Isolation and characterization of a multidrug-resistant Clostridioides difficile toxinotype V from municipal wastewater treatment plant.

PURPOSE: Wastewater treatment plant (WWTP) is regarded as a potential source for transmission of Clostridioides difficile from urban areas into the surface water, through feces of human and animals. The aim of this study was to screen and characterize the C. difficile bacteria in inlet and outlet wastewater of different WWTPs in Tehran, Iran. METHODS: Totally, 72 samples were collected from three different WWTPs (inlet site and outlet sites) during a year. C. difficile was isolated and characterized in terms of toxins, toxinotype, resistance profile and genes, and colonization factors using PCR. RESULTS: One C. difficile toxinotype V was isolated from the outlet samples. The isolate was susceptible to vancomycin but resistant to metronidazole, tetracycline, ciprofloxacin, and moxifloxacin using MIC Test Strips. The isolated C. difficile was toxigenic (tcdA, tcdB, cdtA, cdtB positive and CPE positive) and had tcdC-A genotype. No mutations were found in fliC and fliD. The slpA sequence type was 078 - 01. The C. difficile was positive for tetM, int, but negative for vanA, nim, and tndX genes. Mutations were not observed in gyrA and gyrB genes. CONCLUSIONS: This study provided evidence of presence of a multidrug-resistant C. difficile toxinotype V in one of the municipal WWTP. The transmission of such isolate to the environment and reuse of treated wastewater by human pose a threat to human health and dissemination of antibiotic resistant bacteria which are untreatable.

High prevalence of Clostridiodes diffiicle PCR ribotypes 001 and 126 in Iran.

Clostridium difficile is a leading causative agent of hospital-acquired and community-acquired diarrhea in human. This study aims to characterize the predominant C. difficile strains, RT001 and 126, circulating in Iranian hospitals in relation to resistant phenotypes, the antibiotic resistance genes, and their genetic relatedness. A total number of 735 faecal specimens were collected from patients suspected of CDI in Tehran hospitals. Typing and subtyping of the strains were performed using CE-PCR ribotyping and MLVA, respectively, followed by PCR assays for ARGs and indicators of Tns. Minimum inhibitory concentrations (MICs) of five antibiotics were determined by MIC Test Strips. Among 65 strains recovered from CDI patients, RT001 (32.3%) and RT126 (9.2%) were found as the most frequent ribotypes, and 64 MLVA types were identified. Using MLVA, RT001 and RT126 were subtyped into 6 and 4 groups, respectively. The vanA, nim, tetM, gyrA, gyrB genes were detected in 24.6%, 0%, 89.2%, 95.3%, and 92.3% of the strains, respectively. The indicators of Tns including vanHAX, tndX, and int were found in 0%, 3% and 29.2% of the strains, respectively. The most common amino acid (AA) alterations of GyrA and GyrB were related to substitutions of Thr82 → Val and Ser366 → Val, respectively. Resistance rate to metronidazole, vancomycin, tetracycline, ciprofloxacin, and moxifloxacin was 81.5%, 30.7%, 85%, 79%, and 74%, respectively. This study, for the first time revealed the subtypes of circulating RT001 and RT126 in Iran. It is of importance that the majority of the strains belonging to RT001 were multidrug resistant (MDR). This study also pointed to the intra-hospital dissemination of the strains belonging to RT001 and RT126 for short and long periods, respectively, using MLVA. The most important resistance phenotypes observed in this study was vancomycin-resistant phenotypes. Resistance to metronidazole was also high and highlights the need to determine its resistance mechanisms in the future studies.

First Detection and Characterization of Macrolide-Resistant Mycoplasma pneumoniae from People with Community-Acquired Pneumonia in Iran.

Objectives: Increasing macrolide resistance of Mycoplasma pneumoniae strains is becoming a public health concern worldwide. Nevertheless, no comprehensive genomic background of circulating isolates is available in our region. We aimed to study the genetic diversity of this microorganism using the multiple-locus variable-number tandem-repeat analysis method and to investigate the relationships between MLVA types and macrolide susceptibility profiles of the isolates. Materials and Methods: A total of 270 patients attending Tehran general university hospitals were included in this study. One throat swab was taken from each patient. M. pneumoniae was identified using culture and PCR assay. Macrolide resistance was determined using the broth microdilution method. The MLVA was performed by amplification of four variable-number tandem-repeat loci. Results: Of 270 specimens, M. pneumoniae was detected in 25.2% (n = 68) and 21.8% (n = 59) samples using PCR and culture, respectively. Approximately 56.9% of isolates were resistant to macrolides. Fifty-one of 59 M. pneumoniae isolates were divided into 6 distinct MLVA types. Conclusion: The macrolide-resistant M. pneumoniae (MRMP) rate in this study was relatively high and most of the MRMP isolates were assigned into the type 4/5/7/2. Since a significant association between MLVA type 4/5/7/2 and macrolide resistance of M. pneumoniae isolates was observed, further monitoring of genetic diversity of MRMP isolates might facilitate better understanding of epidemiology of this microorganism. Besides surveillance of the antibiotic susceptibility might be helpful to make necessary reconsiderations on guidelines for treatment of M. pneumoniae infection.

Characterization of Clostridioides difficile isolates recovered from hospitalized patients and the hospitals environment and air: A multicenter study.

In healthcare settings, contamination of environment with toxigenic and hypervirulent Clostridioides difficile strains is a serious concern. Here, we assessed whether patients with C. difficile have a role to play in the dissemination of C. difficile in our settings or other sources are implicated in its circulation. A total of 700 fecal specimens and 1435 environmental samples from surfaces, equipment and air of rooms occupied by patients suspected of C. difficile infection were taken from 4 tertiary hospitals in Tehran, Iran between April 2016 and August 2017. Antibiotic susceptibility testing and detection of resistance genes were performed for the environmental isolates. The clinical and environmental isolates of C. difficile were subjected to Pulsed Field Gel Electrophoresis (PFGE) analysis. Forty three (6.14%) and 2 (0.13%) isolates of C. difficile were recovered from the clinical and environmental samples, respectively. In the clinical settings, 2 patients were suspected of recurrent C. difficile infection. Thirty distinct pulsotypes were found among the C. difficile isolates including 28 singletons and 2 common types. One of the two environmental isolates was isolated from floor in the Medical ward, of pulsotype/ribotype/toxinotype PT10/New ribotype/toxinotype V, harbored cdtA/B and tcdC-A, and resistant to ciprofloxacin. The other one was isolated from air of a room in ICU, assigned to PT11/RT001/toxinotype 0, belonged to tcdC-sc3 genotypes and resistant to metronidazole. The environmental isolates did not generate amplicons in PCR assays targeting vanA and nim genes. This study provided evidence for dissemination of genetically diverse strains of C. difficile in hospitalized patients, presence of C. difficile in hospital air, existence of binary toxin positive/antibiotic-resistant isolate on the floor and intra-hospital dissemination of this pathogen.

Tetracycline and ciprofloxacin multiresidues in beef and chicken meat samples using indirect competitive ELISA.

In livestock and poultry, broad-spectrum antibiotics such as tetracyclines and fluoroquinolones are widely used; thus, controlling food productions made from these animals is a necessary task. Meat may contain residues of antibiotics, even in low concentrations, which can cause a selection pressure for antibiotic resistance. Therefore, measurement of amounts of antibiotics in meat is of major importance. A total of 41 beef and 41 chicken meat samples were collected for 1 year. Tetracycline and ciprofloxacin were extracted from samples and tested by an indirect competitive enzyme-linked immunosorbent assay. Overall, 100% of the beef and more than 95% of the chicken meat samples were positive for ciprofloxacin. Only one of the chicken meat samples had concentrations of ciprofloxacin higher than maximum residue limit (MRL). For ciprofloxacin, none of the beef meat samples exceeded the MRL. For tetracycline, 75% of the beef and 58% of the chicken meat samples were positive. All of the samples had concentrations of tetracycline lower than MRL. It was revealed that the chicken meat samples had higher levels of both antibiotics than those of beef samples. The amounts of tested antibiotics were not high in the meat samples, consequently using of beef and chicken meat by consumers in Iran is not resulted in entrance of high amounts of the antibiotics into human body.

Comparison of the prevalence of enteroviruses in blood samples of patients with and without unstable angina.

BACKGROUND: Although the role of enteroviruses has been proved in heart diseases, extensive information is not available on the association between enteroviruses and unstable angina. In the present study, the authors compared the prevalence of enteroviruses in patients with and without unstable angina. METHODS: Blood samples were taken from 51 patients with unstable angina and 55 patients without unstable angina or myocardial infarction that were admitted to Imam Reza and Ghaem hospitals (Mashhad, northeast of Iran). Reverse transcription polymerase chain reaction (RT-PCR) was performed using specific primers for the detection of the enteroviruses in blood samples of study subjects. RESULTS: Patients with and without unstable angina were similar in age with mean ± standard deviation of 62.6 ± 12.8 and 59.7 ± 12.7 years, respectively (P = 0.243) and there were no differences in gender in these two groups (P = 0.174). Prevalence of the enteroviruses in patients with unstable angina was higher only in 66-80 years age group compared to the control group (patients without unstable angina, P = 0.032). There was a higher prevalence of enterovirus RNA positivity in the blood samples of women with unstable angina (75.9%) than those without unstable angina (41.7%, P = 0.011), however, no significant difference was observed in men (P = 0.983). CONCLUSION: Our data showed that enteroviral RNA positivity was higher in patients with unstable angina compared to those without unstable angina. However, the differences between the two groups were not statistically significant.

Designing and Construction Pcdna3.1 Vector Encoding Cfp10 Gene of Mycobacterium tuberculosis.

BACKGROUND: Pathogenic mycobacteria are a major cause of human morbidity and mortality. Mycobacterium tuberculosis is an etiological agent of human tuberculosis (TB). Designing new vaccines, including DNA vaccines, may be a useful strategy for preventing TB. OBJECTIVES: The purpose of this study was to design and construct an eukaryotic expression vector containing M. tuberculosis. MATERIALS AND METHODS: Genomic DNA of M. tuberculosis H37Rv cultured on Lowenstein Jensen medium was extracted, and cfp10 was amplified by PCR. After digesting the PCR product and the plasmid, the cfp10 fragment was ligated into the vector pcDNA3.1 (+). Correct insertion was confirmed by colony PCR, restriction enzyme digestion, and sequencing. RESULTS: Electrophoresis of the PCR product on gel showed a 303-bp target fragment. Colony PCR, restriction enzyme digestion, and Sequencing methods confirmed the accuracy of the gene cloning. Colony PCR and restriction enzyme digestion confirmed the cloning. CONCLUSIONS: Cloning of cfp10 of M. tuberculosis into an eukaryotic expression vector was performed successfully. We propose this recombinant plasmid for inducing immunity in animal models in future studies. This recombinant vector can also be used in the construction of fusion proteins.