Subinhibitory Concentrations Research Articles

Interaction of lytic phage T1245 with antibiotics for enhancement of antibacterial and anti-biofilm efficacy against multidrug-resistant Acinetobacter baumannii

Biofilms associated with multidrug-resistant (MDR) Acinetobacter baumannii on medical devices remain a big clinical problem. Antibiotic susceptibility tests were performed with eight commonly employed antibiotics against clinical isolates. The effects of antibiotics in combination with well-characterized lytic phage T1245 were studied to assess their antibacterial and anti-biofilm efficacy. Ceftazidime, colistin, imipenem, and meropenem significantly reduced bacterial density up to approximately 80% when combined with phage T1245, compared with control. Phage T1245 in combination with ceftazidime, colistin, and meropenem at subinhibitory concentrations demonstrated significant reduction in biomass and bacterial viability of 3-day established biofilms, compared with antibiotic alone. In addition, electron microscopy further confirmed the disruption of biofilm structure and cell morphology upon treatment with phage T1245 and antibiotics, including ceftazidime, colistin, and meropenem. Combined treatment of phage T1245 with these antibiotics could be employed for the management of A. baumannii infections and eradication of the bacterial biofilms.

Plasmids Can Shift Bacterial Morphological Response against Antibiotic Stress.

Bacterial cell filamentation is a morphological change wherein cell division is blocked, which can improve bacterial survival under unfavorable conditions (e.g., antibiotic stress that causes DNA damage). As an extrachromosomal DNA molecule, plasmids can confer additionally advantageous traits including antibiotic resistance on the host. However, little is known about whether plasmids could shift bacterial morphological responses to antibiotic stress. Here, it is reported that plasmid-free cells, rather than plasmid-bearing cells, exhibit filamentation and asymmetrical cell division under exposure to sub-inhibitory concentrations of antibiotics (ciprofloxacin and cephalexin). The underlying mechanism is revealed by investigating DNA damage, cell division inhibitor sulA, the SOS response, toxin-antitoxin module (parDE) located on plasmids, and efflux pumps. Significantly higher expression of sulA is observed in plasmid-free cells, compared to plasmid-bearing cells. Plasmid carriage enables the hosts to suffer less DNA damage, exhibit stronger efflux pump activities, and thus have a higher antibiotic tolerance. These benefits are attributed to the parDE module that mediates stress responses from plasmid-bearing cells and mainly contributes to cell morphological changes. Collectively, the findings demonstrate that plasmids can confer additional innate defenses on the host to antibiotics, thus advancing the understanding of how plasmids affect bacterial evolution in hostile environments.

Effect of povidone-iodine and propanol-based mecetronium ethyl sulphate on antimicrobial resistance and virulence in Staphylococcus aureus

BackgroundReports are available on cross-resistance between antibiotics and biocides. We evaluated the effect of povidone-iodine (PVP-I) and propanol-based mecetronium ethyl sulphate (PBM) on resistance development, antibiotics cross-resistance, and virulence in Staphylococcus aureus.MethodsThe minimum inhibitory concentration (MIC) of PVP-I and PBM were determined against S. aureus ATCC 25923 using the agar-dilution method. Staphylococcus aureus ATCC 25923 was subjected to subinhibitory concentrations of the tested biocides in ten consecutive passages followed by five passages in a biocide-free medium; MIC was determined after each passage and after the fifth passage in the biocide-free medium. The developed resistant mutant was tested for cross-resistance to different antibiotics using Kirby-Bauer disk diffusion method. Antibiotic susceptibility profiles as well as biocides’ MIC were determined for 97 clinical S. aureus isolates. Isolates were categorized into susceptible and resistant to the tested biocides based on MIC distribution pattern. The virulence of the biocide-resistant mutant and the effect of subinhibitory concentrations of biocides on virulence (biofilm formation, hemolysin activity, and expression of virulence-related genes) were tested.ResultsPVP-I and PBM MIC were 5000 μg/mL and 664 μg/mL. No resistance developed to PVP-I but a 128-fold increase in PBM MIC was recorded, by repeated exposure. The developed PBM-resistant mutant acquired resistance to penicillin, cefoxitin, and ciprofloxacin. No clinical isolates were PVP-I-resistant while 48.5% were PBM-resistant. PBM-resistant isolates were more significantly detected among multidrug-resistant isolates. PVP-I subinhibitory concentrations (¼ and ½ of MIC) completely inhibited biofilm formation and significantly reduced hemolysin activity (7% and 0.28%, respectively). However, subinhibitory concentrations of PBM caused moderate reduction in biofilm activity and non-significant reduction in hemolysin activity. The ½ MIC of PVP-I significantly reduced the expression of hla, ebps, eno, fib, icaA, and icaD genes. The virulence of the biocide-resistant mutant was similar to that of parent strain.ConclusionPVP-I is a highly recommended antiseptic for use in healthcare settings to control the evolution of high-risk clones. Exposure to PVP-I causes no resistance-development risk in S. aureus, with virulence inhibition by subinhibitory concentrations. Also, special protocols need to be followed during PBM use in hospitals to avoid the selection of resistant strains.

Antibiotics resistance evolution of isolated Vibrio parahaemolyticus from mariculture under the continuous culture of sub-inhibitory concentrations of Ulva fasciata hydroponic solution

The outbreak of vibriosis from Vibrio (V.) parahaemolyticus is widespread in the mariculture, and live macroalgae has been considered to be effective and eco-friendly approach for the control of vibriosis. Three V. parahaemolyticus strains with β-lactam antibiotics resistance (resistant to ampicillin (AM), amoxicillin (AMX)) were isolated from mariculture in study, and the antibiotics resistance evolution mechanism was examined at the sub-inhibitory concentration (SIC) of hydroponic solution of Ulva (U.) fasciata (HSUF). The HSUF with the highest density (20 g fresh weight U. fasciata L−1) demonstrated the strongest inhibitory rates (47.0 %–65.8 %) on the three strains during the stable phase (8–24 h) of growth curve, which indicated that the HSUF (≤20 g L−1) could be considered to be at SIC for V. parahaemolyticus strains. After continuous subculture of V. parahaemolyticus with three dilutes (1/2 (HT), 1/20 (MT) and 1/50 (LT)) of HSUF (20 g L−1), all the strains of 20th generation were still resistant to AM and AMX. However, the LT condition reduced MIC of AM (2–16 times) and AMX (0–2 times) to strains, while MT and HT showed significantly various effect of β-lactam antibiotics resistance on different strains. The biofilm formation and ROS content of V. parahaemolyticus were almost positively correlated to the concentrations of HSUF. Transcriptome sequencing analysis of a representative strain showed that the lower concentrations of HSUF caused more down-regulated DEGs of the strains, and more down-regulated (vmeA, vmeB, sapA, mrdA) DEGs of strains were related to the pathway of β-lactam antibiotics resistance at LT condition. Thus, low concentration of HSUF was seemed to have better improvement for V. parahaemolyticus strains resistant to β-lactam antibiotics, which were mainly related to the impairment of biofilm formation, ROS and efflux pump.

Efficacy of endemic Algerian essential oils against single and mixed biofilms of Candida albicans and Candida glabrata

ObjectivesThis study investigated the in vitro effect of Origanum glandulosum, Ammoides verticillata, and Saccocalyx satureioides essential oils against planktonic cells and biofilms formed by single and mixed species of Candida albicans and Candida glabrata oral isolates. The effect of the essential oils in combination with amphotericin B on planktonic cells was also studied. DesignThe antifungal susceptibility of planktonic cells was evaluated by disc diffusion and broth microdilution methods. Resazurin assay and scanning electron microscopy (SEM) were employed to determine the antibiofilm activity. The combinatory effect was evaluated by the checkerboard method. Essential oils were characterized by gas chromatography–mass spectrometry (GC-MS). ResultsThe minimal inhibitory concentrations (MICs) and the minimum fungicidal concentrations (MFCs) of the studied essential oils were ranged between 250 and 2000 µg/mL. Biofilms were inhibited and eradicated by the essential oils at sub-inhibitory concentrations of 500 and 1000 µg/mL, respectively. SEM studies revealed a reduction in the preformed biofilm as a result of Origanum glandulosum essential oil treatment for single and mixed biofilms. Synergistic activity was found when Origanum glandulosum essential oil was combined with amphotericin B against Candida albicans. GC–MS analysis revealed that thymol was the major compound in Origanum glandulosum (38.36 %) and Ammoides verticillata (48.99 %) essential oils, while Saccocalyx satureioides essential oil was dominated by borneol (27.36 %). ConclusionsThe studied essential oils showed significant antifungal and antibiofilm activities, which support their effectiveness as promising candidates for the management of oral Candida infections.

2,4-Disubstituted pyridine derivatives are effective against intracellular and biofilm-forming tubercle bacilli.

It was recently reported that 4-substituted picolinohydrazonamides carrying hydrophilic cyclic amines, such as morpholine and pyrrolidine, at the end of their thiosemicarbazide chain have potent antimycobacterial activity in vitro at concentrations below 1μg/ml. Here, two selected compounds, 2,4-disubstituted pyridine derivatives 11 and 15, revealed significant bactericidal activity against Mycobacterium tuberculosis localized intracellularly within human macrophages, as well as against biofilm-forming tubercle bacilli. Mutants were selected that were resistant to the investigated compounds at an efficiency similar to that identified in the presence of the first line antituberculosis drug rifampicin. The resistant mutants were viable in the presence of the tested compounds exclusively on solid media. Genome-wide sequencing of the mutants selected in the presence of compound 11 revealed the accumulation of nonsynonymous mutations in the mmpR5 gene encoding a transcriptional repressor of the MmpS5-MmpL5 efflux pump, whose upregulation has been associated with bedaquiline resistance. The depletion of MmpR5 in wild-type M. tuberculosis using CRISPR-Cas9 technology increased the resistance of this strain to compound 11. Mass spectrometry-based proteomics (LC-MS/MS) of wild-type tubercle bacilli growing in subinhibitory concentrations of compounds 11 or 15 revealed 15 overproduced proteins not detectable in the control cells, including virulence-related proteins.

The issue beyond resistance: Methicillin-resistant Staphylococcus epidermidis biofilm formation is induced by subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin.

Staphylococcus epidermis is one of the most frequent causes of device-associated infections due to biofilm formation. Current reports noted that subinhibitory concentrations of antibiotics induce biofilm production in some bacteria. Accordingly, we evaluated the effect of exposure of different subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the biofilm formation of methicillin-resistant S. epidermidis (MRSE). Antimicrobial susceptibility testing and minimum inhibitory/bactericidal concentration of antimicrobial agents were determined. MRSE isolates were selected, and their biofilm formation ability was evaluated. The effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin, antibiotics selected among common choices in the clinic, on MRSE biofilm formation was determined by the microtitre method. Besides, the effect of subinhibitory concentrations of cloxacillin, cefazolin, clindamycin, and vancomycin on the expression of the biofilm-associated genes icaA and atlE was evaluated by Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR). Antimicrobial susceptibility patterns of MRSE strains showed a high level of resistance as follows: 80%, 53.3%, 33.3%, 33.3%, and 26.6%, for erythromycin, trimethoprim-sulfamethoxazole, tetracycline, clindamycin, and gentamicin, respectively. Besides, 73.3% of S. epidermidis strains were Multidrug-resistant (MDR). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were in the range of 0.5 to512 μg/mL and 1 to1024 μg/mL for cloxacillin, 0.125 to256 μg/mL and 1 to512 μg/mL for cefazolin, 0.125 to64 μg/mL and 4 to>1024 μg/mL for clindamycin, and 2 to32 μg/mL and 4 to32 μg/mL for vancomycin, respectively. The findings showed that subinhibitory concentrations of cloxacillin, cefazolin, and clindamycin induce biofilm production in MRSE strains. In particular, the OD values of strains were in the range of 0.09-0.95, 0.05-0.86, and 0.06-1 toward cloxacillin, cefazolin, and clindamycin, respectively. On the other hand, exposure to subinhibitory vancomycin concentrations did not increase the biofilm formation in MRSE strains. The findings also demonstrated that sub-MIC of antibiotics up-regulated biofilm-associated genes. In particular, atlE and icaA were up-regulated 0.062 to 1.16 and 0.078 to 1.48 folds, respectively, for cloxacillin, 0.11 to 0.8, and 0.1 to 1.3 folds for cefazolin, 0.18 to 0.98, and 0.19 to 1.4 folds, respectively, for clindamycin. In contrast, the results showed that sub-MIC of vancomycin did not increase the biofilm-associated genes. These findings overall show that exposure to sub-MIC of traditional antibiotics can cause biofilm induction in MRSE, thereby increasing the survival and persistence on various surfaces that worsen the condition of comorbid infections.

Filamentous Bacteria Resembling Fungal Hyphae: A Diagnostic Pitfall

Abstract Introduction/Objective When exposed to antibiotics or a host immune response, Gram-negative bacilli grow with inhibited septation producing long continuous cells; a phenomenon known as filamentation. Methods/Case Report We present a case of a 68-year-old man with quadriplegia, which was caused by the right vertebral artery dissection and subarachnoid hemorrhage requiring an emergent posterior inferior cerebellar artery direct bypass surgery. Lung lower lobe infiltrates were noted 3 days after the surgery. A bronchial wash culture was negative for growth, and a sputum specimen grew Serratia marcescens. The patient was treated with piperacillin/tazobactam, vancomycin, cefepime, cefazolin. After the surgery, he suddenly developed severe cerebral vasospasm of the posterior circulation with cerebellar strokes requiring emergent posterior fossa decompression. He remained on ventricular drainage by external ventricular drainage. After two days, the patient's cerebrospinal fluid (CSF) was sent for evaluation, on which a high segmented neutrophil count of 35% and Gram-negative filamentous organisms with occasional swellings were noted. Multi-drug resistant Serratia marcescens was isolated from the CSF specimen four days later. Neurosurgical procedures were reported to be highly associated with S. marcescens meningitis. The filamentous organisms noted on Gram-stained direct smear most likely represented Gram-negative S. marcescens with filamentation due to administration of sub-inhibitory concentrations of antibiotics. Results (if a Case Study enter NA) NA. Conclusion Gram-negative bacilli can develop abnormal morphology including elongation, swelling, and filamentation due to stress or sub-inhibitory antibiotic exposure, and it is important for microbiologists and pathologists to be aware of this phenomenon to avoid misinterpretation that may lead to inappropriate treatment.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

Subinhibitory antibiotic concentrations promote the horizontal transfer of plasmid-borne resistance genes from Klebsiellae pneumoniae to Escherichia coli.

Horizontal gene transfer plays an important role in the spread of antibiotic resistance, in which plasmid-mediated conjugation transfer is the most important mechanism. While sub-minimal inhibitory concentrations (sub-MIC) of antibiotics could promote conjugation frequency, the mechanism by which sub-MIC levels of antibiotics affect conjugation frequency is not clear. Here, we used Klebsiella pneumoniae SW1780 carrying the multi-drug resistance plasmid pSW1780-KPC as the donor strain, to investigate the effects of sub-MICs of meropenem (MEM), ciprofloxacin (CIP), cefotaxime (CTX), and amikacin (AK) on conjugational transfer of pSW1780-KPC from SW1780 to Escherichia coli J53. Our results showed that the transfer frequencies increased significantly by treating SW1780 strain with sub-MIC levels of MEM, CIP, CTX and AK. Transfer frequencies at sub-MIC conditions in a Galleria mellonella were significantly higher than in vitro. To investigate gene expression and metabolic effects, RT-qPCR and LC-MS-based metabolome sequencing were performed. Transcript levels of T4SS genes virB1, virB2, virB4, virB8, and conjugation-related genes traB, traK, traE, and traL were significantly upregulated by exposure to sub-MICs of MEM, CIP, CTX, and AK. Metabolome sequencing revealed nine differentially regulated metabolites. Our findings are an early warning for a wide assessment of the roles of sub-MIC levels of antibiotics in the spread of antibiotic resistance.

The Effect Citrox BCL on Legionella pneumophila Mechanisms of Biofilm Formation, Oxidative Stress and Virulence.

Legionella pneumophila is responsible for causing Legionnaires' disease and Pontiac fever, also known as legionellosis. The aim of this study was to investigate the mechanistic effect of a mixture of natural antimicrobials (Citrox BCL) in preventing L. pneumophila biofilm formation and reducing its in vitro virulence. The minimum inhibitory concentrations were detected at 0.06%, and the MBC was established at 0.125%. Based on the growth curve profile, the sub-inhibitory concentration of 0.02% was further used to study the mechanistic implications in the absence of a cytotoxic effect on A549 cells. At 24 h post-infection, Citrox BCL reduced (p = 0.005) the intracellular growth of L. pneumophila when the A549 cells or the bacteria were pre-treated with 0.02% Citrox BCL. This result was replicated when Citrox BCL was added during the 24 h infection assay leading to a reduction in intracellular growth (p = 0.003). Herein we show that at the sub-inhibitory concentration of 0.02%, Citrox CBL lowers the ROS levels in infected A549 cells and causes a 45% reduction in L. pneumophila EPS production, a reduction associated with the decline in biofilm formation. Overall, our results corroborate the low c-di-GMP production with the decrease in biofilm formation and low EPS levels. The low EPS levels seemed to be caused by the downregulation of the tatB and tatC gene expressions. Moreover, inhibition of pvcA and pvcB gene expressions, leading to lower siderophore levels, suggests that Citrox BCL reduces the ability of L. pneumophila to sequester iron and reduce biofilm formation through iron starvation.

Sennoside A inhibits quorum sensing system to attenuate its regulated virulence and pathogenicity via targeting LasR in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic pathogen, and the emergence of drug resistance greatly increased the difficulty of treating its infection. Cell density-dependent quorum sensing (QS) system not only regulates the virulence but also associates with the drug resistance of P. aeruginosa. Screening for agents targeting QS to inhibit bacterial virulence and pathogenicity is considered a promising strategy to combat P. aeruginosa infection. In the present study, sennoside A was found to be able to inhibit the QS expression of P. aeruginosa at subinhibitory concentrations. The QS-regulated virulence factors, including protease, elastase, rhamnolipid, and pyocyanin, were also inhibited by sennoside A at both transcriptional and translational levels. Moreover, sennoside A could suppress the motility of twitching, swimming, and swarming as well as the biofilm formation, which is associated with the acute and chronic infections of P. aeruginosa in a dose-dependent manner. The attenuated pathogenicity of P. aeruginosa by sennoside A was further verified by Chinese cabbage, Drosophila melanogaster, and Caenorhabditis elegans infection analysis. Further study found that sennoside A might target the las system, mainly LasR, to interfere with QS. All the results indicate that sennoside A could inhibit the QS system to attenuate its regulated virulence and pathogenicity via mainly targeting LasR in P. aeruginosa and further research to identify its anti-QS activity for other Gram-negative bacteria is warranted.

Concentration-Dependent Global Quantitative Proteome Response of Staphylococcus epidermidis RP62A Biofilms to Subinhibitory Tigecycline.

Staphylococcus epidermidis is a leading cause of biofilm-associated infections on implanted medical devices. During the treatment of an infection, bacterial cells inside biofilms may be exposed to sublethal concentrations of the antimicrobial agents. In the present study, the effect of subinhibitory concentrations of tigecycline (TC) on biofilms formed by S. epidermidis strain RP62A was investigated using a quantitative global proteomic technique. Sublethal concentrations of TC [1/8 (T1) and 1/4 minimum inhibitory concentration (MIC) (T2)] promoted biofilm production in strain RP62A, but 1/2 MIC TC (T3) significantly inhibited biofilm production. Overall, 413, 429, and 518 proteins were differentially expressed in biofilms grown with 1/8 (T1), 1/4 (T2), and 1/2 (T3) MIC of TC, respectively. As the TC concentration increased, the number of induced proteins in each Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway increased. The TC concentration dependence of the proteome response highlights the diverse mechanisms of adaptive responses in strain RP62A biofilms. In both COG and KEGG functional analyses, most upregulated proteins belong to the metabolism pathway, suggesting that it may play an important role in the defense of strain RP62A biofilm cells against TC stress. Sub-MIC TC treatment of strain RP62A biofilms led to significant changes of protein expression related to biofilm formation, antimicrobial resistance, virulence, quorum sensing, ABC transporters, protein export, purine/pyrimidine biosynthesis, ribosomes, and essential proteins. Interestingly, in addition to tetracycline resistance, proteins involved in resistance of various antibiotics, including aminoglycosides, antimicrobial peptides, β-lactams, erythromycin, fluoroquinolones, fusidic acid, glycopeptides, lipopeptides, mupirocin, rifampicin and trimethoprim were differentially expressed. Our study demonstrates that global protein expression profiling of biofilm cells to antibiotic pressure may improve our understanding of the mechanisms of antibiotic resistance in biofilms.

Review of the Distribution and Influence of Antibiotic Resistance Genes in Ballast Water

The misuse of antibiotics causes antibiotic resistance genes (ARGs) in bacteria to be gradually enriched by environmental selection, resulting in increased tolerance and resistance in bacteria to antibiotics. Ballast water is a mobile carrier for the global transfer of bacteria and genes, thus posing a certain risk of ARGs spreading into the global ocean. Therefore, it is important to investigate the current status of ARGs in ballast water, as well as control the abundance of ARGs. Herein, we attempt to comprehensively summarize the distribution and abundance of ARGs in ballast water from different sea areas and analyze the influencing factors (such as physical factors, chemical factors, temperature, pH, etc.) on the distribution of ARGs. Furthermore, we seek to review the changes in ARGs after differential disinfection technology treatment in ballast water (including chlorination, ultraviolet, ozone, and free radical technology), especially the enhancing effect of subinhibitory concentrations of disinfectants on ARGs transfer. Overall, we believe this review can serve as a guide for future researchers to establish a more reasonable standard of ballast water discharge that considers the pollution of ARGs and provide new insight into the risk of vertical and horizontal ARG transfer in ballast water after disinfection.

Rhus coriaria extracts inhibit quorum sensing-related virulence and biofilm production in drug-resistant Pseudomonas aeruginosa recovered from burn wounds.

Numerous studies have confirmed sumac's ability to inhibit pathogens and even eradicate chronic drug-resistant infections. Current research was conducted to demonstrate the action of various sumac extracts at sub-inhibitory concentrations in modulating pathogen-related characteristics instead of killing them. The influence of sumac extracts on the quorum sensing dependent virulence of multidrug-resistant isolates of Pseudomonas aeruginosa recovered from burn wounds was considered by detecting the effect on biofilm development, various virulence factors, and expression of bacterial exotoxin A and quorum sensing related genes. Experiments to characterize and measure sumac extract's impact on the P. aeruginosa growth, biofilm, exoproteases, pyocyanin, motility, and the quorum sensing networks revealed that all studied characteristics were reduced by concentrations below inhibition without affecting bacterial growth. Furthermore, the expression of exotoxin A, rhl, and las glucons was declined or even inhibited by lower levels of sumac fruit fractions. The findings revealed that sumac fights infections either by its inhibitory effect on the bacterial cells or by reducing bacterial signaling and virulence by disruption of the bacterial signal system.

Molecular antibiotic resistance mechanisms and co-transmission of the mcr-9 and metallo-β-lactamase genes in carbapenem-resistant Enterobacter cloacae complex.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) has increasingly emerged as a major cause of healthcare-associated infections, with colistin being one of the last-resort antibiotics of treatment. Mobile colistin resistance (mcr)-9 is a member of a growing family of mcr genes and has been reported to be an inducible gene encoding an acquired phosphoethanolamine transferase. Here, we collected 24 ECC strains from Chongqing, China from 2018 to 2021. Subsequently, antibiotic resistance genes and the transmission dynamics of the strains were determined by PCR, whole-genome sequencing, and bioinformatic analysis. The mcr-9 was identified in IncHI2/2A or IncHI2/2A + IncN plasmids from six CRECC strains and was co-located with bla NDM-1 or bla IMP-4 in 2/6 plasmids. The genetic environment of mcr-9.1 was composed of IS903B-mcr-9.1-wbuC-IS26 in the five mcr-9.1-harboring-plasmid, but IS1B was located downstream of mcr-9.2 in the pECL414-1 sequence. We also found that the pNDM-068001 plasmid carrying mcr-9.1 could be a hybrid plasmid, formed by a Tn6360-like bla NDM-1 region inserted into an mcr-9.1-positive IncHI2/2A plasmid. A conjugation assay showed that plasmids mediated the co-dissemination of mcr-9 and metallo-β-lactamase (MBL) genes. In addition, we performed induction assays with sub-inhibitory concentrations of colistin and found an increase in the relative expression levels of the mcr-9.2, qseC, and qseB genes, as well as an increase in the minimum inhibitory concentration values of colistin in the CRECC414 strain. These findings provide a basis for studying the regulatory mechanisms of mcr-9 expression and highlight the importance of effective monitoring to assess the prevalence of MBL and mcr-9 co-existing plasmids.

The Effect of Selected Bee Products on Adhesion and Biofilm of Clostridioides difficile Strains Belonging to Different Ribotypes.

There is an ongoing search for alternative treatments for Clostridioides difficile infections. The aim of the study was to investigate the antibacterial and antibiotic activity of bee products against C. difficile strains with different polymerase chain reaction ribotypes (RTs). The minimum inhibitory concentration (MICs) of Manuka honey 550+, goldenrod honey, pine honey, and bee bread were determined by the broth dilution method. C. difficile adhesion to HT-29, HT-29 MTX, and CCD 841 CoN cell lines was assessed. Biofilm was cultured in titration plates and visualized by confocal microscopy. The MICs of Manuka honey for C. difficile 630 and ATCC 9689 strains and control strain, M 120, were 6.25%, 6.25%, and 1.56% (v/v), respectively; of goldenrod honey, 50%, 50%, and 12.5%, respectively; of pine honey, 25%, 25%, and 25%, respectively; and of bee bread, 100 mg/L, 50 mg/L, and 100 mg/L, respectively. Manuka honey (1%) increased adhesion of C. difficile RT176 strains, and one strain of RT023, to the CCD 841 cell line. Pine honey (1%) increased RT027 adhesion to the HT-29 cell line. Manuka honey, pine honey, and bee bread at subinhibitory concentrations increased the adhesion of C. difficile. Our research proved that bee products are active against the tested strains of C. difficile.

Ability of Garlic and Ginger Oil to Reduce Salmonella in Post-Harvest Poultry

Approximately 1.35 million human salmonellosis cases are reported in the United States every year, resulting in over 26,000 hospitalizations and 400 deaths. Consumption of contaminated poultry products is one of the leading causes of human salmonellosis. Poultry meat becomes contaminated when feces from an infected bird comes into contact with the carcass during processing. Additional carcasses can then become cross-contaminated along the processing line. While chemicals such as peracetic acid are currently used to kill microbes such as Salmonella, consumers are increasingly calling for more natural alternatives. Our objective for this study was to determine the ability of the phytochemicals garlic and ginger oil to reduce Salmonella prevalence in the processing environment. In a simulated scalding tank environment, dipping contaminated chicken skin samples in a solution containing both garlic and ginger oil reduced Salmonella by up to 2 log CFU. Furthermore, the oils prevented Salmonella growth in the tank solution. The mechanism of action of garlic and ginger was evaluated using the sub-inhibitory concentration of each oil individually. While both were found to decrease autoinducer-2 (AI-2) levels, no effect was seen on expression of 10 genes involved in Salmonella virulence and survival. In total, this work demonstrates the potential of garlic and ginger to reduce Salmonella prevalence in the post-harvest environment. However, more work remains to be done to understand the mechanism of action.

Conjugative transfer of multi-drug resistance IncN plasmids from environmental waterborne bacteria to Escherichia coli.

Watersheds contaminated with municipal, hospital, and agricultural residues are recognized as reservoirs for bacteria carrying antibiotic resistance genes (ARGs). The objective of this study was to determine the potential of environmental bacterial communities from the highly contaminated La Paz River basin in Bolivia to transfer ARGs to an Escherichia coli lab strain used as the recipient. Additionally, we tested ZnSO4 and CuSO4 at sub-inhibitory concentrations as stressors and analyzed transfer frequencies (TFs), diversity, richness, and acquired resistance profiles. The bacterial communities were collected from surface water in an urban site close to a hospital and near an agricultural area. High transfer potentials of a large set of resistance factors to E. coli were observed at both sites. Whole-genome sequencing revealed that putative plasmids belonging to the incompatibility group N (IncN, IncN2, and IncN3) were predominant among the transconjugants. All IncN variants were verified to be mobile by a second conjugation step. The plasmid backbones were similar to other IncN plasmids isolated worldwide and carried a wide range of ARGs extensively corroborated by phenotypic resistance patterns. Interestingly, all transconjugants also acquired the class 1 integron intl1, which is commonly known as a proxy for anthropogenic pollution. The addition of ZnSO4 and CuSO4 at sub-inhibitory concentrations did not affect the transfer rate. Metal resistance genes were absent from most transconjugants, suggesting a minor role, if any, of metals in the spread of multidrug-resistant plasmids at the investigated sites.

In vitro activities of licochalcone A against planktonic cells and biofilm of Enterococcus faecalis

This study aims to evaluate the in vitro antibacterial and anti-biofilm activities of licochalcone A on Enterococcus faecalis and to investigate the possible target genes of licochalcone A in E. faecalis. This study found that licochalcone A had antibacterial activities against E. faecalis, with the MIC50 and MIC90 were 25 μM. Licochalcone A (at 4 × MIC) indicated a rapid bactericidal effect on E. faecalis planktonic cells, and killed more E. faecalis planktonic cells (at least 3-log10 cfu/ml) than vancomycin, linezolid, or ampicillin at the 2, 4, and 6 h of the time-killing test. Licochalcone A (at 10 × MIC) significantly reduced the production of E. faecalis persister cells (at least 2-log10 cfu/ml) than vancomycin, linezolid, or ampicillin at the 24, 48, 72, and 96 h of the time-killing test. Licochalcone A (at 1/4 × MIC) significantly inhibited the biofilm formation of E. faecalis. The RNA levels of biofilm formation-related genes, agg, esp, and srtA, markedly decreased when the E. faecalis isolates were treated with licochalcone A at 1/4 × MIC for 6 h. To explore the possible target genes of licochalcone A in E. faecalis, the licochalcone A non-sensitive E. faecalis clones were selected in vitro by induction of wildtype strains for about 140 days under the pressure of licochalcone A, and mutations in the possible target genes were detected by whole-genome sequencing. This study found that there were 11 nucleotide mutations leading to nonsynonymous mutations of 8 amino acids, and among these amino acid mutations, there were 3 mutations located in transcriptional regulator genes (MarR family transcriptional regulator, TetR family transcriptional regulator, and MerR family transcriptional regulator). In conclusion, this study found that licochalcone A had an antibacterial effect on E. faecalis, and significantly inhibited the biofilm formation of E. faecalis at subinhibitory concentrations.