Effects of sub-inhibitory concentrations of antibiotics and oxidative stress on the expression of type II toxin-antitoxin system genes in Klebsiella pneumoniae

Objectives: Sub-inhibitory concentrations (sub-MICs) of antibiotics reflect the conditions that bacteria encounter in tissues and the natural environment. Sub-MICs of antibiotics can induce stress and alter the expression of different bacterial genes. Bacteria react to stress conditions using different mechanisms, one of which is the toxin-antitoxin (TA) system. This study investigated the expression of the TA system genes under oxidative and antibiotic stresses in Klebsiella pneumoniae (K. pneumoniae). Methods: To determine the effects of sub-MICs of gentamicin, nalidixic acid, ceftazidime, and certain concentrations of H2O2 on bacterial survival and growth, colony forming units were quantitated and turbidity was assessed following the treatment of K. pneumoniae with ½ MICs of antibiotics and 5 mM H2O2 at different time intervals. The expression of TA system genes in K. pneumoniae was evaluated 1 h after treatment using the quantitative real-time PCR (qRT-PCR) method. Results: The results revealed reduced K. pneumoniae growth in the presence of sub-MICs of antibiotics and 5 mM H2O2 compared to the control. Furthermore, according to the results of the qRT-PCR assay, only the presence of gentamicin could increase the expression of TA system genes. Conclusion: Although the exact role of the TA systems in response to stress is still unclear, this study provided information on the effect of the type II TA systems under oxidative and antibiotic stress conditions. © 2019 International Society for Antimicrobial Chemotherap

Persister cells formation and expression of type ii toxin-antitoxin system genes in brucella melitensis (16m) and brucella abortus (b19)

Background & Objective: Persister cells are defined as a subpopulation of bacteriathat are capable of reducing their metabolism and switching to dormancy in stress conditions. Persister cells formation has been attributed to numerous mechanisms, including stringent response and Toxin-Antitoxin (TA) systems. This study aimed to investigate the hypothetical role of TA systems in persister cells formation of Brucella strains by evaluating toxins of type II TA systems (RelE, Fic, Brn T, cogT) expression. Methods: Brucella strains treated with a lethal dose of gentamicin and ampicillin and to determine the number of surviving cells, bacterial colonies were counted at different time intervals. The role of TA systems in persister cell formation was then determined by toxin expression levels using qRT- PCR method. Results: Our results showed the viability of persister cells after 7 h. The results of relative qRT-PCR showed higher levels of toxin gene expression due to stress conditions, suggesting the possible role of TA systems in persister cells formation and antibiotics tolerance.Conclusion: The results of this study showed that considering the importance of persistenc and the tolerance to antibiotics, further studies on persister cells formation and related genes such as the TA system genes in Brucella strains might help us to identify the precise mechanisms leading to persister cells formation. © 2020, Iranian Society of Pathology. All rights reserved

Biofilm establishment, biofilm persister cell formation, and relative gene expression analysis of type II toxin-antitoxin system in Klebsiella pneumoniae

The biofilms forming ability in Klebsiella pneumoniae isolates leads to treatment failure and chronic infections through protecting bacteria against antibiotics. Persister cells are a small fraction of the bacterial population that survives by entry into dormancy state after treatment with high dose antibiotics and are mainly responsible for a high level of biofilm-mediated tolerance to antibiotics. Toxin-antitoxin (TA) systems widely distributed among bacteria. Activation of the TA system is believed to result in the bacterial entrance to the dormant state, inducing persistence, and the formation of biofilms. In this study after biofilm formation and the induction of persister cells in biofilms, the relative expression level of type II TA system genes (relE1/relB1, relE2/relB2, hipA/hipB, vapB/vapC, phd/doc, mazF/mazE) in K. pneumoniae isolates were examined by Real Time PCR. Findings showed an increase in the expression levels of type II TA system genes in biofilm formers and an upregulation of all genes studied in biofilm persister cells except the phd/doc system. Our results can provide information about the potential role of the TA systems in biofilm production and also the biofilms persister cell formation. © 202

Evaluation of gene expression and protein structural modeling involved in persister cell formation in Salmonella Typhimurium

Persisters are phenotypic variants of the bacterial population that survive against lethal doses of bactericidal antibiotics.These phenotypes are created in numerous bacterial species, including those of clinical significance, such as Salmonella Typhimurium. Since persister cells are associated with the failure of antibiotic treatment and infection recurrence, it is crucial to identify the mechanisms that influence the formation of these cells. The aim of this study is to investigate the persister cell formation and expression analysis as well as to predict the 3D structure of the genes involved in the production of persister cells. The presence of persisters in S. Typhimurium was determined by time dependent killing of different types of bactericidal antibiotics and expression of genes associated with persister cell formation which was assessed five hours after the addition of antibiotics by the qRT-PCR. Indeed, the 3D structural model of the proteins studied was predicted by performing several computational methods of retrieved primary protein sequences. The results of the study showed that the S. Typhimurium produced high levels of persister cells in the exposure of bactericidal antibiotics. Furthermore, qRT-PCR resulted in the fact that the expression of related genes was different depending on the type of antibiotic. Overall, this study provides information on the creation of persister cells and the role of different genes in the formation of these cells and structure of proteins involved in the production of persister cells in S. Typhimurium

Combination of Antibiotics-Nisin Reduces the Formation of Persister Cell in Listeria monocytogenes

Persister cells are a subpopulation of bacteria with the ability of survival when exposed to lethal doses of antibiotics, and are responsible for antibiotic therapy failure and infection recurrences. In this study, we investigated persister cell formation and the role of nisin in combination with antibiotics in reducing persistence in Listeria monocytogenes. We also examined the expression of toxin-antitoxin (TA) systems in persister cells of L. monocytogenes to gain a better understanding of the effect of TA systems on persister cell formation. To induce persistence, L. monocytogenes were exposed to high doses of different antibiotics over a period of 24 hr, and the expression levels of TA system was genes were measured 5 hr after the addition of antibiotics by the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) method. To investigate the effect of nisin, L. monocytogenes was exposed to a combination of nisin and antibiotics. According to our results, L. monocytogenes was highly capable of persister cell formation, and the combination of nisin and antibiotics resulted in reduced persistence. qRT-PCR results showed a significant increase in GNAT/RHH expression among the studied systems. Overall, our results demonstrated the potential of the combination of nisin and antibiotics in reducing persister cell formation, and emphasized the role of the GNAT/RHH system in bacterial persistence

Persister cells formation and expression of type II Toxin-Antitoxin system genes in Brucella melitensis (16M) and Brucella abortus (B19)

Background & Objective: Persister cells are defined as a subpopulation of bacteria that are capable of reducing their metabolism and switching to dormancy in stress conditions. Persister cells formation has been attributed to numerous mechanisms, including stringent response and Toxin-Antitoxin (TA) systems. This study aimed to investigate the hypothetical role of TA systems in persister cells formation of Brucella strains by evaluating toxins of type II TA systems (RelE, Fic, Brn T, cogT) expression. Methods: Brucella strains treated with a lethal dose of gentamicin and ampicillin and to determine the number of surviving cells, bacterial colonies were counted at different time intervals. The role of TA systems in persister cell formation was then determined by toxin expression levels using qRT- PCR method. Results: Our results showed the viability of persister cells after 7 h. The results of relative qRT- PCR showed higher levels of toxin gene expression due to stress conditions, suggesting the possible role of TA systems in persister cells formation and antibiotics tolerance. Conclusion: The results of this study showed that considering the importance of persistence and the tolerance to antibiotics, further studies on persister cells formation and related genes such as the TA system genes in Brucella strains might help us to identify the precise mechanisms leading to persister cells formation

Biofilm establishment, biofilm persister cell formation, and relative gene expression analysis of type II toxin-antitoxin system in Klebsiella pneumoniae

The biofilms forming ability in Klebsiella pneumoniae isolates leads to treatment failure and chronic infections through protecting bacteria against antibiotics. Persister cells are a small fraction of the bacterial population that survives by entry into dormancy state after treatment with high dose antibiotics and are mainly responsible for a high level of biofilm-mediated tolerance to antibiotics. Toxin-antitoxin (TA) systems widely distributed among bacteria. Activation of the TA system is believed to result in the bacterial entrance to the dormant state, inducing persistence, and the formation of biofilms. In this study after biofilm formation and the induction of persister cells in biofilms, the relative expression level of type II TA system genes (relE1/relB1, relE2/relB2, hipA/hipB, vapB/vapC, phd/doc, mazF/mazE) in K. pneumoniae isolates were examined by Real Time PCR. Findings showed an increase in the expression levels of type II TA system genes in biofilm formers and an upregulation of all genes studied in biofilm persister cells except the phd/doc system. Our results can provide information about the potential role of the TA systems in biofilm production and also the biofilms persister cell formation. © 202

Effects of sub-inhibitory concentrations of antibiotics and oxidative stress on the expression of type II toxin-antitoxin system genes in Klebsiella pneumoniae

OBJECTIVES: Sub-inhibitory concentrations (sub-MICs) of antibiotics reflect the conditions that bacteria encounter in tissues and the natural environment. Sub-MICs of antibiotics can induce stress and alter the expression of different bacterial genes. Bacteria react to stress conditions using different mechanisms, one of which is the toxin-antitoxin (TA) system. This study investigated the expression of the TA system genes under oxidative and antibiotic stresses in Klebsiella pneumoniae (K. pneumoniae). METHODS: To determine the effects of sub-MICs of gentamicin, nalidixic acid, ceftazidime, and certain concentrations of H2O2 on bacterial survival and growth, colony forming units were quantitated and turbidity was assessed following the treatment of K. pneumoniae with (1/2) MICs of antibiotics and 5 mM H2O2 at different time intervals. The expression of TA system genes in K. pneumoniae was evaluated 1 h after treatment using the quantitative real-time PCR (qRT-PCR) method. RESULTS: The results revealed reduced K. pneumoniae growth in the presence of sub-MICs of antibiotics and 5 mM H2O2 compared to the control. Furthermore, according to the results of the qRT-PCR assay, only the presence of gentamicin could increase the expression of TA system genes. CONCLUSION: Although the exact role of the TA systems in response to stress is still unclear, this study provided information on the effect of the type II TA systems under oxidative and antibiotic stress conditions

Inhibitory Effects of Lactococcus lactis and its Supernatant on Listeria monocytogenes

Background: The application of biological compounds generated by lactic acid bacteria, especially Lactococcus lactis, is recently considered to be a natural preservative for improving quality and health of food. The purpose of this study is to investigate the inhibitory potential of L. lactis supernatant on the expression of inlA, plc, and hly genes related to L. monocytogenes virulence capacity. Methods: L. lactis was cultured under anaerobic conditions for 16 -18 hours. The supernatant and live bacteria were then separated by centrifuge. The anti-listeria effects of L. lactis and supernatant were measured using the agar diffusion technique, and the effect on the expression of the virulence-related genes was calculated by real-time PCR. Also, the effects of live bacteria and its supernatant on the microbial count of milk and sausage infected by L. monocytogenes was evaluated by the colony count assay. Results: After 24 hours, the highest non-growing hole diameter was obtained in the presence of acidic supernatant (pH = 3.5). The microbial count showed the inhibitory effect on the eighth day after incubation with L. lactis. qPCR data revealed a down-regulation of virulence-related genes of inlA (8 fold), hly (6 fold), and plc (1 fold) in L. monocytogenes after 24-hour incubation with the supernatant. Conclusions: Our findings showed that the supernatant of L. lactis has an effective inhibitory role in the growth of L. monocytogenes. In the presence of supernatant, among plc, inlA and hly genes, the expression of inlA and hly genes decreased after 2 hours, which could indicate the molecular inhibitory mechanism of L. lactis in L. mono-cytogenes

The expression of type II TA system genes following exposure to the sub-inhibitory concentration of gentamicin and acid stress in Brucella spp.

Background: Brucellosis is one of the most common diseases that afflicts both humans and animals. Bacteria react to stress conditions using different mechanisms one of which is Toxin-Antitoxin (TA) systems. It is believed that the Toxin-Antitoxin (TA) systems have a key role in the chronicity of the disease. This study investigated the expression of TA system genes under acid and antibiotic stresses in Brucella spp. Methods: Fifty Brucella isolates (17 isolated from animals and 31 isolated from human specimens, and two standard strains) were analyzed using PCR (using two pairs of primers). Then, to determine the effects of sub-MIC of gentamicin on bacterial survival and growth, colony forming unit was quantitated and turbidity was assessed following the treatment of Brucella spp, with ½ MIC of gentamicin at different time intervals. Furthermore, the colony forming unit of Brucella spp, was assessed under acid stress (pH = 5.5) compared to the control (pH = 7.6). Moreover, the expression of TA system genes in Brucella spp, was evaluated 1 h after treatment using qRT-PCR method. Results: A total of 50 isolates, including 41 (82) Brucella melitensis and 7 (14) Brucella abortus with two standard strains Brucella melitensis (16 M) and Brucella abortus (B19) were investigated. Our results revealed the reduced growth of Brucella spp. in the presence of sub-MIC of gentamicin compared to the control. Furthermore, according to the results of qRT-PCR assay, gentamicin could increase the expression of TA system genes. Also, results of qRT-PCR showed that under acid stress, the expression of TA system gene COGT/COGAT decreased compared to the control. Conclusion: Although the exact role of the TA systems in response to stress is still unclear, our study provided information on the effect of the type II TA systems under the acid and antibiotic stress conditions. However, further studies are still required. © 2020 Elsevier Lt