Quinolone resistance is transferred horizontally via uptake signal sequence recognition in Haemophilus influenzae

The presence of Haemophilus influenzae strains with low susceptibility to quinolones has been reported worldwide. However, the emergence and dissemination mechanisms remain unclear. In this study, a total of 14 quinolone-low-susceptible H. influenzae isolates were investigated phylogenetically and in vitro resistance transfer assay in order to elucidate the emergence and dissemination mechanisms. The phylogenetic analysis based on gyrA sequences showed that strains with the same sequence type determined by multilocus sequence typing were classified into different clusters, suggesting that H. influenzae quinolone resistance emerges not only by point mutation, but also by the horizontal transfer of mutated gyrA . Moreover, the in vitro resistance transfer assay confirmed the horizontal transfer of quinolone resistance and indicated an active role of extracellular DNA in the resistance transfer. Interestingly, the horizontal transfer of parC only occurred in those cells that harbored a GyrA with amino acid substitutions, suggesting a possible mechanism of quinolone resistance in clinical settings. Moreover, the uptake signal and uptake-signal-like sequences located downstream of the quinolone resistant-determining regions of gyrA and parC , respectively, contributed to the horizontal transfer of resistance in H. influenzae . Our study demonstrates that the quinolone resistance of H. influenzae could emerge due to the horizontal transfer of gyrA and parC via recognition of an uptake signal sequence or uptake-signal-like sequence. Since the presence of quinolone-low-susceptible H. influenzae with amino acid substitutions in GyrA have been increasing in recent years, it is necessary to focus our attention to the acquisition of further drug resistance in these isolates.

Emerging Risks in Food: Probiotic Enterococci Pose a Threat to Public Health through the Food Chain

Probiotics have been associated with clinical infections, toxicity, and antimicrobial resistance transfer, raising public concerns. Probiotic enterococci are emerging food risks as opportunistic pathogens, yet little attention has been paid to them. Herein, we collected 88 enterococcal isolates from probiotic products used for humans, companion animals, livestock, and aquaculture. Results showed that all 88 probiotic enterococcal isolates harbored diverse virulence genes, multiple antimicrobial resistance genes, and mobile genetic elements. Notably, 77 isolates were highly resistant to gentamicin. Representative enterococcal isolates exerted toxic activities in both in vitro and in vivo models. Collectively, our findings suggest that probiotic enterococci may be harmful to hosts and pose a potential threat to public health.

Molecule transfer mechanism in 2D heterostructured lamellar membranes: The effects of dissolution and diffusion

Two-dimensional (2D) lamellar membranes are promising for efficient molecule transfer, while the underlying molecule transfer mechanism is rarely elucidated. Herein, heterostructured nanosheets are prepared by self-assembling small-sized hydrophilic cyanuric acid melamine (CMN) and hydrophobic g-C3N4 nanosheets. The resultant lamellar membranes show comparable affinity to both polar and nonpolar solvents, allowing them to dissolve on membrane surface and diffuse through membrane channels. Permeance results demonstrate that the transfer of polar solvents is controlled by dissolution and diffusion processes, while that of nonpolar solvents is governed by dissolution process. And the corresponding equations are established. Importantly, polar solvents are induced to form ordered arrangement in hydrophilic nanodomains and then maintain this state in hydrophobic nanodomains, affording low-resistance transfer thus high permeance: 1025 L m-2 h-1 bar-1 for acetonitrile. In contrast, nonpolar solvents with disordered arrangement acquire lower permeance than that of polar ones, but with comparable diffusion ability in these membranes.

Probiotic Bacillus Affect Enterococcus faecalis Antibiotic Resistance Transfer by Interfering with Pheromone Signaling Cascades

Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis natto is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from cCF10 without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30-50 kDa extracellular protease present in B. subtilis natto. Nattokinase of B. subtilis natto was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into “LVTL + VFV” fragments. Moreover, the cleavage product “LVTL” (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis natto can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis natto and L peptide to mitigate E. faecalis antibiotic resistance transfer. Importance Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis natto cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.

A Novel Biofilm Model System to Visualise Conjugal Transfer of Vancomycin Resistance by Environmental Enterococci

Enterococci and biofilm-associated infections are a growing problem worldwide, given the rise in antibiotic resistance in environmental and clinical settings. The increasing incidence of antibiotic resistance and its propagation potential within enterococcal biofilm is a concern. This requires a deeper understanding of how enterococcal biofilm develops, and how antibiotic resistance transfer takes place in these biofilms. Enterococcal biofilm assays, incorporating the study of antibiotic resistance transfer, require a system which can accommodate non-destructive, real-time experimentation. We adapted a Gene Frame® combined with fluorescence microscopy as a novel non-destructive platform to study the conjugal transfer of vancomycin resistance in an established enterococcal biofilm.A multi-purpose fluorescent in situ hybridisation (FISH) probe, in a novel application, allowed the identification of low copy number mobile elements in the biofilm. Furthermore, a Hoechst stain and ENU 1470 FISH probe identified Enterococcus faecium transconjugants by excluding Enterococcus faecalis MF06036 donors. Biofilm created with a rifampicin resistant E. faecalis (MW01105Rif) recipient had a transfer efficiency of 2.01 × 10−3; double that of the biofilm primarily created by the donor (E. faecalis MF06036). Conjugation in the mixed enterococcal biofilm was triple the efficiency of donor biofilm. Double antibiotic treatment plus lysozyme combined with live/dead imaging provided fluorescent micrographs identifying de novo enterococcal vancomycin resistant transconjugants inside the biofilm. This is a model system for the further study of antibiotic resistance transfer events in enterococci. Biofilms promote the survival of enterococci and reduce the effectiveness of drug treatment in clinical settings, hence giving enterococci an advantage. Enterococci growing in biofilms exchange traits by means of horizontal gene transfer, but currently available models make study difficult. This work goes some way to providing a non-destructive, molecular imaging-based model system for the detection of antibiotic resistance gene transfer in enterococci.

Transfer of Antibiotic Resistance Plasmid from Commensal E. coli Towards Human Intestinal Microbiota in the M-SHIME: Effect of E. coli dosis, Human Individual and Antibiotic Use

Along with (in) direct contact with animals and a contaminated environment, humans are exposed to antibiotic-resistant bacteria by consumption of food. The implications of ingesting antibiotic-resistant commensal bacteria are unknown, as dose-response data on resistance transfer and spreading in our gut is lacking. In this study, transfer of a resistance plasmid (IncF), harbouring several antibiotic resistance genes, from a commensal E. coli strain towards human intestinal microbiota was assessed using a Mucosal Simulator of the Human Intestinal Ecosystem (M-SHIME). More specifically, the effect of the initial E. coli plasmid donor concentration (105 and 107 CFU/meal), antibiotic treatment (cefotaxime) and human individual (n = 6) on plasmid transfer towards lumen coliforms and anaerobes was determined. Transfer of the resistance plasmid to luminal coliforms and anaerobes was observed shortly after the donor strain arrived in the colon and was independent of the ingested dose. Transfer occurred in all six simulated colons and despite their unique microbial community composition, no differences could be detected in antibiotic resistance transfer rates between the simulated human colons. After 72 h, resistant coliform transconjugants levels ranged from 7.6 × 104 to 7.9 × 106 CFUcefotaxime resistant/Ml colon lumen. Presence of the resistance plasmid was confirmed and quantified by PCR and qPCR. Cefotaxime treatment led to a significant reduction (85%) in resistant coliforms, however no significant effect on the total number of cultivable coliforms and anaerobes was observed.

Antimicrobial Susceptibility of Bacteroides fragilis Group Isolates to Clindamycin, Tetracycline and Tigecycline, and Their Possession of tet and ermF Genes, which are Responsible for Resistance

Objective: We aimed to determine the resistance of Bacteroides fragilis group (BFG) bacteria to clindamycin, tetracycline and tigecycline and establish the distribution of related resistance genes. Method: In total 82 BFG strains, isolated from different clinical samples between January 2017 and December 2018, were identified by MALDI-TOF MS. Their antimicrobial sensitivities to were determined using agar dilution methodology (CLSI; M11-A7). The tetM, tetQ, tetX, tetX1, tet36, and ermF genes were investigated by PCR. Results: Eighty-two strains of BFG bacteria, isolated from intra-abdominal abscess (n=36), tissue biopsy (n=16), blood (n=14) and other sterile body fluids (n=12), were identified as Bacteroides fragilis (n=48), Bacteroides thetaiotaomicron (n=17), Bacteroides vulgatus (n=5), Bacteroides ovatus (n=4), Bacteroides caccae (n=1), Bacteroides uniformis (n=1) and Parabacteroides distasonis (n=6). The resistance rates to clindamycin, tetracycline and tigecycline were 54.9%, 84.1%, 4.9%, respectively. Non-B. fragilis isolates were more resistant than B. fragilis strains. In total, 57.3% of the isolates were ermF gene positive, while B. thetaiotaomicron had the highest rate (70.6%). The tet gene positivity ranged from 18.8% to 66.7% among species. The tetQ gene positivity was higher than other tet genes. The 92.7% of the isolates were resistant to at least one antibiotic, while 94% had at least one resistance gene. Conclusion: This study provided data on antimicrobial resistance of our BFG isolates to clindamycin, tetracycline and tigecycline and the related resistance genes. However, our information obtained could also be a starting point for further investigation of the antibiotic resistance mechanisms of Bacteroides species, as well as, resistance transfer among BFG isolates and other bacteria.

Genes on the Move: In Vitro Transduction of Antimicrobial Resistance Genes between Human and Canine Staphylococcal Pathogens

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) between people and pets, and their co-carriage, are well-described. Potential exchange of antimicrobial resistance (AMR) genes amongst these staphylococci was investigated in vitro through endogenous bacteriophage-mediated transduction. Bacteriophages were UV-induced from seven donor isolates of canine (MRSP) and human (MRSA) origin, containing tet(M), tet(K), fusB or fusC, and lysates filtered. Twenty-seven tetracycline- and fusidic acid- (FA-) susceptible recipients were used in 122 donor-recipient combinations (22 tetracycline, 100 FA) across 415 assays (115 tetracycline, 300 FA). Bacteriophage lysates were incubated with recipients and presumed transductants quantified on antimicrobial-supplemented agar plates. Tetracycline resistance transduction from MRSP and MRSA to methicillin-susceptible S. pseudintermedius (MSSP) was confirmed by PCR in 15/115 assays. No FA-resistance transfer occurred, confirmed by negative fusB/fusC PCR, but colonies resulting from FA assays had high MICs (≥32 mg/L) and showed mutations in fusA, two at a novel position (F88L), nine at H457[Y/N/L]. Horizontal gene transfer of tetracycline-resistance confirms that resistance genes can be shared between coagulase-positive staphylococci from different hosts. Cross-species AMR transmission highlights the importance of good antimicrobial stewardship across humans and veterinary species to support One Health.

Dogs and Their Owners Have Frequent and Intensive Contact

Contact and interactions between owners and their pets may have beneficial physical and social effects on people, but may also facilitate the transmission of zoonotic agents and resistant bacteria. To estimate the risk of these contacts, more information regarding the frequency and intensity of this physical contact is required. Therefore, an online survey was conducted among pet owners resulting in 701 completed questionnaires. Questions regarding the interactions between dogs and owners were linked with a score from 1 (limited interactions) to 3 (highly intense interactions). After scoring these self-reported interactions, a contact intensity score was calculated for each respondent by summing up the different allocated scores from all questions. This contact intensity score was used to identify predictors of more intense contact based on a multivariable linear regression model. Interactions between dogs and their owners were widespread (e.g., 85.3% of the dogs licked their owner’s hand) and intense (e.g., 49.3% of owners reported being licked in the face). The gender, age, and place of residence (city, village, or countryside) of the respondent, together with the size and age of the dog, were significantly associated with the contact intensity score in the multivariable model. On average, female respondents younger than 65 years who lived in the city and had a small young dog had the most intense contact with it. Further research is necessary to evaluate the risk of these interactions in light of zoonotic and antimicrobial resistance transfer.