxqA (37.03%), in addition to quaternary ammonium compound (QAC) resistance genes qacA/B (11.11%) and qacE (7.40%). Antibiotic resistance genes detected included blaCTX-M-2 (22.22%), blaTEM (3.70%), blaPSE (3.70%), tet(A) (29.63%), tet(B) (29.63%), tet(C) (7.40%), tet(E) (11.11%), aac(6′)-Ib (3.70%), sul1 (14.81%), dfrA12 (3.70%) and dfrA15 (3.70%). Most isolates (96.30%) carried more than one genetic determinant of resistance. The most frequent combinations were efflux pump components acrB and mdfA with tetracycline resistance genes (33.33% of isolates). Isolates carrying QAC resistance genes also carried between 4 and 8 of the additional antimicrobial resistance genes investigated. Regardless of biocide tolerance and antibiotic resistance, all isolates were sensitive to carvacrol (0.25%), thymol (0.125%) and trisodium phosphate (1 to 1.5%), but they exhibited a heterogeneous response to sodium lactate and lysozyme-EDTA combinations that apparently were not related with antibiotic resistance. Results from the study reveal not only a low incidence of biocide tolerance but also the presence of multiple resistance strains carrying multiple genetic determinants of resistance.