Understanding Stimulation of Conjugal Gene Transfer by Nonantibiotic Compounds: How Far Are We?

A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance.

Persistent ciprofloxacin exposure induced the transformation of Klebsiella pneumoniae small colony variant into mucous phenotype.

Introduction: Small colony variant (SCV) is a bacterial phenotype closely related to persistent and recurrent infections. SCVs are mutations that occur within bacterial populations, resulting in a change in bacterial morphology and the formation of small colonies. This morphological change may enhance bacterial resistance to antibiotics and contribute to persistent and recurrent infections. Methods: We isolated Klebsiella pneumoniae (KPN) and its SCV from a child with recurrent respiratory tract infections. KPN and SCV were treated with subinhibitory concentrations of antibiotics. growth curves, serum resistance experiments, macrophage phagocytosis experiments and whole genome sequencing were used to characterize KPN and SCV. Results: After treating KPN and SCV with subinhibitory concentrations of antibiotics, we found that ciprofloxacin induced the SCV transition to the mucoid phenotype. We found that the growth of mucoid Klebsiella pneumoniae was significantly slower than maternal strain and SCV though growth curves. Serum resistance experiments showed that mucoid strains had significantly higher serum resistance compared to maternal strain and SCV. Macrophage phagocytosis experiments revealed that SCV had significantly higher intracellular survival rates compared to maternal strain and mucoid strains. Differential gene analysis of three strains revealed that the mucoid strain contained DNA polymerase V subunit UmuC gene on the plasmid, while the SCV strain had an additional IcmK family IV secretion protein on its plasmid. Discussion: Our study showed the SCV of KPN changed to a mucoid colony when exposed to subinhibitory concentrations of ciprofloxacin. The higher resistance of serum of mucoid colonies was possibly related to the UmuC gene, while the increased intracellular survival of SCV may be related to the IcmK family type IV secretion proteins.

Influence of Nisin-Biogel at Subinhibitory Concentrations on Virulence Expression in Staphylococcus aureus Isolates from Diabetic Foot Infections.

A new approach to diabetic foot infections (DFIs) has been investigated, using a nisin-biogel combining the antimicrobial peptide (AMP) nisin with the natural polysaccharide guar-gum. Since in in vivo conditions bacteria may be exposed to decreased antimicrobial concentrations, known as subinhibitory concentrations (sub-MICs), effects of nisin-biogel sub-MIC values corresponding to 1/2, 1/4 and 1/8 of nisin's minimum inhibitory concentration (MIC) on virulence expression by six Staphylococcus aureus DFI isolates was evaluated by determining bacteria growth rate; expression of genes encoding for staphylococcal protein A (spA), coagulase (coa), clumping factor A (clfA), autolysin (atl), intracellular adhesin A (icaA), intracellular adhesin D (icaD), and the accessory gene regulator I (agrI); biofilm formation; Coa production; and SpA release. Nisin-biogel sub-MICs decreased bacterial growth in a strain- and dose-dependent manner, decreased agrI, atl and clfA expression, and increased spA, coa, icaA and icaD expression. Biofilm formation increased in the presence of nisin-biogel at 1/4 and 1/8 MIC, whereas 1/2 MIC had no effect. Finally, nisin-biogel at sub-MICs did not affect coagulase production, but decreased SpA production in a dose-dependent manner. Results highlight the importance of optimizing nisin-biogel doses before proceeding to in vivo trials, to reduce the risk of virulence factor's up-regulation due to the presence of inappropriate antimicrobial concentrations.

Hormetic dose responses induced by antibiotics in bacteria: A phantom menace to be thoroughly evaluated to address the environmental risk and tackle the antibiotic resistance phenomenon.

The environmental contamination of antibiotics caused by their over or inappropriate use is a major issue for environmental and human health since it can adversely impact the ecosystems and promote the antimicrobial resistance. Indeed, considering that in the environmental matrices these drugs are present at low levels, the possibility that bacteria exhibit a hormetic response to increase their resilience when exposed to antibiotic subinhibitory concentrations might represent a serious threat. Information reported in this review showed that exposure to different types of antibiotics, either administered individually or in mixtures, is capable of exerting hormetic effects on bacteria at environmentally relevant concentrations. These responses have been reported regardless of the type of bacterium or antibiotic, thus suggesting that hormesis would be a generalized adaptive mechanism implemented by bacteria to strengthen their resistance to antibiotics. Hormetic effects included growth, bioluminescence and motility of bacteria, their ability to produce biofilm, but also the frequency of mutation and plasmid conjugative transfer. The evaluation of quantitative features of antibiotic-induced hormesis showed that these responses have both maximum stimulation and dose width characteristics similar to those already reported in the literature for other stressors. Notably, mixtures comprising individual antibiotic inducing stimulatory responses might have distinct combined effects based on antagonistic, synergistic or additive interactions between components. Regarding the molecular mechanisms of action underlying the aforementioned effects, we put forward the hypothesis that the adoption of adaptive/defensive responses would be driven by the ability of antibiotic low doses to modulate the transcriptional activity of bacteria. Overall, our findings suggest that hormesis plays a pivotal role in affecting the bacterial behavior in order to acquire a survival advantage. Therefore, a proactive and effective risk assessment should necessarily take due account of the hormesis concept to adequately evaluate the risks to ecosystems and human health posed by antibiotic environmental contamination.

Decreased biofilm formation by planktonic cells of Listeria monocytogenes in the presence of sodium hypochlorite.

The objective of this study was to determine if the adaptation at planktonic stage to subinhibitory concentrations (SIC) of sodium hypochlorite (NaOCl) could modulate the biofilm forming ability of five Listeria monocytogenes strains V7, Scott A, FSL-N1-227, FSL F6-154 and ATCC 19116 representing serotypes 1/2a, 4b and 4c. Biofilm formation by NaOCl nonadapted and adapted L. monocytogenes planktonic cells was measured in the presence or absence of SIC of NaOCl. The biofilm formation ability of NaOCl nonadapted and adapted L. monocyotgenes planktonic cells was reduced only in the presence of NaOCl (P < 0.05). Scanning electron microscopy revealed that the continuous exposure of NaOCl induced morphological changes in the L. monocytogenes biofilm structure and reduced its attachment to polystyrene surface. The qRT-PCR results also showed that the subinhibitory NaOCl reduced biofilm formation related gene expression such as motility and quorum sensing signals (P < 0.05). These findings indicate that subinhibitory NaOCl can reduce the ability of L. monocytogenes planktonic cells to form biofilms on polystyrene surface.

Detection of ERG11 Overexpression in Candida albicans isolates from environmental sources and clinical isolates treated with inhibitory and subinhibitory concentrations of fluconazole.

BACKGROUND: Candida species can cause serious infection in patients with changes in defence mechanisms and/or when anatomical barriers are compromised. Mutations and overexpression in the ERG11 gene are described as molecular mechanisms of azole resistance. Information is limited on these mechanisms in the presence of subinhibitory concentrations of fluconazole. OBJECTIVES: This study aimed to evaluate the expression of ERG11 gene from Candida albicans isolates, from clinical and hospital environments, in the absence and presence of inhibitory and subinhibitory concentrations of fluconazole. METHODS: The American Type Culture Collection 10231 strain, five clinical isolates and three isolates from hospital environment colonisation were exposed to inhibitory and subinhibitory concentrations of fluconazole. Susceptibility tests were performed according to EUCAST 7.1 guidelines, and the relative expression analysis of ERG11 was performed by qPCR. RESULTS: Differences in response to fluconazole concentrations were observed, with the exception only one clinical isolate when treated with 1/4 of the FLU-minimum inhibitory concentration (MIC). All the other isolates, regardless of the isolation source, had an increase in expression. The overexpression occurred in a very broad range, from 1.086 to 126.105 times. In general, treatment with the highest dose of fluconazole (MIC) was the one that most influenced the ERG11 expression, followed by treatments with 1/2 and 1/4 MIC. CONCLUSIONS: The increased expression of ERG11 by C albicans in the presence of different concentrations of fluconazole is relevant, raising concerns in the care and cleaning of the hospital environment and the prophylactic use of fluconazole that could lead to the selection of potential azole-resistant isolates.

In vitro activities of telithromycin against Staphylococcus aureus biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin.

Introduction. Staphylococcus aureus biofilms are difficult to treat and the effect of telithromycin treatment is still unclear.Aim. This study aimed to explore the effect of telithromycin against Staphylococcus aureus biofilms compared with azithromycin, clindamycin, vancomycin and daptomycin.Methodology. Eight methicillin-susceptible and eight methicillin-resistant S. aureus isolates (MSSA and MRSA, respectively) were used for this study. Biofilm biomasses were detected by crystal violet staining and the adherent cells in the established biofilms were quantified by determination of colony-forming units (c.f.u.). The RNA levels of biofilm formation-related genes were determined by RT-qPCR.Results. Telithromycin [8× minimum inhibitory concentration (MIC)] eradicated more established biofilms than azithromycin or clindamycin in the four MSSA isolates, and eliminated the established biofilms of six MRSA isolates more effectively than vancomycin or daptomycin. Telithromycin (8× MIC) killed more adherent cells in the established biofilms than azithromycin or clindamycin in the six MSSA isolates, and killed more adherent cells than vancomycin in all eight MRSA isolates. Daptomycin also showed an excellent effect on the adherent cells of MRSA isolates, with similarresults to telithromycin. The effect of a subinhibitory concentration of telithromycin (1/4× MIC) was significantly superior to that of azithromycin or clindamycin, inhibiting the biofilm formation of six MSSA isolates and seven MRSA isolates more effectively than vancomycin or daptomycin. The RNA levels of agrA, agrC, clfA, icaA and sigB decreased when treated with telithromycin (1/4× MIC).Conclusions. Telithromycin is more effective than azithromycin, clindamycin, vancomycin, or daptomycin against S. aureus biofilms.

Effect of subinhibitory concentrations of ampicillin on Listeria monocytogenes.

Listeria monocytogenes is an important cause of meningoencephalitis associated with high mortality. The treatment of choice for listeriosis is ampicillin alone or in combination with gentamicin or penicillin G. However, only low ampicillin concentrations are recorded in the central nervous system (CNS). In this study, we analysed the effect of subinhibitory concentrations of ampicillin on the morphology, growth and survival of L. monocytogenes. The non-inhibitory concentration (NIC), the minimum inhibitory concentration (MIC) and the MIC/NIC ratio were determined. Gram and Live/Dead staining showed aggregates of L. monocytogenes cells when grown at subinhibitory concentrations of ampicillin, with >90% of viable cells. The L. monocytogenes strains tested showed an intermediate heteroresistance to ampicillin, characterised by a MIC/NIC ratio of 4. Our results seem to indicate that both intermediate heteroresistance and the formation of aggregates could play a role in the clinical failure of ampicillin in the treatment of CNS infections caused by L. monocytogenes. However, more studies are necessary to elucidate this question.

Effect of subinhibitory concentrations of imipenem and piperacillin on Pseudomonas aeruginosa toxA and exoS transcriptional expression.

Subinhibitory concentrations (subMIC) of antibiotics, although not able to kill bacteria, can modify their physicochemical characteristics and may interfere with some bacterial functions. This study aimed to investigate the effect of subMIC of imipenem and piperacillin on the transcriptional expression of virulence-related genes toxA and exoS in Pseudomonas auroginosa. Five clinical isolates of P. aeruginosa were screened for the presence of toxA and exoS genes and MICs of imipenem and piperacillin were determined using broth macrodilution. The expression levels of toxA and exoS at subMIC concentrations of antibiotics were measured by real-time PCR. Our results showed that the expression of toxA decreased at all subinhibitory concentrations of imipenem, especially at concentrations 2, 4 and 8 mg/L (p < 0.05). Whereas, exoS expression was increased 4.1- to 7-fold at subinhibitory concentrations of imipenem. The increase of toxA expression was measured at concentrations 16, 4, 2, 0.25 and 0.125 mg/L of piperacillin. However, piperacillin had no significant influence on exoS expression (p > 0.05). Further studies will be required to assess whether subMIC of imipenem can improve the outcomes of severe and serious infections caused by P. aeruginosa.

Changes in the composition and architecture of staphylococcal biofilm by nisin.

Bacterial biofilms are involved in various medical infections and food contamination episodes and, for this reason, it is of great importance to developing new strategies of its prevention and control. The subinhibitory concentration of nisin was determined, and its effect against Staphylococcus aureus and Staphylococcus epidermidis biofilms was evaluated. Results obtained by confocal laser microscopy demonstrated morphological changes in the architecture of the structure of biofilms. The main components (polysaccharides, proteins, and extracellular DNA (eDNA)) of the biofilm matrix were determined by spectrophotometry and showed that the formation of staphylococcal biofilms in the presence of nisin results in a less dense matrix structure with modification in its constituents. These results contribute to increase the knowledge of the composition and architecture of the extracellular matrix of biofilms of S. aureus, as well as evidence that the investigation of alternative products to assist in the control and combat of biofilms is a promising strategy.

Influence of subinhibitory antifungal concentrations on extracellular hydrolases and biofilm production by Candida albicans recovered from Egyptian patients.

BACKGROUND: Extracellular hydrolases (phospholipase, aspartyl protease and haemolysin) and biofilm production are considered as major virulence factors of the opportunistic pathogenic fungus Candida albicans. However, the impact of antifungal therapy on such virulence attributes is not well investigated. The common antifungal agents may disturb the production of secreted hydrolases as well as biofilm formation. Accordingly, this study addressed the effect of subinhibitory concentrations (sub-MICs) of selected antifungal agents on some virulence factors of C. albicans clinical isolates. METHODS: C. albicans isolates (n = 32) were recovered from different clinical samples and their identification was confirmed to the species level. Antifungal susceptibility profiles of isolates were determined against (nystatin, fluconazole and micafungin) and minimum inhibitory concentrations (MICs) were interpreted according to Clinical and Laboratory Standards Institute guidelines. Virulence determinants comprising secreted hydrolases (phospholipase, aspartyl protease and haemolysin) and biofilm formation were investigated in the presence of the sub-MICs of the tested antifungal agents. RESULTS: Treatment of clinical C. albicans isolates with subinhibitory nystatin, fluconazole and micafungin concentrations significantly decreased production of extracellular hydrolases. Nystatin had the greatest inhibitory effect on phospholipase and aspartyl protease production. However, micafungin showed the highest reducing effect on the hemolytic activity of the treated clinical isolates. Moreover, nystatin and micafungin, but not fluconazole, had a noticeable significant impact on inhibiting biofilm formation of C. albicans clinical isolates. CONCLUSION: Our findings highlighted the significant influences of commonly prescribed antifungal agents on some virulence factors of C. albicans. Accordingly, antifungal therapy may modulate key virulence attributes of C. albicans.

Subinhibitory Concentrations of Amoxicillin, Lincomycin, and Oxytetracycline Commonly Used to Treat Swine Increase Streptococcus suis Biofilm Formation.

Streptococcus suis is a bacterial swine pathogen with a significant economic burden. It typically colonizes the tonsil and nasal cavity of swine causing a variety of symptoms ranging from asymptomatic carriage to lethal systemic disease. A key barrier toward the development of improved vaccines or interventions for S. suis infections is a gap in our understanding of the mechanisms contributing to persistence in the host, in which colonized pigs continue to shed and transmit S. suis. We hypothesized that exposure to sub-MICs of antibiotics commonly used by the swine industry would increase the biofilm capacity of S. suis strains. Using a 96-well plate MIC protocol, we experimentally determined the MIC for each of 12 antibiotics for a virulent strain of S. suis strain that consistently formed biofilms using a standard crystal violet assay. Using this static biofilm assay, we demonstrated that sub-MICs of bacitracin, carbadox, chlortetracycline, enrofloxacin, gentamicin, neomycin, sulfadimethoxine, tiamulin, and tylosin did not increase S. suis biofilms. In contrast, we demonstrated that sub-MICs of amoxicillin, lincomycin, and oxytetracycline increased overall biofilm formation under both static and flow conditions. The biofilm formation of 11 additional clinical isolates were measured using the relevant concentrations of amoxicillin, lincomycin, and oxytetracycline. Eight of the eleven strains increased the biofilm formation with lincomycin, seven with amoxicillin, and three with oxytetracycline. Collectively, our data demonstrate that exposure to sub-MICs of these commonly used antibiotics contributes to increased biofilm formation of S. suis, thereby potentially increasing survival and persistence within the respiratory tract of swine.

Effect of antibiotics on bacterial populations: a multi-hierachical selection process.

Antibiotics have been widely used for a number of decades for human therapy and farming production. Since a high percentage of antibiotics are discharged from the human or animal body without degradation, this means that different habitats, from the human body to river water or soils, are polluted with antibiotics. In this situation, it is expected that the variable concentration of this type of microbial inhibitor present in different ecosystems may affect the structure and the productivity of the microbiota colonizing such habitats. This effect can occur at different levels, including changes in the overall structure of the population, selection of resistant organisms, or alterations in bacterial physiology. In this review, I discuss the available information on how the presence of antibiotics may alter the microbiota and the consequences of such alterations for human health and for the activity of microbiota from different habitats.

Subinhibitory concentrations of phloretin repress the virulence of Salmonella typhimurium and protect against Salmonella typhimurium infection.

Phloretin, a natural component of many fruits, exhibits anti-virulence effects and provides a new alternative to counter bacterial infection. The aim of this study was to determine the effect of subinhibitory concentrations of phloretin on the virulence of Salmonella typhimurium. At concentrations where growth of Salmonella was not inhibited, phloretin significantly inhibited bacteria biofilm formation and motility. Subinhibitory concentrations of phloretin repressed eight genes involved in the Salmonella pathogenicity island 1 and 3 genes involved in flagella production. Furthermore, subinhibitory concentrations of phloretin inhibited the adhesion and invasion of Salmonella in IEC-6 cells and reduced the LDH levels of S. typhimurium-infected IEC-6 cells. Additionally, phloretin significantly decreased the cecum bacterial loads of the mice infected with live S. typhimurium containing subinhibitory concentrations of phloretin by gavage. These results suggested that subinhibitory concentrations of phloretin attenuate the virulence of S. typhimurium and protect against S. typhimurium infection.

Subinhibitory antimicrobial concentrations: A review of in vitro and in vivo data.

Antimicrobial activity is not an 'all or none' effect. An increase in the rate and extent of antimicrobial action is usually observed over a wide range of antimicrobial concentrations. Subinhibitory antimicrobial concentrations are well known to produce significant antibacterial effects, and various antimicrobials at subinhibitory concentrations have been reported to inhibit the rate of bacterial growth. Bacterial virulence may be increased or decreased by subinhibitory antimicrobial concentrations by changes in the ability of bacteria to adhere to epithelial cells or by alterations in bacterial susceptibility to host immune defences. Animal studies performed in rats, hamsters and rabbits demonstrate decreased bacterial adherence, reduced infectivity and increased survival of animals treated with subinhibitory antimicrobial concentrations compared to untreated controls. The major future role of investigation of subinhibitory antimicrobial concentrations will be to define more fully, at a molecular level, how antimicrobials exert their antibacterial effects.