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Background and Objectives: Of the genes conferring resistance to carbapenems in Acinetobacter baumannii, the bla OXA-23 gene is the most widely found across the world. The gene carrying bla OXA-23 transposons in A. baumannii isolates of global clones GC1 and GC2 is found worldwide. Here, we examined whether transposons play a role in the dissemination of the bla OXA-23 in globally distributed clones, GC1 and GC2 A. baumannii isolates from Iraq., Materials and Methods: The 119 non-repetitive A. baumannii isolates including 94 recovered from clinical specimens and 25 isolates from hospital environment between September 2021 and April 2022 from different medical centers located at various regions in Baghdad, Iraq. The global clones (GC) and the genes encoding carbapenem resistance, including bla OXA-23 , bla OXA-24 , and bla OXA-58 were identified using multiplex PCR assays. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion susceptibility method. The transposons carrying bla OXA-23 were examined using PCR mapping. In cases when carbapenem susceptible A. baumannii isolates were found, they were subjected to E test, full length sequencing of bla OXA-Ab (bla OXA-51-like ) and Institut Pasteur multi-locus sequence typing scheme., Results: All but two isolates (92 clinical and 25 environmental) were identified carbapenem-resistant A. baumannii (CRAB). Of 117 CRAB isolates, 20 belong to GC1, 19 contained bla OXA-23 ; of them, 17 isolates harbored the bla OXA-23 located on Tn2006. Among the 46 CRAB belonging to GC2, 39 contained bla OXA-23 ; of them, 34 carried the bla OXA-23 located on Tn2006. The remaining GC1 and GC2 isolates, one GC1 as well as one GC2 isolate, were susceptible to imipenem, doripenem, and meropenem and considered carbapenem-susceptible A. baumannii (CSAB). Full-length sequencing of the bla OXA-Ab and MLST for the two CSAB isolates belonging to GC1 and GC2 confirmed that the GC1 isolate belongs to ST 623 and contained an allele that encodes an bla OXA-69 variant of the bla OXA-Ab while the GC2 belong to ST2 and carried an bla OXA-66 variant., Conclusion: This study provides evidence for the dissemination of bla OXA-23 on the Tn2006 in CRAB isolates in Baghdad, Iraq. It appears that this transposon is widespread in GC1 and 2 isolates as in the other parts of the world. Interestingly, one GC1 and one GC2 isolate from Iraq were found to be susceptible to carbapenem while the isolates belonging to GC1 and GC2 have so far rarely been found to be susceptible to carbapenem globally., (© 2024. The Author(s).)

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background & Objective: Cerebrospinal fluid (CSF) analysis is helpful in the diagnosis of infections of the central nervous system (CNS), especially after neurosurgical procedures. This study aimed to evaluate the diagnostic value of CSF markers for diagnosis of post-neurosurgical meningitis (PNM)., Methods: Patients with neurosurgical procedures whose CSF was obtained for any reason (meningitis and non-meningitis) during 2020 and 2022, at Imam Khomeini Hospital Complex, Tehran, Iran, were included. Serum and CSF lactate dehydrogenase (LDH), glucose, protein, white blood cells (WBC), red blood cells (RBC), and CSF/serum glucose and LDH ratio were compared between the patients who were diagnosed with PNM and those without meningitis., Results: A total of 115 patients were included, of whom 23 patients were diagnosed with PNM and 92 with non-meningitis. No significant differences were observed in patients' age, gender, and underlying diseases between the two groups. Findings showed a significantly ( P =0.029) lower level of the mean CSF glucose (59.5 mg/dL ±33.9) in patients with meningitis than in patients without meningitis (76.8 mg/dL ± 37.5). The mean CSF/serum glucose ratio was 43.7% in the meningitis group and 56.3% in the non-meningitis group ( P =0.008). The mean WBC count and neutrophil dominance were significantly higher in the meningitis group. No significant differences were observed in CSF LDH, Protein, and RBC between the two groups., Conclusion: A CSF glucose level of less than 60 mg/dL, a CSF/serum glucose ratio of less than 0.44, and a higher CSF WBC and neutrophil count can help diagnose PNM., Competing Interests: The authors report there are no competing interests to declare., (© 2024.)

Purpose: Prior studies have demonstrated that both dietary components and bariatric surgery modify the gut microbiota's composition. However, there is a scarcity of research that has examined the relationship between post-surgical dietary intake and changes in the gut microbiota. The aim of this study was to assess changes in gut microbiota following bariatric surgery and examine their association with postoperative dietary intake., Materials and Methods: The present study involved a sample of 42 adult women who were potential candidates for bariatric surgery, i.e., laparoscopic Roux-en-Y gastric bypass (LRYGB) or sleeve gastrectomy (LSG). The assessment of dietary intake was conducted through the use of three-day food records, both at baseline and six months following the surgical procedure. The gut microbiota was determined through the detection of 16S ribosomal RNA (16S rRNA) gene sequencing., Results: After six months, a significant increase in abundance of Firmicutes (P = 0.01), Bifidobacterium (P = 0.01), and Ruminococcus (P = 0.04) in the LSG group was found. In contrast to the observed rise in Enterobacteria (P = 0.02) levels in the LRYGB group, no significant changes were detected in the composition of other gut microbiota over the 6-month monitoring period subsequent to LRYGB. The results of our study indicate that there is not a statistically significant relationship between dietary consumption and changes in the composition of the gut microbiota in individuals who have undergone LRYGB and LSG., Conclusion: Our findings suggest that there may not be a significant correlation between dietary intake following LRYGB and LSG, and the observed alterations in the gut microbiota during a six-month period of observation. Nevertheless, it is important to acknowledge that the sample size utilized in our study was limited, potentially leading to reduced statistical power and the possibility of yielding findings that do not accurately reflect reality., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acinetobacter baumannii strains belonging to global clone 2 (GC2) contain resistance islands (AbGRIs), which are composed of genes conferring resistance to older and newer antibiotics. Here, to locate these genes in AbGRIs, the GC2 strains from Tehran, Iran were examined. Among the 170 A . baumannii , 90 isolates were identified as GC2. Of the genes that confer resistance to older antibiotics, tetA(B ), tetR(B) (tetracyclines), strA , and strB (aminoglycosides) were located in AbGRI1 of 65 GC2 isolates (72.2%). Of the other aminoglycosides, the aphA1b was located in AbGRI2-12b (63.6%), AbGRI2-12a (21.2%), or AbGRI2-1 (15.1%). The aacC1 and aadA1 genes were co-located within AbGRI2-1 (5.5%). The armA was located in AbGRI3-4 (77.7%) and AbGRI3 ABI221 (22.2%). Of sulfonamides, the sul1 was located within AbGRI2-1 (5.5%). Of beta-lactams, the bla TEM was located in AbGRI2-12b (42%), AbGRI2-12a (14%), AbGRI2-1 (10%), or AbGRI2 ABI257 (34%). The oxa23 gene conferring resistance to newer antibiotics (carbapenems) was located in AbaR4 (81.1%); of them, the AbaR4 was located within AbGRI1 in 45.2% of the isolates. This study showed that the GC2 isolates, which contained at least one AbGRI, disseminate in the hospital. Hence, it is likely that the AbGRIs play a significant role in conferring resistance to older and newer antibiotics in GC2 isolates from Iran. IMPORTANCE The majority of Acinetobacter baumannii isolates that are resistant to multiple antibiotics belong to one of the two major global clones, namely global clone 1 (GC1) and global clone 2 (GC2). The resistance islands, which contain variable assortments of transposons, integrons, and specific resistance genes, have been characterized in the genome of these GCs. In GC2 A. baumannii, the chromosomally located A. baumannii genomic resistance islands (AbGRIs) carry the genes conferring resistance to older and newer antibiotics. In this context, we tested whether GC2 isolates collected from a referral hospital carry the AbGRIs containing these genes. This study provided evidence for the circulation of the GC2 A. baumannii strains harboring AbGRI resistance islands between different wards of a referral hospital.

Aim: The Acinetobacter baumannii genomic resistance islands (AbGRIs), which were characterized in the genome of the global clone 2 (GC2) A. baumannii contain resistance genes. Here, we aimed to determine the occurrence of AbGRIs in GC2 A. baumannii obtained from COVID-19 patients in a referral hospital in Tehran, Iran., Methods: A total of 19 carbapenem-resistant A. baumannii (CRAB) isolates belonging to GC2 and sequence type 2 (ST2), including 17 from COVID-19 patients and two from the devices used in the ICU that the COVID-19 patients were admitted, were examined in this study. Antibiotic susceptibility testing was performed by the disk diffusion method. PCR and PCR mapping, followed by sequencing, were performed to characterize the structure of AbGRI resistance islands in the isolates tested., Results: The AbGRI3 was the most frequent resistance island (RI) detected, present in all the 19 isolates, followed by AbGRI1 (15 isolates; 78.9%) and AbGRI2 (three isolates; 15.8%). Notably, AbGRIs were identified in one of the A. baumannii strains, which was isolated from a medical device used in the ICU where COVID-19 patients were admitted. Furthermore, new structures of AbGRI1 and AbGRI3 resistance islands were found in this study, which was the first report of these structures., Conclusions: The present study provided evidence for the circulation of the GC2 A. baumannii strains harboring AbGRI resistance islands in a referral hospital in Tehran, Iran. It was found that resistance to several classes of antibiotics in the isolates collected from COVID-19 patients is associated with the resistance genes located within AbGRIs., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Pertussis, or whooping cough, is a highly contagious respiratory disease caused by Bordetella pertussis (BP). Pertactin (PRN) is one of the main immunogenic components of BP and is employed in many commercialized acellular pertussis vaccines (aPVs). Purification of this protein by conventional chromatography methods is challenging and commonly requires multiple laborious processes with low recovery. Using specific monoclonal antibodies (mAbs) for the purification of PRN antigen is expected to yield high purity and recovery of the target molecule., Methods: Recombinant PRN antigen was used to produce mouse mAbs using hybridoma technology. Structural and functional characteristics of the mAbs were assessed by ELISA, immunoblotting, and flow cytometry. Selected mAbs were employed to purify PRN by affinity chromatography, and the purity and recovery of the purified protein were analyzed by ELISA, SDS-PAGE, and immunoblotting. Moreover, ELISA and flow cytometry techniques were designed using these mAbs to detect PRN in different strains of BP., Results: Five mAbs were produced and selected based on their reactivity with native PRN. Our results demonstrate that purification of PRN by affinity chromatography resulted in a highly pure antigen with 75-85 percent recovery. In addition, ELISA and flow cytometry results indicated that these mAbs could recognize PRN in the bacterial cell walls of different BP strains., Conclusion: We successfully produced PRN-specific mAbs and designed an affinity chromatography method to purify PRN antigen with higher purity and recovery than conventional methods. These mAbs could be employed as valuable tools for the detection and purification of PRN for vaccine manufacturing., Competing Interests: Declaration of Competing Interest The authors reported no declarations of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Immigration is considered as a risk factor of tuberculosis (TB). Qom province receives millions of pilgrims and significant numbers of immigrants each year. Most of the immigrants to Qom, arrive from neighboring TB-endemic countries. This study aimed to identify the current circulating Mycobacterium tuberculosis genotypes in Qom province using 24-locus MIRU-VNTR genotyping., Methods: Eighty six M. tuberculosis isolates were collected during 2018-2022 from patients referring to Qom TB reference laboratory. The DNA of isolates was extracted and followed by 24 loci MIRU-VNTR genotyping which performed using the web tools available on MIRU-VNTRplus., Results: Of 86 isolates, 39 (45.3%) were of Delhi/CAS genotype, 24 (27.9%) of NEW-1, 6 (7%) of LAM, 6 (7%) of Beijing, 2 (2.3%) of UgandaII, 2 (2.3%) of EAI, 1 of S (1.2%) and 6 (7%) did not match with profiles present in MIRUVNTRplus database., Conclusions: About half of the isolates belong to Afghan immigrants; which warns health policy makers about the future situation of TB in Qom. Also, the similarity of Afghan and Iranian genotypes provides evidence that immigrants partake in the circulation of M. tuberculosis. This study underpin the studies about the circulating M. tuberculosis genotypes, their geographical distribution, the association of TB risk factors with these genotypes and the impact of immigration on the situation of TB in Qom province., (© 2023. The Author(s).)

Aminoglycosides are used to treat infections caused by carbapenem-resistant Acinetobacter baumannii (CRAB) strains. However, resistance to aminoglycosides has increased remarkably in the last few years. Here, we aimed to determine the mobile genetic elements (MGEs) associated with resistance to aminoglycosides in the global clone 2 (GC2) A. baumannii . Among the 315 A. baumannii isolates, 97 isolates were identified as GC2, and 52 of GC2 isolates (53.6%) were resistant to all the aminoglycosides tested. The AbGRI3s carrying armA were detected in 88 GC2 isolates (90.7%), and of them, 17 isolates (19.3%) carried a new variant of AbGRI3 (AbGRI3 ABI221 ). aphA6 was located in Tn aphA6 of 30 isolates out of 55 aphA6 -harboring isolates, and 20 isolates were found to harbor Tn aphA6 on a RepAci6 plasmid. Tn 6020 carrying aphA1b was detected in 51 isolates (52.5%), which was located within AbGRI2 resistance islands. The pRAY * carrying the aadB gene was detected in 43 isolates (44.3%), and no isolate was found to contain a class 1 integron harboring this gene. The GC2 A. baumannii isolates contained at least one MGE carrying the aminoglycoside resistance gene, located mostly either in the chromosome within AbGRIs or on the plasmids. Thus, it is likely that these MGEs play a role in the dissemination of aminoglycoside resistance genes in GC2 isolates from Iran., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Naderi, Talebi, Gheybizadeh, Seifi, Ghourchian, Rahbar, Abdollahi, Naseri, Eslami and Douraghi.)

Since the introduction of the Bacillus Calmette-Guérin (BCG) vaccine, the genomes of vaccine strains have undergone variations due to repeated passages in different laboratories and vaccine production facilities. Genetic variations have been considered as one of the effective factors in the BCG variable protective efficacy. Consecutive subcultures have been shown to play an essential role in causing genetic variations in several microorganisms, including Mycobacterium bovis BCG. Therefore, the world health organization (WHO) recommendation to limit the passages of master seed lot in the BCG vaccine production should be considered. Besides, the role of other external variables such as quality of the raw ingredients of the culture media, the type of the culture medium and the cultivation methods in the vaccine production has been poorly studied. Here, the effect of passages and culture medium on genetic variations in a BCG seed lot was investigated during a year. The findings of this study revealed a total of 19 variants compared to seed lot while the passages were more than the number recommended by WHO. The first culture of seed lot in the Sauton broth and Middlebrook 7H9 media, and the last subculture in Sauton broth had the least and the most variants, respectively. The observation of the higher number of variants in the last cultures on Sauton broth and Middlebrook 7H9 in comparison to the first and the middle cultures may indicate the effect of passages on the genetic variations in BCG. Additionally, more variants in BCG grown in the Sauton broth do not necessarily represent the greater ability of this medium to cause genetic mutations. For a better conclusion, it is required to examine the medium components as independent variables., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Asadian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immunotoxins are regarded as a type of targeted therapy for killing cells by highly potent bacterial, fungal or plant toxins. Shiga like toxins (SLTs) are a group of bacterial AB5 protein toxins that inhibit host cell protein synthesis through the removal of a single adenine residue from the 28S rRNA and lead to apoptosis. Here, we described the design and usage of a Stx-based immunotoxin that can induce the selective cytotoxicity and apoptosis in Fn-14-positive cells related to the colon and lung cancer. In the present study, the Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system when driven from inclusion bodies by 8 M urea. The Stx2a-PE15-P4A8 fusion protein was expressed efficiently in E. coli (DE3) system and then purified. The purified fusion protein could specifically target Fn-14 receptor existed on colon and lung cancer cell lines and suppress these cells in a dose-dependent manner. In addition, the protein was able to nearly 50 % of apoptotic cell death and maintains about 54 % of its stability after 24 h of incubation in mouse serum at 37 °C. Compared to PE38-P4A8 construct in our previous study, these results showed that the Stx2a-PE15-P4A8 construct can be an efficient therapeutic candidate for cancer immunotherapy., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Bacillus Calmette-Guérin (BCG) refers to a group of vaccine strains with unique genetic characteristics. BCG is the only available vaccine for preventing tuberculosis (TB). Genetic and biochemical variations among the BCG vaccine strains have been considered as one of the significant parameters affecting the variable protective efficacy of the vaccine against pulmonary tuberculosis. To track genetic variations, here two vaccine strains (Danish 1331 and Pasteur 1173P2) popularly used according to the BCG World Atlas were subjected to a comparative analysis against the Mycobacterium tuberculosis H37Rv, Mycobacterium bovis AF2122/97, and Mycobacterium tuberculosis variant bovis BCG str. Pasteur 1173P2 reference genomes. Besides, the presence or absence of the experimentally verified human T cell epitopes was examined., Results: Only two variants were identified in BCG Danish 1331 that have not been reported previously in any BCG strains with the complete submitted genome yet. Furthermore, we identified a DU1-like 14,577 bp region in BCG Danish 1331; The duplication which was previously seemed to be exclusive to the BCG Pasteur. We also found that 35% of the T cell epitopes are absent from both strains, and epitope sequences are more conserved than the rest of the genome., Conclusions: We provided a comprehensive catalog of single nucleotide polymorphisms (SNPs) and short insertions and deletions (indels) in BCG Danish 1331 and BCG Pasteur 1173P2. These findings may help determine the effect of genetic variations on the variable protective efficacy of BCG vaccine strains., (© 2022. The Author(s).)

Objectives: We aimed to identify the enterotoxigenic Bacteroides fragilis (ETBF) and bft subtypes among patients with diarrhea. In addition, we assessed whether DNA gyrase subunit B (gyrB) and neuraminidase (nanH) genes are useful determinants for identification of B. fragilis compared to 16S rRNA sequencing as a reference method., Methods: The 530 fecal specimens were cultured on BBE agar. The colonies which supposed to be a member of B. fragilis group were subjected to 16S rRNA gene sequencing and PCR assays targeting the Bacteroides fragilis group (BFG), gyrB and nanH. The B. fragilis toxin (bft) gene and its subtype was detected by PCR. The specificity of PCR assays was calculated considering the 16S rRNA gene sequencing as the reference method., Results: A total of 111 Gram-negative anaerobic coccobacilli were isolated from 530 fecal specimens using BBE agar. Of the 111 isolates, 100 (90.09%) were assumed to be a member of Bacteroides fragilis group as they yielded an amplicon through PCR using the group-specific primers (Bfra-F/g-Bfra-R). However, only 28 isolates out of 100 were definitively identified as species of Bacteroides using16S rRNA gene sequencing; of which 15 isolates were B. fragilis and the remaining 13 isolates were identified as B. thetaiotaomicron (n = 6), Parabacteroides distasonis (n = 3), B. vulgatus (Phocaeicola vulgatus) (n = 1), B. ovatus (n = 1), B. congonensis (n = 1) and B. nordii (n = 1). Among the 15 isolates of B. fragilis, 4 were found to be ETBF. Compared to the reference method, the specificity and accuracy of the PCR targeting gyrB gene (64.7% and 65%) was higher than of nanH (36.4% and 46%, respectively., Conclusions: This study demonstrated that more than one-fourth of B. fragilis isolates harbored bft gene and less than 1% of patients with diarrhea harbored ETBF. The slight agreement between the PCR assays -already used for identification of B. fragilis which targeting gyrB or nanH - and 16S rRNA gene sequencing as the reference method was noted., Competing Interests: Declaration of Competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Objectives: The dysbiosis of bacteria and horizontal transfer of antibiotic resistance genes (ARGs) could be highly problematic particularly in the oral environment. Here, we aimed to identify the anaerobic species from patients with periodontitis and to screen the isolates for the β-lactamase resistance genes, bla TEM , cfxA, its variants, and mobA., Methods: The 129 samples from periodontal pockets were subjected to anaerobic culture, followed by 16S rRNA gene sequencing, PCR assays for the cfxA, bla TEM , and mobA. The minimum inhibitory concentration (MIC) of amoxicillin, ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, and cefixime was determined against CfxA producing isolates using MIC Test Strips., Results: The species with frequency higher than 10% were Lactobacillus spp. (26.3%), Streptococcus spp. (18.8%), Leptotrichia wadei (14%) and Veillonella spp. (11.4%). The bla TEM was not found in any of the isolates whereas cfxA was found in 12.5% of isolates including V. parvula, V. rogosae, Prevotella nigrescens and Campylobacter concisus. Of CfxA variants, CfxA2 (90%) was the most frequent one. Among the CfxA producing isolates, the resistance to ampicillin and amoxicillin was observed only in two isolates of P. nigrescens and V. rogosae., Conclusions: This study showed that various anaerobes species may be involved in the development of periodontitis. Of them, Prevotella and Veillonella species were found to commonly carry cfxA even though they are susceptible to beta-lactams and its combination., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aims: Investigate the genetic stability of the BCG vaccine produced in Iran from different batches compared to the reference strain., Methods and Results: We comparatively analyzed the whole genome sequences of the vaccine batches from different years. Eleven vials of different batches from 2010, 2018, and 2019 were included. Complete genome analyses revealed no difference between the old (2010) and new (2018 and 2019) vaccine batches. Additionally, minor genetic changes include five single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were observed compared to the BCG Pasteur 1173P2 reference strain, which were shared among all batches. Besides, the batches were identical to the reference strain in terms of antibiotic resistance genes, prophage sequences, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems., Conclusions: High genetic stability of the BCG vaccine used in the national immunization program was confirmed, which indicates the optimal conditions in the vaccine production process., Significance and Impact of the Study: Genetic differences within and between vaccine strains have been declared as one of the main parameters related to the BCG vaccine variable protective efficacy. No study has been done to investigate the genetic variations of the vaccine batches at the single-base level., (© 2021 The Society for Applied Microbiology.)

Biofilm formation and antibiotic resistance are the most important ways in which water bacteria such as Pseudomonas aeruginosa are protected against antibacterial agents. The aim of this study was to develop a rapid and cost-effective laboratory method for modeling and optimizing chlorine disinfection conditions. Critical factors (disinfection type, concentration, contact time and pH) were tested on bactericidal effect, inhibition of biofilm formation (IBF) and antibiotic susceptibility (AS) of P. aeruginosa . The central composite face-centered (CCF) design was applied to model the effect of disinfection process on the IBF and AS. The results showed that the IBF response was more affected by the strain type of P. aeruginosa and the type of disinfectant, which may be due to previous species growth conditions of the standard strain and greater durability of CAT in water. Optimization of factors affecting disinfection had a significant effect on the planktonic form, but was not effective in removing the biofilm of P. aeruginosa . Furthermore, the concentration of NaOCl and CAT was more effective than pH on planktonic and biofilm cells inactivation. The model of AS was weaker than other models due to limited contact time and use of high concentrations of disinfectant. The use of chlorine compounds based on the recommended levels in water does not prevent the formation of P. aeruginosa biofilm. According to the optimization findings, although increasing the contact time and concentration of the disinfectant increases the bactericidal effect of chlorine, it can also increase the resistance of P. aeruginosa to some antibiotics., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest., (© Springer Nature Switzerland AG 2021.)

Background and Aims: Acinetobacter baumannii is among the most concerning cause of nosocomial infections due to its high level of antibiotic resistance and high mortality. The aim of this study was to determine the role of efflux pumps in resistance of A. baumannii strains to three disinfectants, including MICROZED ID-MAX, NANOSIL D2, and OPIDEX OPA., Methods: Twenty-eight environmental and clinical isolates of A. baumannii were collected from selected hospitals of central Iran. The minimum inhibitory concentrations of the disinfectants were determined and real time reverse transcriptase-PCR was performed to investigate the expression level of qacEΔ1 , amvA , abeM , and adeB efflux pump genes., Results: Considering both clinical and environmental isolates, there was a significant difference in the mean expression level of qacEΔ1 gene between susceptible and resistant strains to MICROZED ID-MAX disinfectant, of amvA and abeM genes between susceptible and resistant strains to NANOSIL D2 disinfectant and of abeM gene in susceptible and resistant strains to OPIDEX OPA disinfectant (all P  ˂ .05). The expression levels of abeM and amvA genes were higher in the environmental isolates that were resistant to NANOSIL D2 disinfectant compared to those that were susceptible ( P  ˂ .05)., Conclusions: This study provided evidence for the role of abeM and amvA genes in the resistance of environmental isolates to disinfectants, particularly hydrogen peroxide derivatives., Competing Interests: The authors declare that they have no conflict of interest., (© 2021 The Authors. Health Science Reports published by Wiley Periodicals LLC.)

Background: Carbapenem-resistant Acinetobacter baumannii (CRAB) is among the most concerning cause of healthcare-associated infections (HAI) due to its high level of antibiotic resistance and high mortality. In the era of the COVID-19 pandemic, the key priority of infection control committees is to contain the dissemination of antibiotic resistant Gram-negative bacteria. Here, we aimed to timely recognize the emergence of CRAB in COVID-19 cases admitted to the wards of a tertiary referral hospital and to identify the genetic relatedness of the isolates., Methods: From 30 March to 30 May 2020, a total of 242 clinical samples from COVID-19 cases were screened for CRAB isolates using standard microbiologic and antibiotic susceptibility tests. The PCRs targeting oxa23, oxa24, oxa58, bla TEM and bla NDM-1 genes were performed. Two multiplex PCRs for identifying the global clones (GC) of A. baumannii were also performed. The sequence type of CRABs was determined using Institut Pasteur (IP) multilocus sequence typing (MLST) scheme., Results: Eighteen CRAB isolates were recovered from COVID-19 patients with the mean age of 63.94 ± 13.8 years. All but 4 COVID-19 patients co-infected with CRAB were suffering from an underlying disease. Death was recorded as the outcome in ICUs for 9 (50%) COVID-19 patients co-infected with CRAB. The CRAB isolates belong to GC2 and ST2 IP and carried the oxa23 carbapenem resistance gene., Conclusion: This study demonstrated the co-infection of CRAB isolates and SARS-CoV-2 in the patients admitted to different ICUs at a referral hospital in Tehran. The CRAB isolates were found to belong to ST2 IP , share the oxa23 gene and to have caused several outbreaks in the wards admitting COVID-19 patients., (© 2021. The Author(s).)

Objectives: To analyse the context of genes conferring antibiotic resistance in two carbapenem-resistant Acinetobacter baumannii isolates recovered in Tehran, Iran., Methods: The antibiotic resistance phenotype for 28 antibiotics was determined using disc diffusion. The whole genome sequences of ABH008 and ABS200 were determined using the Illumina HiSeq X Ten platform. Resistance genes were identified using ResFinder and multilocus sequence types were determined using the Oxford and Institut Pasteur schemes., Results: Isolates ABH008 and ABS200, recovered in 2012 and 2013, respectively, in two different Tehran hospitals, belong to the common global clone 1 lineage, ST1 IP and ST231 OX . They are resistant to sulfamethoxazole, tetracycline, gentamicin, amikacin, third-generation cephalosporins and carbapenems. Despite being isolated in different hospitals, phylogenetic analysis indicated they are closely related. Consistent with this, both isolates carry catA1, sul1, aacC1 and aadA1 in a novel variant of the AbaR3-type resistance island, named AbaR31. Both isolates are resistant to amikacin and carbapenems owing to aphA6 and oxa23 , respectively. The oxa23 gene is located in the AbaR4 resistance island, and aphA6 in Tn aphA6 , and both mobile elements are in an ∼90 kbp plasmid encoding the putative RepAci6 replication initiation protein. Resistance to third-generation cephalosporins is due to the acquisition by homologous recombination of a 5 kb DNA segment that contains ISAba1- ampC from a ST623 strain., Conclusions: The resistance gene complements of ABH008 and ABS200 were found in AbaR31 and a plasmid that encodes RepAci6. The close genetic relationship of ABH008 and ABS200, despite each being recovered from different hospitals, indicates transmission between the two hospitals., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Objectives: This study aimed to evaluate antibiotic resistance profiles and presence of virulence genes among Salmonella enterica serovar Enteritidis ( S. Enteritidis) isolated from patients with gastroenteritis in various regions of Iran. Moreover, genetic relatedness among the strains was assessed by pulsed-field gel electrophoresis (PFGE)., Materials and Methods: From April through September 2017, 59 Salmonella strains were isolated from 2116 stool samples. Of these strains, 27 S. Enteritidis were recovered. These strains were subjected to disk diffusion tests, polymerase chain reaction (PCR) for detection of virulence genes ( invA, hilA, pefA, rck, stn, ssrA, ssaR, sefA, spvC, sipA, sipC, sopB, sopE , and sopE2 ), and PFGE., Results: High prevalence of resistance towards cefuroxime (n = 20, 74.1%) and ciprofloxacin (n = 13, 48.2%) were demonstrated. All tested strains possessed invA, hilA, sefA, sipA, sopB , and sopE . The least prevalent virulence gene was rck (n = 6; 22.2%). Based on combinations of virulence genes, 12 virulotypes were observed. The most common virulotype was VP2 (n = 12; 44.4%), harboring all of the virulence genes except for rck . PFGE typing showed only two distinct fingerprints among tested strains. Each fingerprint had completely different virulotypes. Notably, VP4 (harboring all genes except for rck and spvC ) was only presented in pulsotype A, while VP2 was confined to pulsotype B., Conclusion: S . Enteritidis strains were derived from a limited number of clones, suggesting that it is highly homogenous. Future works should consider combinations of other genotyping methods together with larger sample sizes from more diverse sources., Competing Interests: The authors declare that they have no conflicts of interest.

AmpC β-lactamases hydrolyze all β-lactams except cefepime and carbapenems. The study of AmpC-producing E. coli has high priority for the infection control committee. This research is aimed to investigate the resistant urinary AmpC-generating E. coli isolates and identify their genetic variety. Some 230 E. coli isolates from patients suffering urinary tract infection symptoms were studied in 2017-2018 to assess their susceptibility toward antimicrobial agents. AmpC gene was evaluated by PCR and molecular typing using the 10-loci MLVA method. MLVA images were examined by BioNumerics 6.6 software through the use of the UPGMA algorithms. Thirty-eight AmpC-generating E. coli isolates were detected. The most abundant determinant was bla CIT and bla EBC , bla FOX , and bla DHA had the next ranks, respectively. Six major clusters and a singleton were identified by MLVA. AmpC beta-lactamases in urinary isolates of E. coli in the hospital under study and high rate of additional resistance to gentamicin, cotrimoxazole and ciprofloxacin. The most frequent gene determinant of AmpC beta-lactamase was bla CIT and vary depending on time and geographical location., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dolatyar Dehkharghani, Haghighat, Rahnamaye Farzami, Rahbar and Douraghi.)

Stenotrophomonas maltophilia exhibits wide spectrum of fluoroquinolone resistance using different mechanisms as multidrug efflux pumps and Smqnr alleles. Here, the role of smeDEF, smeVWX efflux genes and contribution of Smqnr alleles in the development of fluoroquinolone resistance was assessed. Ciprofloxacin, levofloxacin and moxifloxacin resistance were found in 10.9%, 3.5%, and 1.6% of isolates, respectively. More than four-fold differences in ciprofloxacin MICs were detected in the presence of reserpine and smeD, F, V expression was significantly associated with ciprofloxacin resistance (p = 0.017 for smeD, 0.003 for smeF, and 0.001 for smeV). Smqnr gene was found in 52% of the ciprofloxacin-resistant isolates and Smqnr8 was the most common allele detected. Fluoroquinolone resistance in S. maltophilia clinical isolates was significantly associated with active efflux pumps. There was no correlation between the Smqnr alleles and ciprofloxacin resistance; however, contribution of the Smqnr genes in low-level levofloxacin resistance was revealed.

Objectives: Strain subtyping is an important epidemiological tool to trace contamination, determine clonal relationships between different strains, and the cause of outbreaks. Current subtyping methods, however, yield less than optimal subtype discrimination. Pulsed-field gel electrophoresis is the gold standard method for Escherichia coli and Multiple-Locus Variable-number tandem repeat Analysis is a rapid PCR-based method. The purpose of this study was to evaluate MLVA and PFGE methods for subtyping β -lactamase-producing E. coli strains isolated from urinary tract infections., Materials and Methods: Overall, 230 E. coli isolates from patients with urinary tract infections were examined for antimicrobial susceptibility testing. 10-loci and 7-loci MLVA and PFGE methods were used for molecular typing of β -lactamase-producing E. coli isolates., Results: Out of 230 isolates, 130 (56.5%) β -lactamase-producing E. coli isolates were found in this study. The diversity indices of the VNTR loci showed an average diversity of 0.48 and 0.54 for 7-loci and 10-loci MLVA, respectively. The discriminatory power of PFGE showed a value of 0.87. The discordance between the methods was high., Conclusion: Our study showed that PFGE is more discriminatory than MVLA. MLVA is a PCR- based method and can generate unmistakable data, in contrast to PFGE. Optimization of polymorphic VNTR is essential to improve the discriminatory power of MLVA based on geographical region.

Stenotrophomonas maltophilia isolates are responsible for various hospital-acquired infections and are particularly increasing in the immunocompromised patients. The aim of this study was to determine the clonal relatedness between S. maltophilia isolates originating from the clinic and environment. A total of 150 S. maltophilia isolates from patients and 1108 environmental samples obtained in three hospitals from Tehran. Following molecular identification targeting 23S rRNA gene, the clonal relatedness of the environmental and clinical isolates was determined using pulsed field gel electrophoresis (PFGE). Of the 150 clinical and 18 environmental isolates identified using phenotypic tests, the speciation of 120 and 15 was confirmed by targeting the 23S rRNA gene. The 24 common pulsotypes (PTs) and 32 single PTs were identified by PFGE. Only a small cluster was shared among the clinic and environment within a hospital; therefore, the intra-hospital dissemination of certain isolates of S. maltophilia among the clinic and environment was demonstrated., (© 2020 The Society for Applied Microbiology.)

Today, the targeted therapies like the use of immunotoxins are increased which targeted specific antigens or receptors on the surface of tumor cells. Fibroblast growth factor-inducible 14 (Fn14) is a cytokine receptor which involves several intercellular signaling pathways and can be highly expressed in the surface of cancer cells. Since the cleavage of enzymatic domain of Pseudomonas exotoxin A (PE) occurs in one step by furin protease, we fused enzymatic subunit of Shiga-like toxin type 2a (Stx2a) with domain II and a portion of Ib of PE to increase the toxicity of Stx. Then, we genetically fused the Fv fragment of an anti-Fn14 monoclonal antibody (P4A8) to STX2a-PE15 and evaluated the STX2a-PE15-P4A8 chimeric protein as a new immunotoxin candidate. In silico analysis showed that the STX2a-PE15-P4A8 is a stable chimeric protein with high affinity to the Fn14 receptor. Despite, the STX2a-PE15-P4A8 can be bind to the B cell receptor, but it has been weakly presented by major histocompatibility complex molecules II (MHC-II). So, it may have a little immunogenicity. On the basis of our in-silico studies we predict that STX2a-PE15-P4A8 can be a good candidate for cancer immunotherapy., Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-021-00079-w., Competing Interests: Conflict of interestThe authors declare that they have no competing interests., (© The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature 2021.)

Objectives: The molecular mechanisms involved in biofilm formation inStenotrophomonas maltophilia are poorly understood. Here, we examined whether the presence of smf-1, rmlA, spgM and rpfF genes is associated with biofilm formation and antibiotic resistance in S. maltophilia., Methods: A total of 150 S. maltophilia isolates were collected from three tertiary-care hospitals in Iran and were identified through PCR amplification of the 23S rRNA gene. Biofilm formation was determined by microtitre plate assay. Presence of smf-1, rmlA, spgM and rpfF genes was examined by PCR., Results: Among the isolates examined, 148 (98.7%) were able to produce biofilm, of which 69 (46.0%) were strong biofilm-producers, whereas 32 (21.3%) and 47 (31.3%) were moderate and weak biofilm-producers, respectively. The frequency ofsmf-1, rmlA, spgM and rpfF was 99.3%, 98.0%, 97.3% and 70.0%, respectively. Statistical analysis indicated a direct correlation between presence of the rpfF gene and biofilm formation (P < 0.001). The high prevalence of smf-1 (99.3%) among the isolates is noted and there was a significant association between smf-1 and biofilm-forming ability (P < 0.01), but lower than rpfF. Additionally, a direct association was found between resistance to ticarcillin/clavulanate, ceftazidime, ciprofloxacin and doxycycline and strong biofilm formation in the S. maltophilia isolates (P < 0.01)., Conclusion: This study demonstrated thatS. maltophilia clinical isolates significantly differ in biofilm-forming ability. Moreover, presence of rpfF and smf-1, but not spgM, could be associated with biofilm formation. This study highlights the importance of rpfF in formation of biofilm compared with the other genes involved., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Although the diversity of Clostridioides difficile toxins have been extensively studied, little is known about the variation in the surface associated proteins (SAPs) which are important in early steps of bacterial colonization and infection. Here, we examined 65C. difficile isolates to identify polymorphisms in the genes encoding SAPs., Methods: PCR was used to amplify slpA, fliC, fliD, cwp66 and cwp84 genes, followed by sequencing. In addition, the antigenicity and immunogenicity properties of different types of SlpA, FliC, FliD, Cwp66 and Cwp84 proteins were predicted in-silico by VaxiJen and BcePred online servers., Results: The predominant slpA sequence type was gr-01 (42.37%), followed by hr-01 (11.86%) and 078-01 (10.16%). In addition, two new slpA subtypes of smz (smz-09-Ir and smz-010-Ir) and a new slpA sequence type (Ir-01) were identified among the isolates examined. Analysis of the nucleotide sequences of fliC, fliD, cwp66 and cwp84 genes revealed 7, 5,5,3 different sequence types, respectively. Insilico analysis of antigenicity of SAPs showed that FliC had the highest level of antigenicity whereas SlpA and Cwp66 proteins had the highest level of immunogenicity., Conclusions: This study pointed to the nucleotide polymorphism in SAPs of C. difficile isolates and demonstrated noticeable diversity in antigenicity and immunogenicity of these proteins which need to be taken into consideration as promising therapeutic or vaccine targets., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest., (© Springer Nature Switzerland AG 2020.)

Introduction: Despite the moderate incidence of tuberculosis (TB) in many parts of Iran, Golestan province had a permanently higher TB incidence rate than the national average. Moreover, Golestan province receives immigrants, mainly from TB-endemic areas of Iran and neighbor countries. Here, we aimed to characterize the circulating Mycobacterium tuberculosis complex (MTBC) isolates in terms of the spoligotype and drug resistance patterns, across Golestan province., Materials and Methods: A set of 166 MTBC isolates was collected during July 2014 to July 2015 and subjected to drug susceptibility testing for first- and second-line anti-TB drugs and spoligotyping., Results: Of 166 MTBC isolates, 139 (83.7%) isolates were assigned to 28 spoligotype international types (SITs). The most frequent SITs were SIT127/Ural-2 (n=25, 15.1%), followed by SIT1/Beijing (n=21, 12.7%) and SIT3427/Ural-2 (n=18, 10.8%). The set of 18 isolates (10.8%) showed resistance to at least one drug, which mainly belonged to SIT1/Beijing (n=7, 38.9%), orphan patterns (n=4, 22.2%) and SIT357/CAS1-Delhi (n=3, 16.7%). In addition, four isolates (2.4%) were resistant to pyrazinamide. The analysis of mutation corresponded to resistance to rifampin and isoniazid showed that two isolates had Ser531Leu substitution in rpo B, four isolates had Ser315Thr substitution in kat G and one isolate had [C(-15)T] in inh A locus., Conclusion: High diversity in spoligotypes of the MTBC isolates and lack of dominant genotype might be due to residence of immigrants in this region and consequent reactivation of latent infection. In addition, due to the presence of extensively drug-resistant (XDR) isolates in Golestan province, it is important to conduct future studies to determine transmission pattern of drug-resistant isolates in this region., Competing Interests: The authors declare that there is no conflict of interest., (© 2020 Mansoori et al.)

The worldwide distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) has become a global concern, particularly in countries where antibiotic prescription is not tightly regulated. However, knowledge of the genomic aspects of CRAB from many parts of the world is still limited. Here, 50 carbapenem-resistant A. baumannii isolates recovered at a single hospital in Tehran, Iran, during several outbreaks in 2012 and 2013 were found to be resistant to multiple antibiotics. They were examined using PCR mapping and multilocus sequence typing (MLST). All Iranian strains belonged to sequence type 328 in the Institut Pasteur MLST scheme (ST328 IP ), a single-locus variant of ST81 IP, and all Iranian strains contained two carbapenem resistance genes, oxa23 and oxa24. The oxa23 gene is in the transposon Tn 2006 in AbaR4, which interrupts the chromosomal comM gene. Phylogenetic analysis using whole-genome sequence (WGS) data for 9 isolates showed that they belonged to the same clade, designated the ST81/ST328 clade, within lineage 2 of global clone 1 (GC1). However, there were two groups that included either KL13 or KL18 at the K locus (KL) for capsular polysaccharide synthesis and either a tet39 or an aadB resistance gene, respectively. The genetic context of the resistance genes was determined, and the oxa24 (OXA-72 variant) and tet39 (tetracycline resistance) genes were each in a p dif module in different plasmids. The aadB gene cassette (which encodes gentamicin, kanamycin, and tobramycin resistance) was harbored by pRAY*, and the aphA6 gene (which encodes amikacin resistance) and sul2 gene (which encodes sulfamethoxazole resistance) were each harbored by a different plasmid. The sequences obtained here will underpin future studies of GC1 CRAB strains from the Middle East region. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii strains are among the most critical antibiotic-resistant bacteria causing hospital-acquired infections and treatment failures. The global spread of two clones has been responsible for the bulk of the resistance, in particular, carbapenem resistance. However, there is a substantial gap in our knowledge of which clones and which specific lineages within each clone are circulating in many parts of the world, including Africa and the Middle East region. This is the first genomic analysis of carbapenem-resistant A. baumannii strains from Iran. All the isolates, from a single hospital, belonged to lineage 2 of global clone 1 (GC1) but fell into two groups distinguished by genes in the locus for capsule biosynthesis. The analysis suggests a potential origin of multiply antibiotic-resistant lineage 2 in the Middle East region and highlights the ongoing evolution of carbapenem-resistant GC1 A. baumannii strains. It will enhance future studies on the local and global GC1 population structure., (Copyright © 2020 Douraghi et al.)

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.

Introduction: Salmonella enterica serotype Enteritidis (S. Enteritidis) is a cause of food-borne human illness. Given the prevalence of antibiotic resistance of Salmonella Enteritidis and the lack of antibiotic efficacy in future years, its replacement with other agents is necessary. One of the most useful agents is bacteriophages., Methods: S. Enteritidis was identified using a multiplex polymerase chain reaction assay. The effective bacteriophages were isolated from hospital wastewater samples. The effects of the bacteriophages were evaluated both in vitro and in vivo., Results: The phage SE20 belonged to the Podoviridae family, and the genome size was 40 kb. The evaluation of phage SE20 at variable pH ranges showed its susceptibility to pH < 3 and pH > 12. The animal model showed that mice infected with S. Enteritidis developed hepatomegaly and splenomegaly, but did not experience gastrointestinal complications after receiving the bacteriophages., Conclusions: The results of this study suggest that phage SE20 is a promising candidate for controlling salmonellosis caused by Salmonella Enteritidis.

Purpose: Laparoscopic Roux-en-Y gastric bypass (LRYGB) and laparoscopic sleeve gastrectomy (LSG) are the most common techniques for treatment of morbid obesity. However, a few studies have compared the energy expenditure and plasma amino acid profile after LRYGB and LSG. The present study was conducted to assess the resting metabolic rate (RMR) and plasma amino acid profile in obese patients who underwent LRYGB and LSG before and 6 months after the surgery in order to compare these changes from baseline between the two procedures., Materials and Methods: Forty-three adult obese patients participated in this study (LRYGB = 22 and LSG = 21) and were followed up for 6 months. RMR was measured by indirect calorimetry. The plasma amino acid profile was determined using high-performance liquid chromatography (HPLC)., Results: Mean percent excess weight loss (%WL) were 22.8 ± 4.5% and 23.3 ± 5.7% in LRYGB and LSG, respectively. RMR reduced significantly from baseline by - 459 ± 202 kcal/day in LRYGB and - 500 ± 262 kcal/day in LSG. RMR reduced beyond the expected decrease in both procedures. A decreasing trend was observed in the plasma concentration of branched-chain amino acids (BCAA), aromatic amino acids (AAA), and amino acid index (AAI) in both techniques. There was no significant difference in weight, RMR analysis, and amino acid change from baseline between LRYGB and LSG., Conclusion: Our results showed that the effects of LRYGB and LSG on RMR and amino acid profile were comparable.

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: TnaphA6-carrying repAci6 plasmids have been detected in Acinetobacter baumannii isolates belonging to global clones, GC1 and GC2, worldwide. Here, we examined whether RepAci6 plasmids family play a role in the dissemination of the aphA6 in GC1 A. baumannii isolates from Iran., Results: We found that 22 isolates carried the repAci6 gene, suggesting that they contain a RepAci6 plasmid family. Using the primers linking the aphA6 gene to the backbone of repAci6 plasmid, it was revealed that 16 isolates from different hospitals harbored TnaphA6 on a repAci6 plasmid., Conclusions: This study provides evidence for the dissemination of TnaphA6 on the plasmids encoding RepAci6 in Iranian A. baumannii isolates. Furthermore, it seems that TnaphA6 might be acquired by distinct plasmids separately as it was found to be located on the variants of repAci6 plasmids.

Objectives: Clostridium difficile (C. difficile) is the main causative agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis. The accumulation of antimicrobial resistance in C. difficile strains can drive C. difficile infection (CDI) epidemiology. This study was undertaken to evaluate the antimicrobial resistance patterns of toxigenic C. difficile isolates cultured from diarrhoeal stool samples of hospitalised patients with suspected CDI in three tertiary care hospitals in Tehran, Iran., Methods: Two hundred and fifty diarrhoeal stool samples were investigated by toxigenic culture using cycloserine-cefoxitin-fructose agar and the VERO cell line. Antimicrobial susceptibility to metronidazole, vancomycin, clindamycin, tetracycline, and moxifloxacin was performed by disk diffusion and Etest methods on Brucella Blood Agar supplemented with hemin and vitamin K., Results: Thirty-five stool samples (14.0%) proved positive using C. difficile toxigenic culture. According to Clinical and Laboratory Standards Institute breakpoints, the following resistance was identified in C. difficile isolates: metronidazole (2 of 35); moxifloxacin (7 of 35); clindamycin (18 of 35); and tetracycline (5 of 35). Using European Committee on Antimicrobial Susceptibility Testing breakpoints, three of 35 isolates showed reduced-susceptibility for vancomycin and 14 of 35 for metronidazole. In addition, the results showed a good correlation between the inhibition zone diameter (disk diffusion) and MIC values (Etest); Pearson correlation coefficient 0.7400.95 (P< 0.001)., Conclusions: Multidrug resistance was observed in Iranian clinical toxigenic C. difficile isolates, including reduced susceptibility to first-line CDI treatment drugs. In addition, disk diffusion can be used as a cost-effective option for the antimicrobial susceptibility testing of C. difficile isolates., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Rapid and species-specific detection, and virulence evaluation of opportunistic pathogens such as Pseudomonas aeruginosa, are issues that increasingly has attracted the attention of public health authorities. A set of primers and hydrolysis probe was designed based on one of the P. aeruginosa housekeeping genes, gyrB, and its specificity and sensitivity was evaluated by TaqMan qPCR methods. The end point PCR and SYBR Green qPCR were used as control methods. Furthermore, multiplex RT-qPCRs were developed for gyrB as reference and four virulence genes, including lasB, lasR, rhlR and toxA. Totally, 40 environmental samples, two clinical isolates from CF patients, two standard strains of P. aeruginosa, and 15 non-target reference strains were used to test the sensitivity and specificity of qPCR assays. In silico and in vitro cross-species testing confirmed the high specificity and low cross-species amplification of the designed gyrB418F/gyrB490R/gyrB444P. The sensitivity of both TaqMan and SYBR Green qPCRs was 100% for all target P. aeruginosa, and the detected count of bacteria was below ten genomic equivalents. The lowest M value obtained from gene-stability measurement was 0.19 that confirmed the suitability of gyrB as the reference gene for RT-qPCR. The developed qPCRs have enough detection power for identification of P. aeruginosa in environmental samples including clean and recreational water, treated and untreated sewage and soil. The short amplicon length of our designed primers and probes, alongside with a low M value, make it as a proper methodology for RT-qPCR in virulence genes expression assessment.

Cytotoxin is one of the important pathogenic factors, which plays a role in the virulence of Klebsiella oxytoca . The aim of this study was to investigate molecular typing of clinical isolates of the cytotoxin-producing K. oxytoca using internal transcribed spacer (ITS) PCR. A total of 75 isolates of K. oxytoca were isolated from clinical samples; they were verified as K. oxytoca by standard microbiological tests and PCR. Production of toxin determines the cytotoxic effects on HEp-2 cells. The genetic diversity of isolates of the cytotoxin-producing K. oxytoca were defined by ITS-PCR. Of all the isolates investigated, five K. oxytoca strains isolated from stool cultures, two strains from blood samples, one strain from a wound and one strain isolated from urine had cytotoxic effects on HEp-2 cells. The ITS-PCR patterns showed genetic diversity among cytotoxin-producing isolates. The ITS-PCR method had good discriminatory power; performance of this method and interpretation of the results were easy and repeatable. Five genetic diversity patterns were identified by ITS-PCR.

Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world. We aimed to characterize C. difficile isolates among hospitalized patients, hospital staffs, and hospital environment samples obtained in three tertiary care hospitals of Iran with regard to their molecular types between June 2016 and November 2017. The toxigenicity of C. difficile isolates was determined by toxigenic culture and multiplex-PCR. Toxigenic C. difficile isolates collected were ribotyped using capillary gel electrophoresis-based PCR and the database of WEBRIBO (http://webribo.ages.at). Of 500 clinical and non-clinical samples, toxigenic C. difficile were identified in 35 of 250 stool samples (14%) and in 3 of 250 swabs (1.2%). The most frequently found ribotypes (RTs) were 039, AI-12, and AI-21 (15.8, 10.52, and 10.52% of all isolates, respectively). Further RTs were: 017, 001, AI-3, AI-15, AI-18, AI-10, AI-4, and PR21195 (as new ribotype). The epidemic RTs (027 and 078) seen in the Europe, North America, and Asia were completely absent in this study., (© 2019 APMIS. Published by John Wiley & Sons Ltd.)

Background: Clostridioides difficile is a major cause of nosocomial infectious diarrhea in hospitalized patients throughout the world., Methods: A multiplex real-time PCR assay was developed and evaluated in comparison with toxigenic culture (TC) (as gold standard method) for direct detection of toxigenic C. difficile in fecal specimens. The multiplex real-time PCR assay simultaneously detected glutamate dehydrogenase (gluD), toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtB) genes in stool samples., Results: The results of multiplex real-time PCR were compared to those of the TC method in 250 patients suspected of C. difficile infection. The prevalence of positive TC was 13.6%. Forty-two stool samples (16.8%) were determined to be gluD+ using multiplex real-time PCR. These included 35 (83.3%) toxigenic (32 tcdA+, tcdB+ and three tcdB+) and 7 (20.0%) were cdtB+. The multiplex real-time PCR assay had a sensitivity of 91.45%, specificity of 99.54%, and positive and negative predictive values of 97% and 98.6%, respectively, compared to the TC method for diagnosis of C. difficile. The analytical sensitivity of the multiplex real-time PCR assay was estimated to be 102 CFU/g of stools and 0.0200 pg of genomic DNA from culture. The analytical specificity was determined to be 100% by using enteric and non-C. difficile standard bacterial strains., Conclusions: The molecular method developed in the study was rapid, sensitive, and specific for detection of toxigenic C. difficile. It is applicable to be performed in clinical laboratories and correlated well with the results obtained by TC.

Lack of culturability in the viable but non-culturable (VBNC) bacteria and the ability to regain infectivity in favourable conditions is one of the new challenges of public health providers for Pseudomonas aeruginosa monitoring in environmental samples. Propidium monoazide quantitative polymerase chain reaction (PMA-qPCR) is one of the promising methods for timely detection of VBNC pathogens in environmental samples. We developed and used a method for the first time to detection of VBNC P. aeruginosa in swimming pool water samples using a membrane filter (MF). Moreover, the dominant model of the distribution of colonies on the MF and the effect of the culture medium and MF type on colony recovery by MF were evaluated. Swimming pool samples were subjected to conventional culture-based, qPCR and PMA-qPCR methods and the results were compared for the presence of VBNC P. aeruginosa in the samples. The positivity rate was 21% and 75% for P. aeruginosa in water samples as confirmed by standard culture-based and qPCR methods, respectively. Furthermore, of 24 samples, 9 (37.5%) were positive for VBNC P. aeruginosa . The developed qPCR/PMA-qPCR assay can detect the VBNC bacteria directly from aquatic samples and may result in better monitoring of recreational waters., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest.

This study was set to determine the genetic linkage and the clonal relationship between vancomycin-resistant Enterococcusfaecium (VREfm) isolates in three hospitals of Ahwaz city. In this study, 1050 samples were collected from various rectal swabs, hands of health care workers, environmental surfaces, medical equipment and 146 enterococci isolates from clinical sources of three hospitals from March to September 2015. Antimicrobial resistance patterns in VREfm were detected by disk diffusion method. Genetic linkages of VREfm strains were investigated by pulse field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods. Out of 366 enterococcal isolates, 163 Enterococcus faecium isolates were found to be resistant to vancomycin. PFGE and MLST analysis showed the presence of 79 pulsotypes and 11 sequence types (ST), respectively. In total, 90% of the isolates belonged to clonal complex 17 (CC17). Three new STs were reported for the first time in this study and ST80 was the predominant ST. We found a high prevalence of diverse VREfm with threatening antibiotic resistance patterns in all the studied sources with the dominance of CC17 VREfm strains in Ahwaz hospitals. Also, the results of typing method showed inter- and intra-hospital circulation of VREfm and similar pulsotypes and STs among different sources., (© FEMS 2019.)

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.

Aims: Little is known about the resistance rate and susceptibility profile of Clostridium difficile isolates in Iran. Therefore, the aim of present study is to assess the rate of drug-resistant C. difficile., Methods and Results: During a 6-year period, four hospitals submitted 735 stool specimens from patients suspected for C. difficile infections to the anaerobic bacteriology laboratory. The 46 C. difficile isolates were subjected to disc diffusion and minimum inhibitory concentration (MIC) Test Strips. All isolates were susceptible to vancomycin (VAN) while the highly resistant phenotypes of metronidazole (MTZ) (67·4%), moxifloxacin (78·3%), ciprofloxacin (69·5%) and tetracycline (82·6%) were observed. Of more concern, 67·3% of C. difficile isolates displayed multidrug-resistant phenotypes. More than half of the isolates (n = 27, 58·6%) were coresistant to ciprofloxacin and moxifloxacin. The MIC90 of VAN was ≤2 mg l -1 , whereas this value for MTZ, ciprofloxacin, moxifloxacin and tetracycline was higher than the resistance breakpoints. According to the comparison of interpretive categories for two tests, the categorical agreement was less than 90% for VAN, ciprofloxacin and tetracycline., Conclusions: The disc diffusion method can be used to detect the isolates with reduced susceptibility to MTZ or moxifloxacin. The high rate of resistance to fluoroquinolones highlights the possibility of the emergence of hypervirulent strains in our settings., Significance and Impact of the Study: This study provides data regarding the high level of resistance against multiple antibiotics except VAN., (© 2018 The Society for Applied Microbiology.)

We aimed to determine the genetic diversity, phylogenetic relationship and transmission dynamics of Mycobacterium tuberculosis complex (MTBC) genotypes in an area with high tuberculosis (TB) incidence. A set of 164 MTBC isolates from new TB patients of Golestan province, Iran, were subjected to genotyping using the standard 24-locus MIRU-VNTR method. Recent TB transmission was evaluated and phylogenetic relationships were analysed by minimum spanning tree and cluster-graph methods. Among the 164 isolates, 132 distinct patterns were detected. The 48 clustered isolates (29.3%) were distributed into 16 clusters ranging in size from 2 to 12 isolates. The most frequent genotype was Central Asian Strain/Delhi (CAS/Delhi) (n = 67, 40.8%), followed by NEW-1 (n = 53, 32.3%) and Beijing (n = 19, 11.6%) genotypes. Thirty five (72.9%) of NEW-1 isolates were recovered from immigrant patients and 84.2% (n = 16) of Beijing genotypes recovered from native cases. Statistically significant association was found between clustering and smoking (p = 0.047), drug addiction (p = 0.01) and prison history (p = 0.003). The estimated proportion of recent transmission was 19.5%. Presence of highly diverse MTBC isolates circulating in this province without a dominant genotype might be a consequence of importation of various genotypes in this area., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: To evaluate the impact of the Integrated Pain Team (IPT)-an interdisciplinary chronic pain care intervention embedded in primary care at a large Veterans Affairs health care system. Outcomes evaluated included IPT's impact on the perceived effectiveness of chronic pain care; provider self-confidence; and primary care team satisfaction, stress, and burnout., Method: This mixed-methods quality-improvement study employed: 1) qualitative semistructured interviews of 61 primary care providers, other primary care team members, and organizational stakeholders; and 2) a supplementary quantitative survey of 65 providers, comparing those who had referred patients to IPT with those who had not., Results: Most interview participants reported that IPT improved chronic pain care by providing patients with a comprehensive pain treatment plan, educating them about opioid risks, and introducing multimodal treatment options. Interviewed care team members reported improved patient education and fewer emotionally charged contacts from patients. Interviewed providers felt that IPT allowed them to focus their time on health concerns other than pain. However, our supplemental survey found that IPT-utilizing providers were no more confident than other providers in their own pain care skills or in their relationships with chronic pain patients., Conclusions: Integrating an interdisciplinary chronic pain care intervention into primary care can provide needed support to care teams and may improve chronic pain care. Elements of the IPT model identified as important to its effectiveness include its interdisciplinary biopsychosocial approach and attentive patient follow-up. However, enhancing providers' confidence and self-efficacy in chronic pain care may require educational and support resources beyond the current IPT model.

Little is known about the toxin profiles, toxinotypes and variations of toxin Clostridioides difficile C ( tcdC ) in Iranian C. difficile isolates. A total of 818 stool specimens were obtained from outpatients ( n  = 45) and hospitalized patients ( n  = 773) in Tehran, Iran, from 2011 to 2017. The 44 C. difficile isolates were subjected to PCR of toxin C. difficile A ( tcdA ), toxin C. difficile B ( tcdB ), tcdA 3'-end deletion, toxinotyping and sequencing of the tcdC gene. Thirty-eight isolates (86.36%) were identified as tcdA and tcdB positive, and the remaining six isolates (13.63%) were nontoxigenic. All tcdA - and tcdB -positive isolates yielded an amplicon of 2535 bp by PCR for the tcdA 3' end. Fourteen (36.84%), seventeen (44.73%) and seven (18.43%) isolates belonged to wild-type, toxin C. difficile C subclone3 ( tcdC-sc3 ) and tcdC-A genotype of tcdC , respectively. Thirty-one isolates (81.57%) belonged to toxinotype 0, and seven isolates (18.42%) were classified as toxinotype V. This study provides evidence for the circulation of historical and hypervirulent isolates in the healthcare and community settings. Furthermore, it was also demonstrated that the tcdC-A genotype and toxinotype V are not uncommon among Iranian C. difficile isolates.

In recent years, the increasing resistance of enteropathogenic Escherichia coli (EPEC) to commonly used antibiotics has made it difficult to choose the best treatment option. Bacteriophage therapy could be a potent alternative to antibiotic therapy for antibiotic-resistant bacteria. The aim of the present study was to isolate and identify a specific bacteriophage against EPEC and characterize bacteriophage in vitro and in vivo. The specific bacteriophage was isolated, and the effect of phage therapy on 48 mice (Balb/c) was investigated. Animals were divided into six groups, including A: PBS (negative control); B: bacteria (positive control); C: bacteria + ciprofloxacin (after 24 h); D: bacteria + bacteriophage (after 24 h); E: bacteria + ciprofloxacin + bacteriophage (after 24 h) and F: bacteriophage + bacteria (after 24 h). Specific bacteriophage against EPEC was isolated from hospital sewage. The bacteriophage had an icosahedral head (120 nm) and a tail (138 nm). The single dose of the bacteriophage (2 × 109 pfu ml-1) was able to control the infection. Unfortunately, because of the misuse of antibiotics by EPEC infected patients, the antibiotic resistant bacteria will become prevalent in the future and the treatment of EPEC infection is going to become more difficult than ever., (© FEMS 2018.)

Despite global efforts to tackle resistance in extended-spectrum beta-lactamase (ESBL)-producing isolates via old antibiotics, there are limited data on the efficacy of fosfomycin-an old oral antibiotic-against Enterobacteriaceae in the Middle East. The purpose of this study was to evaluate the in vitro activity of fosfomycin against urinary ESBL-producing isolates of Enterobacteriaceae. Between 2004 and 2015, 363 isolates of ESBL-producing Enterobacteriaceae were recovered from high-risk patients suffering from cardiac disorders and were subjected to polymerase chain reaction using specific primers for the bla TEM , bla SHV , and bla CTX-M genes. Antibiotic susceptibility testing was performed for fosfomycin and other antibiotic comparators. For the isolates considered nonsusceptible to fosfomycin by disk diffusion, the minimum inhibitory concentration (MIC) was determined. The susceptibility rate to fosfomycin remained almost steady (90-100%) over a 12-year period, although it fluctuated vis-à-vis ciprofloxacin (0-54%), trimethoprim/sulfamethoxazole (9.1-31.7%), and nitrofurantoin (41.7-100%). Of all the antibiotics tested, fosfomycin was the most active antimicrobial agent (97%) against the ESBL-positive isolates. Fosfomycin maintained higher activity against ESBL-Escherichia coli than against ESBL-Klebsiella pneumoniae. Only 11 (3%) isolates were not susceptible to fosfomycin according to disk diffusion and they had MICs greater than 1,024 mg/ml. All of the fosfomycin-nonsusceptible isolates were positive for the bla CTX-M gene (100%), while 5 (45.4%) and 3 (27.3%) of the isolates harbored the bla TEM and bla SHV genes, respectively. We showed that fosfomycin had a numerically higher susceptibility rate than the other antibiotics against the ESBL-producing isolates of the most common Enterobacteriaceae. Given its low resistance rate and oral administration, fosfomycin may be deemed a promising antibiotic for the treatment of urinary tract infections caused by ESBL-producing Enterobacteriaceae.

Vaccination with whole-cell or acellular (Ac) vaccines has been very effective for the control of pertussis. The immune response to Ac vaccines has been generally associated with a shift toward the Th2 profile. In the present study, overlapping recombinant fragments of filamentous hemagglutinin (FHA) and pertactin (PRN) were produced in Escherichia coli. BALB/c mice were immunized with recombinant FHA and PRN together with the native pertussis toxin and alum or CpG as adjuvant. Immunized mice were subsequently aerosol challenged with Bordetella pertussis. Bacterial growth was assessed in bronchoalveolar lavage samples and the levels of cytokines were quantitated in supernatants of stimulated splenocytes by enzyme-linked immunosorbent assay. Our results demonstrated that both PRN and FHA antigens were able to induce IFN-γ, IL-4, and to some extent IL-17 cytokines in challenged mice. The level of IFN-γ was higher in response to CpG formulated antigens. These findings indicate immunoprotective efficacy of our recombinant FHA and PRN antigens in mice.