Nanoparticles Impact the Expression of the Genes Involved in Biofilm Formation in S. aureus, a Model Antimicrobial-Resistant Species

Background:     Infection with resistant bacteria are still reported in hospitals despite the routine cleaning of hospital surfaces. Presence of drug-resistant microbes in the on environment of hospitals and on medical equipment is indicative of the need for control measures which could impact the emergence of such microbes. In addition, biofilms are increasingly associated with human infections and it necessitates careful considerations on usage of a diverse range of medical devices, such as catheters, implants and pacemakers in hospitals.  Methods:      This study was designed to compare the effect of silver, ZnO nanoparticles and curcumin on drug-resistant Gram-positive and Gram-negative bacteria which were already isolated from different wards of the hospital. The MIC value were determined for silver, curcumin and ZnO nanoparticles. As the second step, the expression level of the genes involved in biofilm formation in S. aureus, including icaA, icaD, fnbA and fnbB, was studied to analyze the physiological reaction to controlled concentrations of such nanoparticles using RT-qPCR assessments. Results:     In this study, a total of 172 bacterial isolates were recovered from clinical and environmental samples (96 and 76 isolates, respectively). API-20 test revealed that these isolates belonged to 8 species. All antimicrobial resistant isolates were susceptible to the metal oxide nanoparticles. The results of q-PCR in this study showed that the expression of icaA and icaD genes in the presence of silver, curcumin and zinc nanoparticles were not significantly reduced compared to the control samples. But, exposure to nanoparticles reduced the expression of fnbA and fnbB genes from 0.46 to 0.06. Conclusion:  The results of our study showed that nanoparticles are highly effective on antibiotics- resistant isolates and these compounds can be used in the treatment of resistant bacteria. In addition, this study also demonstrates the promising potential of using nanoparticles as anti-biofilm formation agents

Identification of an intestinal microbiota signature associated with hospitalized patients with diarrhea

As an important global health challenge, diarrhea kills nearly two million people each year. Postinfectious irritable bowel syndrome (IBS) usually manifests itself as the diarrhea-predominant subtype. Small intestinal bacterial overgrowth has been observed more frequently in patients with IBS compared to healthy controls. However, the pathophysiology of IBS is not fully understood, and based on recent evidences, altered gut microbiota is involved in the pathogenesis of IBS. Therefore, we aimed to compare the microbiome in hospitalized patients with diarrhea and healthy individuals. Thirty patients and 10 healthy controls were included into this case–control study. Microbial count was performed using quantitative real-time polymerase chain reaction method using bacterial 16S rRNA gene. Clostridium cluster IV and Bacteroides were significantly more frequent in the patients compared with the healthy individuals (p = 0.02 and 0.023, respectively). However, the quantity of Enterococcus and Bifidobacterium groups were significantly higher in healthy controls than in diarrheal group (p = 0.000076 and 0.001, respectively). The results showed that the number of bacteria in all bacterial groups was significantly different between healthy individuals and diabetic group, whereas the difference between the healthy group and IBS was not significant for Bifidobacterium group. The findings of this study outlined the relationship between diarrhea, IBS, and diabetes disease and bacterial composition. It could be concluded that modifying the bacterial composition by probiotics can be helpful in the control and management of the mentioned disease

Study of MazEF, sam, and phd-doc putative toxin–antitoxin systems in Staphylococcus epidermidis

Today, to replace the antibacterial targets to overcome antibiotic resistance, toxin–antitoxin (TA) system is noticeable, where the unstable antitoxin neutralizes the stable toxin and protects the bacteria against the toxic effects. The presence and expression of TA genes in clinical and non-clinical strains of Staphylococcus epidermidis were investigated in this study. After identification of three TA pairs (mazEF, sam, and phd-doc) via existing databases (earlier, there has been no information in the case of S. epidermidis isolates), the presence and expression of these pairs were investigated by PCR and q-PCR, respectively. We detected three TA modules in all antibiotic sensitive and resistant isolates. In addition, q-PCR analysis revealed that the transcripts were produced from the three TA modules. This study showed the significant prevalence of these systems in pathogenic bacteria and they were equally found in both oxacillin-resistant and oxacillin-susceptible bacteria. The high prevalence of three systems can make them suitable as potential targets for antibiotic therapy

Time-variable expression levels of mazF, atlE, sdrH, and bap genes during biofilm formation in Staphylococcus epidermidis

Staphylococcus epidermidis is an opportunistic pathogen causing infections related to the usage of implants and medical devices. Pathogenicity of this microorganism is mainly linked to its capability to form biofilm structures. Biofilm formation vastly depends on several factors including different proteins. We studied the expression levels of three proteins including SdrH, Bap, AtlE, and MazF at different time intervals during the course of biofilm formation. In this study, a catheter-derived S. epidermidis isolate with strong ability of biofilm formation was selected. PCR assay was used to detect sdrH, bap, atlE, and mazF genes in this isolate. Real-time PCR was used to determine the expression levels of these genes after 4, 8, and 20 h during the course of biofilm formation. The studied genes showed different expression levels at different time intervals during biofilm formation by real-time PCR method. Expression levels of atlE and sdrH genes were the highest at 4 h, whereas bap gene showed the highest expression level at 8 h during the course of biofilm formation. In addition, the expression level of mazF gene peaked at 4 h and then progressively decreased at 8 and 20 h. Our results suggest the importance of AtlE, SdrH, and MazF proteins in the establishment and development of the biofilm structure. In addition, our results showed the important role of protein Bap in the accumulation of biofilm structure. Future studies are required to understand the exact role of MazF in the process of biofilm formation

Genotypic characterization, invasion index and antimicrobial resistance pattern in Listeria monocytogenes strains isolated from clinical samples

Objective: To evaluate antimicrobial resistance, invasion index and genetic profile in Listeria monocytogenes isolated from clinical samples. Methods: At all, 170 clinical samples were collected from patients with spontaneous abortions hospitalized in Shariati hospital in Tehran during June 2010 to August 2013. Invasion index was determined using HeLa cells. The multiple-locus variable-number tandem-repeats analysis (MLVA) was used for evaluation of genetic relatedness. Results: Out of 14 L. monocytogenes isolates, 4 (28.57%), 2 (14.28%), 0 (0%), 5 (35.71%) and 3 (21.42%) were isolated from placental tissue, urine, blood, vaginal and rectal swabs, respectively. High resistance to penicillin and multidrug resistant were found amongst isolates. The invasion index was in the range of 0.001–0.007. Seven different types were obtained by MLVA assay and type 2 and 3 with 4 strains were the most frequent type. Strains isolated from the vagina and the placenta of the same type were also more resistant to penicillin. Conclusions: Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to accomplish, rapid, and trustworthy, it is well suited to interlaboratory comparisons during epidemiological investigations. Also further studies of larger samples from a variety of sources such as food and animal specimens recommended comparing by MLVA method

The frequency of Listeria monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR

Background: Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis which has extensive clinical manifestations. Infections with L. monocytogenes are a serious threat to immunocompromised persons. The aim of this study was to determine the frequency of L. monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR. Materials and Methods: In this study, 617 specimens were analyzed. All specimens were cultured in the specific PALCAM agar. Colonies were initially identified by routine biochemical tests. Finally, PCR assays using primers specific for inlA gene were performed. Results: In all, 46 (8.2%) L. monocytogenes isolates were recovered from 617 specimens. Fourteen (8.2%) strains, including 4 (7.5%), 2 (5.7%), 5 (14.2%) and 3 (8.5%) isolates were obtained from placental tissue, urine, vaginal and rectal swabs, respectively. In addition, 9 (7.4%) strains of L. monocytogenes which were isolated from 107 different dairy products originated from cheese 5 (7.1%), cream 2 (10%) and kashk 2 (11.7%), respectively. Among 11 (5.2%) strains isolated from 210 different meat products, 5 (5.5%), 4 (7.2%) and 2 (3%) strains belonged to sausage, meat and poultry extracts, respectively. Finally, 12 (9.2%) Listeria strains were recovered from 130 animal specimens that included 6 (10%), 4 (8%) and 2 (10%) strains from goat, sheep and cattle, respectively. Furthermore, all Listeria isolates (100%) were found to be carriers of  inlA gene in PCR assay. Conclusion: The present study showed that the clinical and non-clinical specimens were contaminated with L. monocytogenes. So, it seems necessary to use a simple and standard technique such as PCR for rapid detection of this organism from various sources

Evaluation of cell-penetrating peptide–peptide nucleic acid effect in the inhibition of cag A in Helicobacter pylori

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 mu M of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 mu M after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori

Typing and Evaluation of the Genetic Relatedness of Listeria monocytogenes Strains Isolated from Food Samples by the Multiple-Locus Variable number Tandem Repeat Analysis (MLVA)

Background and Aim:Listeria monocytogenes cause listeriosis and fatal infections in humans. The aim of this study was typing and evaluation of the genetic relatedness of L. monocytogenes strains from food samples using MLVA technique. Materials and Methods: 317 food samples were collected from 2009 to 2013 in Tehran,Iran. After final diagnosis of L. monocytogenes DNA was extracted to perform of MLVA technique, and also PCR products were analyzed by Gene Tools software. The number of tandem repeats was determined by using special equation for each selected locus. Also typing of strains was done. Results: 24 samples of 317 food samples were positive for L. monocytogenes using standard laboratory techniques. A total 13 different types were determined by MLVA technique that type 2 and type 3 were the most abundant types by 6 and 4 strains, respectively. Conclusions: The results of this study showed the presence of L. monocytogenes in dairy products and meat samples, therefore all people, especially pregnant women should observe health tips when using these products. The results of typing showed that L. monocytogenes strains from different sources can have the same origin. MLVA technique is easy with high accuracy and this method can be used in typing and evaluation of the genetic relatedness of L. monocytogenes for determination the source of contamination

Molecular typing and antibiotic resistance patterns among clinical isolates of Acinetobacter baumannii recovered from burn patients in Tehran, Iran

Acinetobacter baumannii (A. baumannii) is now considered a highly resistant pathogen to various types of antibiotics. Therefore, tracking the source of its prevalence and continuous control is crucial. This study aimed to determine antibiotic resistance and perform various molecular typing methods on clinical isolates of A. baumannii isolated from hospitalized burn patients in Shahid Motahari Burn Hospital, Tehran, Iran. Hospital isolates were confirmed by phenotypic and molecular methods. Then the sensitivity to different antibiotics was determined using the minimum inhibitory concentration (MIC) method. In order to perform molecular typing, three-locus dual assay multiplex polymerase chain reaction (PCR), multiple-locus variable-number tandem repeat analysis (MLVA), and multilocus sequence typing (MLST) methods were used. Among the 60 isolates collected, the frequencies of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 90 and 10%, respectively. The most effective antibiotics were colistin with 100% and tigecycline with 83.33% sensitivity. Isolates were 100% resistant to piperacillin/tazobactam and cephalosporins, and 68.3% were resistant to carbapenem. The results of multiplex PCR showed five groups that international clone I (IC I) and IC II were the most common. The MLVA method identified 34 MLVA types (MTs), 5 clusters, and 25 singletons. Multilocus sequence typing results for tigecycline-resistant isolates showed seven different sequence types (STs). Increasing antibiotic resistance in A. baumannii isolates requires careful management to control and prevent the occurrence of the pre-antibiotic era. The results of this study confirm that the population structure of A. baumannii isolates has a high diversity. More extensive studies are needed in Iran to better understand the epidemiology of A. baumannii