Antimicrobial investigation on the multi-state outbreak of salmonellosis and shigellosis in Iran.

Background: Foodborne diseases are caused by indigestion of contaminated food. In some cases they may result in either hospitalization or death. The Centers for Disease Control (CDC) and Prevention in 2017 stated that 10% reduction in foodborne illness would prevent nearly five million illnesses every year. Approximately one out of six Americans become ill from contaminated foods or beverages every year. Another problem is drug resistance which is responsible for approximately 2 million illnesses and around 23000 dead every year. Nearly 400,000 Americans acquire antibiotic-resistant Salmonella or Campylobacter each year. The aim of this study was to evaluate the outbreak of salmonellosis and shigellosis along with their antibiotic susceptibility patterns in different provinces of Iran. Methods: Over a period of 2 years from 2015 to 2016, a total of 1055 cases in 249 outbreaks reported in 20 provinces of Iran, as a part of surveillance by the National Institute of Health (NIH). The stool samples of patients were taken and tested for Salmonella spp. and Shigella spp. by conventional standard techniques. Disk diffusion was used for the antibiotic sensitivity test. Results: Of 1055 cases, 118 (11.2%) contained Shigella and 74 (7%) contained Salmonella. Antibiotic susceptibility tests showed that entirely 100% of Salmonella and Shigella isolates were susceptible to ciprofloxacin; whereas 12.2% of Salmonella and 98.2% of Shigella were resistant to cotrimoxazole. Conclusion: Our results show that there is a need for more food handling practices to minimize the exposure of consumers to Salmonella and Shigella , at all points along the distribution chain.

Sarcocystis infection in beef and industrial raw beef burgers from butcheries and retail stores: A molecular microscopic study.

Sarcocystis is a genus of eucoccidian parasites, which globally infects humans and various animals. In addition to economic losses in livestock industries, the parasite is a zoonosis that infects humans through contaminated beef and pork with the parasite sarcocysts. Therefore, this study was carried out to assess Sarcocystis contamination in beef and industrial raw beef burger samples from butcheries and retail stores in Tehran, Iran. Overall, 180 samples of 90 beefs and 90 raw industrial beef burgers with at least 80% meat were randomly collected in Tehran, Iran. Samples were studied microscopically after peptic digestion. Furthermore, sample genomic DNAs were used in conventional polymerase chain reaction (PCR) to amplify approximately 900-bp fragments from 18S ribosomal DNA. Of 180 samples, 170 samples (94.4%) were microscopically and 161 samples (89.44%) were molecularly positive for Sarcocystis spp. Eucoccidial DNA fragments were detected in 161 samples (89.4%), including 78 (86.6%) beef and 83 (92.2%) beef burger samples. No significant differences were found between the beef and beef burger infestations by Sarcocystis bradyzoites using statistical analysis (P > 0.05). Statistically significant differences were seen between the sample type and the intensity of parasites in samples (P = 0.003). Furthermore, differences between the conventional PCR results (positive/negative) and the intensity of parasites in samples were statistically significant (P < 0.001). The considerable prevalence of Sarcocystis spp. in beef and beef burger samples reflects high transmission of the parasite in meat producing cattle, which is important due to food hygiene. Although the most prevalent bovine species, S. cruzi, is not a zoonosis, it is highly recommended to follow guidelines on the parasite transmission prevention due to the existence of S. hominis as a zoonotic bovine species.

The rK39 Antigen from an Iranian Strain of Leishmania infantum: Detection of Anti-Leishmania Antibodies in Humans and Dogs.

BACKGROUND: Visceral leishmaniasis (VL) is the most severe form of leishmaniasis in Iran with high mortality rates in the case of inaccurate diagnosis and treatment. This study aimed to prepare and evaluate a new rk39 recombinant antigen from an Iranian strain of Leishmania infantum for diagnosis of VL in humans and dogs. METHODS: rK39-based enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for the detection of anti L. infantum antibodies. We screened 84 human sera and 87 dog sera from clinical cases in the endemic area of Meshkin-Shahr, Iran along with 176 sera from healthy controls (collected from 86 humans and 90 dogs) during 2013-2016. RESULTS: Using the rK39 ELISA, a sensitivity of 85.7% (95% CI, 95-99%) and a specificity of 86.0% (95% CI, 95%-99%) were detected in human sera at a 1:800 (cut-off) titer when DAT-confirmed cases were compared with healthy controls; a sensitivity of 96.6% (95% CI, 95%-99%) and specificity of 94.4% (95% CI, 95%-99%) were found at a 1:80 (cut-off) titer compared with DAT. Kappa analysis indicated agreement between the rK39 ELISA and DAT (0.718) when using human sera at a 1:800 (cut-off) titer as well as (0.910) at a 1:80 (cut-off) titer when using dog sera (P<0.05). CONCLUSION: New rk39 recombinant antigen from an Iranian strain of Leishmania infantum seems to be used for diagnosis of VL in humans and dogs. Further extended field studies are recommended.

In Vitro Effects of Pumpkin (Cucurbita moschata) Seed Extracts on Echinococcus granulosus Protoscoleces.

BACKGROUND: Echinococcus granulosus parasite causes a zoonotic disease which is important for public and veterinary health. Since pumpkin seeds (Cucurbita sp.) are used as traditional vermifuge in Iran, they may be a potential herbal anthelmintic. METHODS: This study was designed in 2016 to evaluate the in vitro scolicidal effect of Cucurbita moschata seeds form northern part of Iran. Hydroalcoholic and petroleum ether extracts were prepared by maceration and soxhlet respectively. Both extracts with four different concentrations (100, 10, 1, 0.1 mg/ml) were incubated against protoscoleces in 5, 15, 30 and 60 min. RESULTS: Maximum mortality was 16% with 1% hydroalcoholic extract in 60 min. The highest mortality with organic extract was 4% with 10% concentration in 60 min (P=0.015). CONCLUSION: Since highest mortality was 16%, the extract did not reach to LD50 (50% mortality). Therefore, the potency of the total extract is not sufficient as potential scolicidal drug.

Investigation of the antimicrobial activity of a short cationic peptide against promastigote and amastigote forms of Leishmania major (MHRO/IR/75/ER): An in vitro study.

Cutaneous leishmaniasis is one of the most endemic global health problems in many countries all around the world. Pentavalent antimonial drugs constitute the first line of leishmaniasis treatment; however, resistance to these drugs is a serious problem. Therefore, new therapies with new modes of action are urgently needed. In the current study, we examined antimicrobial activity of CM11 hybrid peptide (WKLFKKILKVL-NH2) against promastigote and amastigote forms of L. major (MHRO/IR/75/ER). In vitro anti-leishmanial activity was identified against L. major by parasite viability and metabolic activity after exposure to different peptide concentration. In the presentt study, we demostrated that different concentrations of CM11 result in dose dependent growth inhibition of Leishmania promastigotes. Furthermore, we demostrated that CM11 peptide has significant anti-leishmanial activities on amastigotes. Our results demonstrated that CM11 antimicrobial peptide may provide an alternative therapeutic approach for L. major treatment.

Molecular analysis of integrons and antimicrobial resistance profile in Shigella spp. isolated from acute pediatric diarrhea patients.

Introduction:Shigella spp. is a growing global health concern due to increasing multiple drug resistance, commonly resulting in therapeutic failure. Integrons are gene expression systems run by integrase genes. The aims of this study were detection of class I, II and III integrons and assessment of antimicrobial resistance in Shigella spp. isolated from acute pediatric diarrhea patients. Materials and methods: From January to December 2015, 16 Shigella spp. were isolated from 310 non-duplicative diarrheal stool samples in Children's Medical Center, Tehran, Iran. The isolates were analyzed for their antibiotic susceptibility using CLSI guidelines M100-S14. Multiplex PCR was used for amplification of I, II and III integron-associated integrase (intl) genes. Results: Of 310 stool samples, 16 (5.2%) were positive for Shigella spp., in 7 of them S. sonnei and in 9 of them S. flexneri were identified. Results of the antimicrobial susceptibility test showed that 6.2%, 50%, 31.2%, 6.2%, 81.2%, 56.2% and 31.2% of the isolates were resistant to gentamicin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, ampicillin and trimethoprim-sulfamethoxazole, respectively. Multiplex PCR results revealed that 6.2% (1/16), 31.2% (5/16), 50% (8/16) of Shigella isolates carried intlI, intlII and both intlI/intllI genes. No class 3 integrons were detected. Discussion: In this study, multidrug resistance was seen in Shigella isolates similar to that in isolates from other geographical areas. This is possible due to inappropriate use of antimicrobials. Furthermore, prevalence of multidrug resistance was significantly linked to the presence of integrin genes. Conclusion: A class 2 integron plays a role in presence of multidrug resistance in Shigella spp. It is vital to prevent the spread of antibiotic resistance through continuous monitoring.

Frequency of Mutations in Quinolone Resistance-Determining Regions and Plasmid-Mediated Quinolone Resistance in Shigella Isolates Recovered from Pediatric Patients in Tehran, Iran: An Overlooked Problem.

Fluoroquinolone (FQ) resistance in clinical isolates of Shigella species has been increasing reported in recent years. This study was carried out to find the mutations within the quinolone resistance-determining regions (QRDRs) and the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants among the clinical isolates of Shigella sp. in Tehran, Iran. A total of 50 Shigella isolates were collected from five teaching therapeutic centers in Tehran, Iran and analyzed for antibiotic susceptibility over a period of 20 months from July 2015 to January 2017. The PCR and direct nucleotide sequencing were used for genetic alterations in the QRDRs. The PMQR genes were detected using PCR. The results revealed four types of mutations in the QRDR of gyrA: 20 (40%) had a S83L mutation, 1 (2%) had a S83A mutation, 2 (4%) had a D87G mutation, and 1 (2%) isolate had a D87Y mutation. Mutations were also found at codon N57D, D200N, and E210K in three isolates. Seven hospitalized children had qnrS determinants, and one isolates had the mutation S83A, while two isolates had double mutations at S83L and/or D87G (Ser83Leu and Asp-87Gly). The PMQR gene-positive isolates had the single replacement of serine with leucine. In hospitalized children, two isolates had two types of PMQR determinants (qnrS and qnrA) and (qnrS and qnrB) at once. The results of this study indicate that the emergence of strains with mutations in the QRDR regions and the capture of PMQR determinants in strains may lead to failure in therapy with FQ and the widespread emergence of strains with high-level FQ resistance.

Antimicrobial activity of an antimicrobial peptide against amastigote forms of Leishmania major.

Zoonotic cutaneous leishmaniasis caused by Leishmania major is a most common type of vector-borne disease in Iran. The pentavalent antimonial drugs have been used in the treatment of cutaneous leishmaniasis for a long time, but drug resistance and some of serious side effects have been reported. Thus, discovery and development of new therapeutic candidates are needed. The CM11 peptide is one of these peptides that its anti-bacterial activity has been proven. This peptide is a short cecropin-melittin hybrid peptide obtained through a sequence combination approach. The aim of this study was to evaluate in vitro anti-leishmanial activity of CM11 peptide against amastigote forms of Leishmania major. In this study, amastigote forms of Iranian strain of L. major (MRHO/IR/75/ER) were cultured in the presence of different concentrations of meglumine antimoniate (Glucantime®) to find the most appropriate in vitro concentration of Glucantime® against L. major amastigotes. Then, the anti-leishmanial activities of various concentrations of CM11 peptide (8, 16, 32 and 64 µM) were evaluated for 24, 48 and 72 hr by DAPI staining. In addition, MTT assay was used to determine the cytotoxic effects of CM11 peptide on murine fibroblast cell line. The results showed that CM11 peptide has antimicrobial activity against Iranian isolate of L. major in the laboratory conditions. It seems that the CM11 peptide has significant potential to be used as a new anti-leishmanial agent.

Cloning of K26 Hydrophilic Antigen from Iranian Strain of Leishmania infantum.

BACKGROUND: Visceral leishmaniasis (VL) caused by Leishmania infantum is the most severe form of leishmaniasis in Iran, which causes a high mortality rate in the case of inaccurate diagnosis and treatment. This study aimed to clone of K26 gene from Iranian strain of L. infantum and register the sequencing results in Genbank to facilitate the preparation a new K26 antigen for the detection of L. infantum infection. METHODS: L. infantum was obtained from an infected domestic dog in Meshkin-Shahr area from northwestern Iran in 2015. Canine visceral leishmaniasis was confirmed by direct agglutination test (DAT), rK39 dipstick and parasitological methods. L. infantum was confirmed by N-acetyl glucosamine -1-phosphate transferase (nagt)-PCR and its sequencing. The band of interest for k26 form Iranian strain of L. infantum was purified by gel extraction kit after PCR amplification and then ligated into pBluescript II SK (+) and pET-32a (+), respectively. The sequences of recombinant plasmids were analyzed and submitted to Genbank. RESULTS: The submission of rk26 nucleotide sequence was performed to the GeneBank/NCBI Data Base under accession number KY212883. The related gene was showed a homology about 99% to L. chagasi and L. infantum k26 gene, while the level of homology in comparison with different strains of L. donovani ranged from 84-94%. CONCLUSION: The successful rk26 cloning into an expression vector performed in this study could help to produce a new recombinant antigen for serodiagnosis of VL especially in areas where L. infantum is the main causative agent.

Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria.

Lactobacilli are normal microflora of the gastrointestinal (GI) tract and are a heterogeneous group of lactic acid bacteria (LAB). Lactobacillus strains with Probiotic activity may have health Benefits for human. This study investigates the probiotic potential of Lactobacillus strains obtained from the feces of healthy infants and also explores antibacterial activity of Lactobacillus strains with probiotic potential against enteropathogenic bacteria. Fecal samples were collected from 95 healthy infants younger than 18 months. Two hundred and ninety Lactobacillus strains were isolated and assessed for probiotic potential properties including ability to survive in gastrointestinal conditions (pH 2.0, 0.3% oxgall), adherence to HT-29 cells and antibiotic resistance. Six strains including Lactobacillus fermentum (4 strains), Lactobacillus paracasei and Lactobacillus plantarum showed good probiotic potential and inhibited the growth of enteropathogenic bacteria including ETEC H10407, Shigella flexneri ATCC 12022, Shigella sonnei ATCC 9290, Salmonella enteritidis H7 and Yersinia enterocolitica ATCC 23715. These Lactobacillus strains with probiotic potential may be useful for prevention or treatment of diarrhea, but further in vitro and in vivo studies on these strains are still required.

Prevalence of Salmonella spp. in Packed and Unpacked Red Meat and Chicken in South of Tehran.

BACKGROUND: Despite of the advances in infectious diseases prevention and food technology, food-borne diseases are considered major problems in developed and developing countries. Meat plays a key role in transferring zoonotic diseases to human. OBJECTIVES: This study was conducted in south of Tehran, Iran, to investigate the prevalence rate of Salmonella spp. in packed and unpacked red meat and chicken. MATERIALS AND METHODS: A total of 379 packed and unpacked samples including 189 red meat and 190 chicken samples were collected randomly. From each sample, 25 g was separated and treated with 225 mL of buffered peptone water, homogenized and incubated at 37°C for 24 hours. Samples were enriched using Rappaport-Vassiliadis broth and then streaked onto Hektoen enteric agar. RESULTS: Totally, 86 out of 190 chicken and 38 out of 189 red meat samples were contaminated with Salmonella spp. The most isolated serotypes were Salmonella thompson (67.7%), S. heaardt (6.5%), S. enteritidis (4.8%), and S. veyle (4%), respectively. In general, the rate of chicken contamination was higher than meat, as 43.3% of packed and 46% of unpacked chicken samples were contaminated. CONCLUSIONS: These results confirmed the pervious findings, stating that proper packaging of meat products can effectively decreases the rate of microbial contaminations.

Efficacy of immunization with outer membrane proteins for induction of pulmonary clearance of nontypeable Haemophilus influenzae in a rat respiratory model.

Three strains of nontypeable Haemophilus influenzae namely NTHi-I, NTHi-II and NTHi-III were isolated from the sputum of patients with bronchitis and identified by biochemical, serological and electron microscopy. The polypeptide patterns of isolates were compared and found to have similar sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) polypeptide patterns, although some of the bands were specific in some strains. A similar comparison was made on extracted outer membrane proteins (OMPs) on the above mentioned strains, using Triton X-100 and sodium dodecyle sulphate (SDS). It was found that the polypeptides with molecular weights of 70, 42, 33 and 27 KDa were identified as P1, P2, P4 and P5 respectively. The protein estimation of crude OMPs from the three strains were calculated, and OPM-I prepared from NTHi-I showed the highest amount of protein and was chosen for its immunogenicity in a rat respiratory model. The efficacy of immunization with OMP was determined by enhancement of pulmonary clearance of live bacteria in the rat lung. A significant protective immune response induced by OMP was observed by enhanced respiratory clearance of nontypeable H. influenzae following mucosal immunization.

Protective Immune Responses Induced in Chickens by Outer Membrane Proteins Extracted from Different Strains of Escherichia coli.

Different strains of Escherichia coli (E. coli) from human, chickens, and the common strain between human and chickens were isolated and typed with mono-specific antibody. The E. coli strains from each group of human, chicken and common between human and chicken were selected. The polypeptide patterns of selected strains were analyzed and compared with each other by Sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS - PAGE). The SDS - polyacrylamide gel electrophoresis patterns between the strains were very similar, although the densities of some of the peptide bands were different, and some were missing in some strains. A similar comparison was made on extracted outer membrane proteins (OMP) using Triton X-100 and sodium dodecyle sulphate on the above-mentioned strains. The polypeptide patterns between the strains 078 (chicken strain), 06 (human strain) and O2 (the common strain between human and chicken) were also very similar and two major bands with molecular weights of 44 KD and 25 KD were very distinctive, and seen between all the strains.The passive haemoagglutination tests (PHA) in chickens injected OMP from the common strain (O2), showed increased level of antibody after the second injection which remained constant after repeated injections.In the challenge study, with LD100 from homologous strain of O2, a significant protection was observed in the groups injected the OMP (extracted from O2 strain), compared with controls injected saline.The results of this study indicate that purified E. coli outer membrane protein, can induce a significant protection immunity against colibacillus diseases in chickens, and probably in immunocompromised hosts with repeated urinary infections.