Investigating the effect of the inhibitory peptide on L.monocytogenes cell invasion: an in silico and in vitro study.

AIMS: L.monocytogenes monocytogenes is an omnipresent bacterium that causes a fatal food-borne illness, listeriosis. The connection of this bacterium to E-cadherin through internalin A plays a significant role in the internalization of the bacteria. In this study, this interaction has been investigated for the design of an inhibitory peptide. METHODS: The interaction of the proteins involved in the entry of bacteria was evaluated by molecular docking. According to their interactions, an inhibitory peptide was designed to bind to internalin A by server peptiderive. Its effects on L.monocytogenes invasion on the Caco-2 cell line and biofilm formation were also assessed. FINDINGS: Docking results showed that the peptide has a high affinity for binding to Internalin A. The synthesized peptide at a concentration of 64 µg/ml inhibited 80% of the invasion of L.monocytogenes into the Caco-2 cell line. Furthermore, the studied peptide at the highest concentration had a slight inhibitory effect on biofilm formation. CONCLUSION: These results reveal that short polypeptides can impede the invasion of target cells by L. monocytogenes in vitro and could be advantageous as restoring agents in vivo.

Evaluation of Genetic Content of the CRISPR Locus in Listeria monocytogenes Isolated From Clinical, Food, Seafood and Animal Samples in Iran.

CRISPR arrays, which are organized to fight against non-self DNA elements, have shown sequence diversity that could be useful in evolution and typing studies. In this study, 55 samples of L. monocytogenes isolated from different sources were evaluated for CRISPR sequence polymorphism. The CRISPR loci were identified using CRISPR databases. A single PCR assay was designed to amplify the target CRISPRs using an appropriate universal primer. Sequencing results were analyzed using CRISPR databases and BLASTn, and the CRISPR locus was present in all the strains. Three hundred repeats including 55 terminal repeats were identified. Four types of consensuses direct repeat (DR) with different lengths and sequences were characterized. Sixty repeat variants were observed which possessed different polymorphisms. Two hundred and fifty spacers were identified from which 35 consensus sequences were determined, indicating the high polymorphism of the CRISPR spacers. The identified spacers showed similarities to listeria phage sequences, other bacterial phage sequences, plasmid sequences and bacterial sequences. In order to control the bacterial outbreaks, a robust and precise system of subtyping is required. High levels of polymorphism in the CRISPR loci in this study might be related to the origin and time of the samples' isolation. However, it is essential to assess, on a case-by-case basis, the characteristics of any given CRISPR locus before its use as an epidemiological marker. In conclusion, the results of this study showed that the use of sequence content of CRISPR area could provide new and valuable information on the evolution and typing of the L. monocytogenes bacterium.

Distribution and expression of virulence genes (hlyA, sat) and genotyping of Escherichia coli O25b/ST131 by multi-locus variable number tandem repeat analysis in Tehran, Iran.

Escherichia coli ST131 is a pandemic clone with high antibiotic resistance, and it is a major causative agent of urinary tract infection (UTI) and bloodstream infections. This study evaluated the distribution and expression of virulence genes and genotyping of E. coli O25b/ST131 by Multi-locus variable number tandem repeat analysis (MLVA) method among UTI in patients at Tehran hospitals, Iran.A total of 107 E. coli isolates were collected from UTI patients. Polymerase chain reaction (PCR) amplification of the pabB gene was used to identify E. coli O25b/ST131 and the prevalence of sat and hlyA virulence genes was also analyzed. The microtiter method quantified biofilm formation ability in E. coli O25b/ST131. The Real-Time PCR (qRT-PCR) was performed to evaluate the expression of sat and hlyA genes. Finally, MLVA was performed for E. coli O25b/ST131 genotyping by targeting seven tandem repeats. SPSS-16 software was used for statistical analysis. Molecular study showed that 71% of isolates carried the pabB gene and were considered E. coli O25b/ST131 strains. Also, 45.8% and 17.8% of isolates carried sat and hlyA genes, respectively. The 57.9% isolates had biofilm formation ability. Expression of the studied virulence genes showed an increase in strong biofilm producing E. coli O25b/ST131 strains. A total of 76 (100%) E. coli O25b/ST131 strains were typed by the MLVA method.High prevalence of E. coli O25b/ST131 isolates in UTI patients can be a serious warning to the treatment due to the high antibiotic resistance rate, expression of virulence genes, and biofilm formation.

The expression of type II TA system genes following persister cell formation in Pseudomonas aeruginosa isolates in the exponential and stationary phases.

Failure of infection therapy in the presence of antibiotics has become a major problem which has been mostly attributed to the ability of bacterial persister cell formation. Bacteria use various mechanisms to form persister cells in different phases, among which is the toxin-antitoxin (TA) systems. This study aimed at investigating the expression of type II TA system genes under the stress of ciprofloxacin and colistin antibiotics in the exponential and stationary phases. To determine the effects of ciprofloxacin and colistin on persister cell formation in the exponential and stationary phases of Pseudomonas aeruginosa strains, colony counting was performed at different time intervals in the presence of fivefold MIC of ciprofloxacin and colistin. In addition, the expression of relBE, Xre-COG5654, vapBC, and Xre-GNAT genes in P. aeruginosa isolates was assessed 3.5 h after antibiotic treatment in the exponential and stationary phases using qRT-PCR. Our results indicated the presence of persister phenotype of P. aeruginosa strains in the presence of fivefold MIC of ciprofloxacin and colistin compared to the control after 3.5 h of incubation in the exponential and stationary phases. Also, the number of persister cells in the stationary phase was higher than that of the exponential phase. According to the results of qRT-PCR, ciprofloxacin and colistin may induce persister cells by increasing the expression of type II TA systems in stationary and exponential phases. Ciprofloxacin and colistin may increase the formation of persister cells by affecting the expression of type II TA systems.

Prevalence of bacteriocin genes in Lactobacillus strains isolated from fecal samples of healthy individuals and their inhibitory effect against foodborne pathogens.

OBJECTIVES: Foodborne diseases are considered as an important public health issue. The purpose of the current study was to isolate Lactobacillus spp. strains from fecal samples, investigate their antimicrobial properties, and assess the expression of genes encoding bacteriocin in co-culture of Lactobacillus with enteric pathogens. MATERIALS AND METHODS: Fecal samples of healthy people were collected. Human colon adenocarcinoma cell line Caco-2 was used to examine Lactobacillus strains adherence capacity. Quantitative real-time reverse transcription PCR (qRT-PCR) was used to determine bacteriocin-encoding genes expression in co-culture of the selected Lactobacillus strain with Salmonella, Shigella, and two diarrheagenic Escherichia coli serotypes during 4, 6, and 24 hr of incubation. RESULTS: The selected L. plantarum strain was able to inhibit four foodborne pathogens in both methods. L. plantarum No.14 exhibited the highest ability to adhere to Caco-2 cells. In this study, pln F, sak P, pln I, pln B, and pln J genes of L. plantarum No.14 were upregulated in co-culture of L. plantarum No.14 with diarrheagenic E. coli serotypes. In addition, acd, Lactacin F, sak P, pln J, pln EF, and pln NC8 genes as well as pln NC8 and pln A genes mRNA levels were significantly increased in co-culture of L. plantarum No.14 with Shigella dysenteriae, and Salmonella typhi, respectively, during 24 hrs of incubation. CONCLUSION: Other studied genes were down-regulated during the incubation time. The selected L. plantarum strains could be served as alternative antimicrobial agents against pathogens which could contaminate foodstuffs and are responsible for human diseases.

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.

Evaluation of the genetic relatedness of Bacteroides fragilis isolates by TRs analysis.

OBJECTIVES: Human gastrointestinal tract harbors a variety of bacteria with vital roles in human health. Bacteroides fragilis is considered one of the dominant constituents of gut microflora which can act as an opportunistic pathogen leading to various diseases, including colon cancer, diarrhea, uterine and intrathecal abscesses, septicemia, and pelvic inflammation. In this study, multiple locus variable number of tandem repeats analysis (MLVA) was performed to genetically differentiate 50 B. fragilis isolates. MATERIALS AND METHODS: Eight suitable tandem repeats (TRs) were selected by bioinformatics tools and were then subjected to PCR amplification using specific primers. Finally, MLVA profiles were clustered using BioNumerics 7.6 software package. RESULTS: All VNTR loci were detected in all isolates using the PCR method. Overall, B. fragilis isolates were differentiated into 27 distinct MLVA types. The highest diversity index was allocated to TR1, TR2, TR5, TR6, and TR8; with this taken into account, strain type 14 was the most prevalent with 12 strains belonging to this type. Clustering revealed three major clusters of A, B, and C. With regards to the pathogenicity of B. fragilis and the outcomes of infections related to this microorganism, it is imperative to study this microorganism isolated from both patients and healthy individuals. CONCLUSION: This study aimed at evaluating the efficiency of MLVA for the genetic differentiation of B. fragilis. The results of this study indicate the promising efficiency of MLVA typing for cluster detection of this bacterium.

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.

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.

Prevalence of vaginolysin, sialidase and phospholipase genes in Gardnerella vaginalis isolates between bacterial vaginosis and healthy individuals.

Background: Gardnerella vaginalis is considered as the predominant microorganism found in bacterial vaginosis (BV). The aim of this study was to evaluate the prevalence of virulence factors in G. vaginalis associated with BV or non-BV cases and their correlations with this disorder. Methods: A total of 102 vaginal specimens were collected from patients during their visit to Akbar Abadi hospital in Tehran, Iran. Bacterial vaginosis was determined by Nugent score and Amsel's criteria. Polymerase chain reaction (PCR) was used for the detection of G. vaginalis 16S rRNA, vaginolysin, sialidase and phospholipase genes. To evaluate the association between the presence of vly, pho, and sld genes and BV. Pearson Chi-square test was applied using SPSS software. P-value ≤0.05 was considered as significant. Results: Totally, 27.4% of the patients were suffering from BV. Gardnerella vaginalis was found in 100% women with BV and in 56.7% women with normal vaginal discharge. The prevalence of vly, sld and pho genes in BV-associated G. vaginalis was 10 (35.7%) (95% CI [0.18, 0.53]), 19 (67.8%) (95% CI [0.51, 0.85]) and 6 (21.4%) (95% CI [0.06, 0.37]), respectively. The prevalence of the aforementioned genes in non-BV associated G. vaginalis was 20 (47.6%) (95% CI [0.33, 0.63]), 28 (66.6%) (95% CI [0.52, 0.81]), and 5 (11.9%) (95% CI [0.02, 0.22]), respectively. Our results showed no statistically significant association between the presence of the virulence genes and BV associatedness of this microorganism. Conclusion: Our results showed the presence of G. vaginalis in all BV patients and relatively high prevalence in healthy individuals. The prevalence rates of the three virulence genes were different in BV and non-BV associated G. vaginalis; however, the differences were not statistically significant.

Does biofilm formation have different pathways in Staphylococcus aureus?

OBJECTIVES: Biofilm formation is one of the most important factors in the development of infections caused by Staphylococcus aureus. In this study, the expression levels of genes responsible for biofilm formation were studied in methicillin sensitive and methicillin resistant S. aureus. MATERIALS AND METHODS: A total of 100 meticillin-resistant s.aureus (MRSA) and meticillin-sensetive s.aureus (MSSA) isolates were studied. Bacterial biofilm formation was evaluated phenotypically using microtiter plate method. Real-time PCR tests were conducted to determine the expression levels of genes involved in biofilm formation. RESULTS: Quantitative biofilm formation test was repeated three times for each specimen. The prevalence of weak, medium, and strong biofilm producers were 16%, 49%, and 35%, respectively. In MSSA isolates, expression levels of ica genes increased compared to the fnbA, fnbB, clfA and clfB genes. These results were different in MRSA isolates, and ica genes showed a decreased gene expression levels compared to the aforementioned genes. CONCLUSION: Considering the results of this study, clf genes probably contribute to the same extent in both MRSA and MSSA isolates, and there is probably no significant difference in the role of these genes in these isolates. In addition, the results of this study indicated that MRSA may not use the conventional route for biofilm formation and may use independent pathways through Polysaccharide intercellular adhesion (PIA).

Genotyping and Phylogenetic Analysis of Group B Streptococcus by Multiple Locus Variable Number Tandem Repeat Analysis in Iran.

BACKGROUND: Group B streptococcus (GBS), also known as Streptococcus agalactiae, is well known as a causative agent for neonatal invasive diseases; it is also a major pathogen in adults. Analytic epidemiology is required to monitor the clinical isolates of GBS. However, there is insufficient information on the genetic background of GBS in Iran, and this information is needed to guide and develop a GBS vaccine. MATERIALS AND METHODS: In total, 90 well-char - acterized GBS isolates were collected from April 2014 to August 2015. In this study, molecular typing was used to disclose a relationship between the multiple-locus variable number tandem repeat analysis (MLVA) types, serotyping, and pilus islands. The isolates were characterized by the types of capsular polysaccharides and pilus islands and were examined by MLVA to study the epidemiological relationship of isolates. RESULTS: The results indicate that there is a significant relationship between the distribution of serotypes and pilus island genes; GBS isolates were differentiated into 12 types by capsular polysaccharides and pilus islands analysis. The discriminatory power of an MLVA analysis was high based on the five most variable numbers of tandem repeat loci and 44 MLVA types that were identified. CONCLUSION: This study has provided useful insights into the genetic heterogeneity of GBS isolates in Tehran and Alborz, Iran. The extensive distribution of pilus islands in various serotypes and MLVA types throughout the GBS population refers to the advancement of the pilus-based GBS vaccines.

Relationship between γ-interferon gene polymorphisms and susceptibility to brucellosis infection.

Interferon-gamma (IFN-γ) is a pro-inflammatory cytokine that plays a pivotal role in the defense mechanism against Brucella infection. It was hypothesized that the IFN-γ in (+874 A/T in intron 1) TT and +5644 T/A, TT genotypes, which are reportedly associated with high IFN production, are associated with susceptibility to brucellosis in Iranian subjects. Genotyping of these IFN-γ variants by an allele-specific polymerase chain reaction method was performed in 281 subjects, comprising 153 patients with active brucellosis and 128 healthy controls. It was found that the +874 minor allele (A) and homozygote genotype (AA) were significantly more frequently present in brucellosis patients than in controls (OR = 2.588; 95% CI, 1.313-5.104; P = 0.006 for the AA genotype; OR = 1.575; 95% CI, 1.124-2.216; P = 0.010 for the A allele). However, the allelic and genotypic distribution of the IFN-γ polymorphism at position UTR5644 A>T did not differ significantly between patients and controls (P > 0.05). The distribution of haplotypes in this study suggests that the T/A haplotype (+874/UTR5644), which was present more frequently in controls than in patients, may protect subjects against Brucella infection. It is suggested that IFN-γ +874 AA genotype and A allele are risk factors for developing brucellosis infection in Iranian subjects.

The association of interleukin-18 promoter polymorphisms and serum levels with duodenal ulcer, and their correlations with bacterial CagA and VacA virulence factors.

BACKGROUND: We analyzed the impact of interleukin (IL)-18 promoter polymorphisms on IL-18 serum levels in Helicobacter pylori-infected duodenal ulcer (DU) patients and healthy asymptomatic (AS) carriers. We also aimed to determine the association of the H. pylori virulence factors CagA and VacA antibodies with serum concentrations of IL-18 in order to elucidate any correlation between them. METHODS: Three groups of patients were enrolled: DU patients (67 individuals), AS carriers (48 individuals), and H. pylori-negative subjects (26 individuals). Serum concentrations of IL-18 were determined by ELISA. Patient sera were tested by Western blot method to determine the presence of serum antibodies to bacterial CagA and VacA. Genotyping of IL-18 promoter polymorphisms at positions - 137G/C and - 607C/A were performed by allele-specific primer PCR protocol. RESULTS: Our study revealed that serum IL-18 levels are positively influenced by CagA-positive H. pylori strains, so that maximum levels of IL-18 were detected in DU patients with the CagA(+) phenotype, regardless of the presence of the anti-VacA antibody. Regarding IL-18 promoter polymorphisms, the AA genotype and A allele at position - 607C/A were found to be significantly lower in DU patients than in AS carriers and H. pylori-negative subjects (p = 0.032 and 0.043, respectively). CONCLUSIONS: The IL-18 - 607C variant was associated with higher levels of serum IL-18 and an increased risk of DU. Moreover, our findings indicated that serum concentrations of IL-18 were influenced by CagA factor, irrespective of the VacA status, suggesting that high levels of IL-18 in CagA-positive subjects predisposes to susceptibility to DU.

Circulating levels of interleukin (IL)-12 and IL-13 in Helicobacter pylori-infected patients, and their associations with bacterial CagA and VacA virulence factors.

OBJECTIVE: The aim of this study was to determine the association of the Helicobacter pylori virulence factors, cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) antibodies, with serum levels of interleukin (IL)-12 and IL-13 in H. pylori-infected duodenal ulcer (DU) patients and H. pylori-infected asymptomatic (AS) carriers in order to elucidate any correlation between them. METHODS: A total of 67 DU patients, 48 AS individuals, and 26 healthy H. pylori-negative subjects were enrolled in this study. Serum concentrations of IL-12 and IL-13 were determined by enzyme-linked immunosorbent assay (ELISA) method. Patient sera were tested by Western blot method to determine the presence of serum antibodies to bacterial virulence antigens p120 (CagA) and p95 (VacA). Serum concentrations of IL-12 and IL-13 were compared in 9 groups, including 4 AS phenotypes (CagA⁺VacA⁺, CagA⁺VacA⁻, CagA⁻VacA⁺, CagA⁻VacA⁻), 4 DU phenotypes (CagA⁺VacA⁺, CagA⁺VacA⁻, CagA⁻VacA⁺, CagA⁻VacA⁻), and 1 control group. RESULTS: The results revealed that DU patients positive for CagA, independent of the anti-VacA antibody status, showed drastically elevated levels of IL-12 (251 ± 43 pg/ml) when compared with the other groups (p = 0.0001). No significant difference was found between groups regarding levels of IL-13 (p > 0.05). CONCLUSIONS: Our findings indicate that in the DU group, the serum concentrations of IL-12 but not of IL-13 were influenced by bacterial CagA, independent of the VacA status, suggesting that high IL-12 levels may contribute to susceptibility to DU in CagA-positive individuals. These findings could possibly be considered to improve the predictive or prognostic values of inflammatory cytokines for DU, and also to design possible novel therapeutic approaches.