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.

Interesting probiotic traits of mother's milk Lactobacillus isolates; from bacteriocin to inflammatory bowel disease improvement.

AIMS AND BACKGROUND: Lactobacillus spp. are an important element in breast milk. This component has a beneficial effect on the composition of the intestinal microflora and the intestinal immune system. The aim of this study was to isolate and identify Lactobacillus strains in breast milk and evaluate some of their probiotic properties, such as presence of bacteriocin genes, adhesion to HT-29 cell line, competition with enteropathogens in cell culture, and effect on serum level of lipids and digestive enzymes, and mice model of inflammatory bowel disease (IBD). MATERIALS AND METHODS: A total of 323 lactic acid bacteria (LAB) were isolated from breast milk samples of healthy mothers with the age ranges from 21 to 45 years old. These isolates were subjected to phenotypic and molecular experiments. The frequency of bacteriocin genes was determined by polymerase chain reaction (PCR). Adhesion of Lactobacillus isolates to HT-29 cells was measured based on the number of attached bacterial cells in 20 fields of the light microscopy. Competition test was done by colony count and real-time PCR procedures. Five strongly adhesive Lactobacillus strains were selected and administered orally to the treatment groups. After 8 days, the serum level of digestive enzymes and improvement in induced IBD, and after 14 days, the serum level of lipids (triglycerides, total cholesterol, HDL, and LDL) in treated mice were surveyed compared to the control groups. RESULTS: Based on the phenotypic and molecular experiments, L. casei, L. plantarum, L. rhamnosus, and L. acidophilus strains were isolated and identified in the breast milk samples. The highest frequency of bacteriocin genes belonged to Plantaricin B (100%), followed by Plantaricin D (84.7%), Plantaricin G (84.7%), and Plantaricin EF (54.3%). Also, 71.8% of the isolates were strongly adhesive, 21.8% were non-adhesive, and 6.4% were adhesive. Lactobacillus strains had a significant effect on the displacement of enteropathogens. The in vitro cholesterol-removing ability of L. casei (L1), L. casei (L2), L. casei (L3), L. plantarum (L4), and L. rhamnosus (L5) was 3.5, 31.5, 21.3, 18.7, and 27.3%, respectively. The serum level of total cholesterol in the L. plantarum (L4) group as well as LDL in the L. casei (L3) (p = .0108) and L. rhamnosus (L5) (p = .0206) groups decreased significantly compared to the control group. The serum level of lipase increased in all the treatment groups compared to the control group, which was significant in the L. plantarum (L4) group (p = .0390). Disease activity index (DAI) scores were improved significantly in L. casei (L3) group compared to the IBD control group (p < .0001). CONCLUSION: It could be concluded that lactobacilli strains isolated from the breast milk samples had good probiotic properties, such as presence of bacteriocin genes, attaching to enterocyte-like HT-29 cells, competing with intestinal pathogens, lowering cholesterol, and improving IBD. Thus, after further studies, they could be considered as probiotic strains.

Analysis of virulence genes and molecular typing of Listeria monocytogenes isolates from human, food, and livestock from 2008 to 2016 in Iran.

The frequency of Listeria monocytogenes isolates collected from a total of 1150 samples including food (n = 300), livestock (n = 50), and human clinical (n = 800) was evaluated during 2008-2016. Antimicrobial resistance patterns, virulence factors, and molecular characteristics of these isolates were analyzed using disk diffusion method, sequencing, serotyping, and pulsed-field gel electrophoresis (PFGE). The analysis of 44 L. monocytogenes isolates showed that 72.7% (32 of 44) of all the isolates belonged to Serotype 1/2c, and 15.9% (7 of 44) belonged to Serotype 3c. All 44 isolates were resistant to one or more antimicrobial agents with the most frequent resistance to penicillin (75%) and tetracycline (47.7%). Of the 44 L. monocytogenes strains, 100, 69.2, and 62.5% of livestock, human, and food strains were resistant to penicillin, respectively. Using pulsed-field gel electrophoresis (PFGE) technique, the isolates' genetic diversity was determined, and 28 PFGE patterns with 8 common (CT) and 20 single types (ST) were identified. This study highlights the high prevalence of Serotype 1/2c in clinical and livestock samples, while different serotypes were observed in food samples. The presence of rare serotypes such as 4c, belonging to the Lineage III, as well as 4e and 1/2c which are infrequent in Iran indicates that paying attention to uncommon serotypes, especially 1/2c, during the listeriosis outbreaks is necessary.

The effect of improved formulation of chitosan-alginate microcapsules of Bifidobacteria on serum lipid profiles in mice.

BACKGROUND: Probiotics have been associated with many beneficial effects in human digestive physiology. The aim of this study was to evaluate the effect of improved formulation of chitosan-alginate microcapsules of Bifidobacterium strains on serum triglycerides, cholesterol, HDL, and LDL in mice. METHODS: Five approved probiotic strains of Bifidobacterium were tested for anti-proliferative effect and interleukin-8 induction on HT-29 cell lines. Bifidobacterium strains plus five approved Lactobacillus were encapsulated in chitosan-alginate microcapsules and tested for its survival in simulated gastrointestinal conditions. These microcapsules were administered to 4 groups of mice (including 1. Bif (Bifidobacterium strains), 2. Lac (Lactobacillus strains), 3. Bif-Lac (Bifidobacterium plus Lactobacillus strains) and 4. Control) for 8 days. At eighth day, the blood of mice were taken and serum levels of triglycerides, cholesterol, HDL, and LDL of them were determined. RESULTS: All of the Bifidobacterium strains significantly (P < 0.001) reduced secretion of IL-8 in HT-29 cells as well as maximum antiproliferative effects (P < 0.001). In addition, all microcapsules showed impressive survival rate in bile (>%94.1) and gastrointestinal (>%78.28) conditions (P < 0.05). Only Bif-Lac group displayed significantly lower serum cholesterol and LDL levels than control group (P < 0.05). Besides, all groups indicate statistically significant weight loss of mice during the 8 days in comparison with the control group (P < 0.05). CONCLUSION: The results of this study showed that the microencapsulated probiotics with alginate and chitosan had an effective mean of delivery of viable bacterial cells and non-pharmacological interventions use to reduce serum cholesterol and LDL levels in in-vivo condition.

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.

Brief outcome of five decades of battle with infectious diseases in Iran.

According to WHO health profile, Iran has better situation in controlling some infection disease like leprosy, dengue fever, tularemia and hepatitis B than United States, even though Iran is in a more dangerous area than the USA. Achieving optimum control for infectious disease in the Middle East requires huge financial costs, equipment and a great time. Some of Iran's actions to control infectious diseases include: special attention of the Iran government to the health issue, training and developing human resources, membership and close cooperation with international organizations like WHO, detecting and monitoring emerging diseases before their arrival and distribution in Iran, expanding and updating national immunization and vaccination program since 1992, national project implementation titled "Health system development plan", supplying and manufacturing most of drugs required in Iran by the Iranian companies as a strategic planning, great coordination between different departments of the MOHME and other relevant institutions to Iran Army and Ministry of Intelligence to prevent the emergence of bioterrorism, and etc. We believe that Iran has obtained an acceptable score in the control of infectious diseases; but it still has very big challenges to reach the ideal level.

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.

The effects of probiotics on reducing the colorectal cancer surgery complications: A periodic review during 2007-2017.

BACKGROUND & AIMS: To assess the effects of pro-/synbiotic treatment on patients with colorectal cancer (CRC), a systematic review was conducted on randomized controlled trials. METHODS: International databanks (ISI Web of Science, PubMed, Scopus, and Google Scholar) were searched from January 2007 to December 2017 using the following keywords: 'colorectal cancer' and 'probiotics'. The search was restricted to original articles published in English. Reference lists of all related studies were also reviewed to find other relevant publications. The statistical analysis was performed using SPSS software version 18.0 (IBM, NY, USA). Also, p < .05 was regarded as statistically significant. RESULTS: A total of 21 clinical trials were retrieved, involving 1831 patients subjected to elective colorectal surgery. The studies included in this review have investigated the effects of probiotics on different aspects of colorectal cancer treatment (p < .05). According to the present study results, probiotics could significantly decrease inflammatory factors, chemotherapy side effects, severe diarrhea, postoperative infectious complications, and duration of antibiotic therapy; shift fecal microbiota in favor of Actinobacteria; and change the tumor tissue microbiota (p < .05). CONCLUSION: Based on the present review, the preoperative use of pro-/synbiotics as prophylaxis for patients with CRC could improve clinical outcomes. More detailed data about the types of probiotic species and the optimal consumption dose of pro-/synbiotics should be taken in to account in future meta-analysis reviews.

High incidence of virulence determinants, aminoglycoside and vancomycin resistance in enterococci isolated from hospitalized patients in Northwest Iran.

BACKGROUND: Multidrug resistant (MDR) enterococci are important nosocomial pathogens causing serious problem in hospitalized patients. The aim of present study was to investigate the frequency of high-level aminoglycoside-resistant and vancomycin-resistant enterococci (VRE) and virulence encoding genes in enterococci isolated from hospitalized patients. METHODS: A total of 100 enterococci isolated from urine samples of hospitalized patients with symptomatic urinary tract infections were investigated for antimicrobial susceptibility, the frequency of aminoglycoside and vancomycin resistance genes (including aac (6')-Ie-aph (2")-Ia, aph (3')-IIIa, ant (4')-Ia, aph (2")-Ic, aph (2")-Ib, aph (2")-Id, ant (3″)-III, ant (6')-Ia, vanA, vanB and vanC) and virulence encoding genes (including gelE, PAI, esp, ace, cyl, hyl and sprE). RESULTS: Enterococcus faecalis species was identified as predominant enterococci (69%), followed by "other" Enterococcus species (21%) and E. faecium (10%). Ninety three percent of isolates were resistant to one or more antimicrobial agents, with the most frequent resistance found against tetracycline (86%), ciprofloxacin (73%) and quinupristin-dalfopristin (53%). Gentamicin and streptomycin resistance were detected in 50 and 34% of isolates, respectively. The most prevalent aminoglycoside resistance genes were ant (3″)-III (78%) and aph (3')-IIIa (67%). Vancomycin resistance was detected in 21% of isolates. All E. faecium isolates carried vanA gene, whereas, the vanB gene was not detected in Enterococcus species. The most frequent virulence gene was ace (88.6%), followed by esp (67.1%), PAI (45.5%) and sprE (41.7%). CONCLUSION: Our study revealed the high frequency of gentamycin resistance and VRE in E. faecium isolates, with a high prevalence and heterogeneity of virulence and resistance genes. Due to high frequency of MDR enterococci, it seems that the appropriate surveillance and control measures are essential to prevent the emergence and transmission of these isolates in hospitals.

Therapeutic bacteria to combat cancer; current advances, challenges, and opportunities.

Successful treatment of cancer remains a challenge, due to the unique pathophysiology of solid tumors, and the predictable emergence of resistance. Traditional methods for cancer therapy including radiotherapy, chemotherapy, and immunotherapy all have their own limitations. A novel approach is bacteriotherapy, either used alone, or in combination with conventional methods, has shown a positive effect on regression of tumors and inhibition of metastasis. Bacteria-assisted tumor-targeted therapy used as therapeutic/gene/drug delivery vehicles has great promise in the treatment of tumors. The use of bacteria only, or in combination with conventional methods was found to be effective in some experimental models of cancer (tumor regression and increased survival rate). In this article, we reviewed the major advantages, challenges, and prospective directions for combinations of bacteria with conventional methods for tumor therapy.

High Rate of Serotype Switching and Genetic Variations Indicates Widespread Recombination Between Clinical and Commensal Penicillin-Nonsusceptible Streptococcus pneumoniae in Tehran.

A total of 161 Streptococcus pneumoniae were collected between 2013 and 2015 in Tehran, Iran. The strains were tested for antimicrobial susceptibility and minimum inhibitory concentrations, serotyped, and genotyped by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Penicillin-binding proteins (PBPs) were also typed by restriction fragment length polymorphism (PBP-RFLP). Out of 161 strains, 32 isolates (20%) were highly resistant to penicillin. The most frequent serotypes among the penicillin-nonsusceptible S. pneumoniae (PNSP) were 14 (24%), 23F (18%), and 19F (17%). RFLP of pbp2b, pbp2x, and pbp1a genes revealed 8, 6, and 7 different patterns, respectively. Analysis of 93 PNSP isolates displayed 80 PFGE types with 8 common types constituting 21 (23%) isolates. The remaining 72 isolates (77%) were single types. MLST indicated a high degree of genetic diversity among the 93 PNSP with 36 different sequence types. Six internationally known penicillin resistant clones were identified in our isolates among which Spain23F-1 (ST81), Spain6B-2 (ST90), and Spain9V-3 (ST156) were the predominant clones. The results indicated international identifiable clones of S. pneumoniae, especially Spain23F-1 with high penicillin resistance could play a major role in spread of antimicrobial resistance in Iran. The extensive sequence variation in PBP2x, PBP2b, and PBP1a in resistant strains of clinical and commensal S. pneumoniae was suggestive of a widespread homologous recombination within S. pneumoniae populations.

Novel vaccine candidates against Mycobacterium tuberculosis.

Tuberculosis (TB) is now among the top ten causes of mortality worldwide being resulted in 1.7 million deaths including 0.4 million among people with HIV in 2016. The Bacille Calmette-Guerin (BCG) is the only available TB vaccine which fails to provide consistent protection against pulmonary TB in adults and adolescents despite being efficacious at protecting infants and young children from the most severe, often deadly forms of TB disease. To achieve the goal of global TB elimination by 2050 we will need new interventions including more improved vaccines that are effective in adult individuals who have not been infected with Mycobacterium tuberculosis as well as latently infected or immunocompromised subjects. In recent decades, multiple new vaccine candidates including whole cell vaccines, adjuvanted proteins, and vectored subunit vaccines have entered into the clinical trials. These new TB vaccines are hoped to provide encouraging safety and immunogenicity under various conditions including prevention of TB disease in adolescents and adults, as BCG replacement/boosters, or as therapeutic vaccines to reduce the duration of TB therapy. In this review, we will discuss the status of novel TB vaccine candidates currently under development in preclinical or clinical phases.

Trends in the discovery of new drugs for Mycobacterium tuberculosis therapy with a glance at resistance.

Despite the low expensive and effective four-drug treatment regimen (isoniazid, rifampicin, pyrazinamide and ethambutol) was introduced 40 years ago, TB continues to cause considerable morbidity and mortality worldwide. In 2015, the WHO estimated a total of 10.4 million new tuberculosis (TB) cases worldwide. Currently, the increased number of multidrug-resistant (MDR-TB), extensively-drug resistant (XDR-TB) and in some recent reports, totally drug-resistant TB (TDR-TB) cases raises concerns about this disease. MDR-TB and XDR-TB have lower cure rates and higher mortality levels due to treatment problems. Novel drugs and regimens for all forms of TB have emerged in recent years. Moreover, scientific interest has recently increased in the field of host-directed therapies (HDTs) in order to identify new treatments for MDR-TB. In this review, we offer an update on the discovery of new drugs for TB therapy with a glance at molecular mechanisms leading to drug resistance in Mycobacterium tuberculosis.