Publisher Correction: Genomic insights into the 2016-2017 cholera epidemic in Yemen.

In the HTML version of this Letter, the affiliations for authors Andrew S. Azman, Dhirendra Kumar and Thandavarayan Ramamurthy were inverted (the PDF and print versions of the Letter were correct); the affiliations have been corrected online.

Genomic insights into the 2016-2017 cholera epidemic in Yemen.

Yemen is currently experiencing, to our knowledge, the largest cholera epidemic in recent history. The first cases were declared in September 2016, and over 1.1 million cases and 2,300 deaths have since been reported1. Here we investigate the phylogenetic relationships, pathogenesis and determinants of antimicrobial resistance by sequencing the genomes of Vibrio cholerae isolates from the epidemic in Yemen and recent isolates from neighbouring regions. These 116 genomic sequences were placed within the phylogenetic context of a global collection of 1,087 isolates of the seventh pandemic V. cholerae serogroups O1 and O139 biotype El Tor2-4. We show that the isolates from Yemen that were collected during the two epidemiological waves of the epidemic1-the first between 28 September 2016 and 23 April 2017 (25,839 suspected cases) and the second beginning on 24 April 2017 (more than 1 million suspected cases)-are V. cholerae serotype Ogawa isolates from a single sublineage of the seventh pandemic V. cholerae O1 El Tor (7PET) lineage. Using genomic approaches, we link the epidemic in Yemen to global radiations of pandemic V. cholerae and show that this sublineage originated from South Asia and that it caused outbreaks in East Africa before appearing in Yemen. Furthermore, we show that the isolates from Yemen are susceptible to several antibiotics that are commonly used to treat cholera and to polymyxin B, resistance to which is used as a marker of the El Tor biotype.

Genetic approach toward linkage of Iran 2012-2016 cholera outbreaks with 7th pandemic Vibrio cholerae.

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012-2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods - Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)-and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.

Antimicrobial, anti-adhesive, and anti-invasive effects of condition media derived from adipose mesenchymal stem cells against Shigella flexneri.

The objective of the current study was to examine the antimicrobial, anti-adhesion, and anti-invasion properties of various concentrations of condition media obtained from adipose mesenchymal stem cells (AD-MSCs CM) against Shigella flexneri (S. flexneri). AD-MSCs characterization and antimicrobial assay were performed using flow cytometry and microdilution by colony counting, respectively. For evaluating adhesion and invasion, Caco-2 cells were infected by S. flexneri at three different multiplicities of infection (MOIs of 1, 10, and 50) and then treated with DMEM medium and AD-MSCs CM. The inhibitory effect of AD-MSCs CM was assessed after 24 and 48 h of treatment by CFU (colony-forming unit) counting. A total of 84, 65, and 56% reduction in the adhesion rate of S. flexneri to Caco-2 cells treated with AD-MSCs CM were observed at MOIs of 1, 10, and 50, respectively. While S. flexneri at MOI:1 had no invasive effect on Caco-2 cells, convincing invasion was detected at MOIs of 10 and 50, showing a significant decrease following treatment with AD-MSCs CM. The current study results open new insights into AD-MSCs CM as a new non-antibiotic therapeutic candidate for S. flexneri infections.

Characteristics of multiresistant Pseudomonas aeruginosa isolates from burn patients in Iran.

Infections caused by multidrug resistant (MDR) Pseudomonas aeruginosa isolates in burn patients restrict therapeutic strategies. The current study aimed to analyze antibiotic resistance genes and multilocus sequence typing (MLST) of P. aeruginosa strains isolated from burn patients in Shahid Motahari hospital in Tehran, Iran.Altogether 63 P. aeruginosa isolates were characterized in this study. Antibiotic susceptibility testing was performed by disc diffusion method. PCR was performed to determine the frequency of resistance genes. The expression rates of mexB, mexY genes were evaluated by Real-Time PCR. Genotyping of isolates was performed by MLST analysis. All isolates were MDR in this study. The highest resistance was detected against gentamicin, tobramycin, and cefoxitin (100%), while all isolates were susceptible to colistin. Altogether 14 resistance profiles were determined, and profile 1 included more than 50% of the isolates with the highest resistance. In this study blaampC, blaVIM-2, blaOXA-10, and aac(6')-Ib resistance genes were detected in all isolates. The expression levels of mexB and mexY genes were upregulated in 66.6 and 88.8% of MDR isolates, respectively. Overexpression of both genes was detected in 55.5% of the isolates.MLST analysis revealed five sequence types (STs), including ST235, ST664, ST532, ST2637, and ST230, which showed a significant relationship with antibiotic resistance profiles. The present study indicates an increase in antibiotic resistance against different antibiotic families among P. aeruginosa isolates. We describe the circulation of globally distributed STs among hospitalized patients, and we report ST235 as the most common MDR clone in our study.

Phenotypic and genotypic changes of Staphylococcus aureus in the presence of the inappropriate concentration of chlorhexidine gluconate.

BACKGROUND: Chlorhexidine gluconate (CHG) is a disinfectant agent with different applications in health care. Improper use of CHG causes antimicrobial resistance in bacteria as a public health threat. Since Staphylococcus aureus is a common bacteria, it is expected usually exposed to CHG in the hospital and community. The present study aimed to correlate the phenotypic and genotypic changes in a S. aureus strain upon serial adaptation with supra-inhibitory CHG concentration for 50 days. RESULTS: After in vitro serial culture of 5 × 105 CFU/ml of a clinical vancomycin-susceptible S. aureus strain (VAN-S) into brain heart infusion (BHI) broth containing CHG 1/4, 1/2, 1, and 2 × minimal inhibitory concentration (MIC) values of VAN-S in 37 °C during 50 days, we isolated a S. aureus strain (CHGVan-I) with a ≥ twofold decrease in susceptibility to CHG and vancomycin. CHG-induced CHGVan-I strain was considered as a vancomycin-intermediate S. aureus (VISA) strain with a VAN MIC of 4 µg/ml using the broth macro dilution method. However, reduced resistance was observed to tetracycline family antibiotics (doxycycline and tetracycline) using a modified Kirby-Bauer disk diffusion test. Moreover, a remarkable reduction was detected in growth rate, hemolysis activity (the lysis of human red blood cells by alpha-hemolysin), and colony pigmentation (on BHI agar plates). Biofilm formation (using the Microtiter plate method and crystal violet staining) was significantly increased upon CHG treatment. Adaptive changes in the expression of a set of common genes related to the development of VISA phenotype (graTSR, vraTSR, walKR, agr RNAIII, sceD, pbpB, and fmtA) were analyzed by Reverse Transcription quantitative PCR (RT-qPCR) experiment. Significant changes in vraTSR, agr RNAIII, sceD, and pbpB expression were observed. However, gene sequencing of the two-component system vraTSR using the Sanger sequencing method did not detect any non-synonymous substitution in CHGVan-I compared to wild-type. The clonality of VAN-S and CHGVan-I strains was verified using the pulsed-field gel electrophoresis (PFGE) method. CONCLUSIONS: The importance of the present study should be stated in new detected mechanisms underlying VISA development. We found a link between the improper CHX use and the development of phenotypic and genotypic features, typical of VISA clinical isolates, in a CHG-induced strain. Since disruption of the cell wall biosynthesis occurs in VISA isolates, our CHG-induced VISA strain proved new insights into the role of CHG in the stimulation of the S. aureus cell wall.

Tropheryma whipplei in the stool samples of children with acute diarrhea: a study from Tehran, Iran.

BACKGROUND: Recently, Tropheryma whipplei has been suggested as one of the causative agents of diarrhea among children worldwide. Limited data is available on the prevalence of T. whipplei among children with diarrhea in most countries such as Iran. This study was conducted to evaluate the prevalence of T. whipplei in children with acute diarrhea in Iran. METHODS: In this study, the stool samples were collected from 130 children under 10 years old with acute diarrhea from children's hospitals in Tehran city. Genomic DNA was extracted from stool samples and was tested for the presence of DNA of T. whipplei using the SYBR Green Real-time PCR method. Positive T. whipplei samples were finally confirmed by PCR Product sequencing. RESULTS: The mean age of participants was 32.5 months, and 54.6% of children were female. Using the SYBR Green Real-time PCR, 9.23% (12/130) of samples were positive for T. whipplei, which were confirmed by sequencing. 66.67% of positive cases were males. The duration of diarrhea in infected children with T. whipplei (83.3%) was significantly longer (OR: 5.93, 95% CI 1.24-28.22) compared to children with negative results (45.8%). Other demographic factors and clinical signs had not a statistically significant relationship with T. whipplei infection. CONCLUSIONS: In this study, T. whipplei was detected in stool samples of children with acute diarrhea. The results indicated that T. whipplei could be associated with childhood diarrhea in Iran. The health care system and physicians should be aware of the presence of T. whipplei infection in Iran, especially in childhood diarrhea.

Investigation of the Antibacterial and Antibiofilm Activity of Selenium Nanoparticles against Vibrio cholerae as a Potent Therapeutics.

Vibrio cholerae is a major cause of severe diarrhea, which is ecologically flexible, and remains as a major cause of death, especially in developing countries. Consecutive emergence of antibiotic-resistant strains is considered to be as one of the major concerns of the World Health Organization (WHO). Nanoparticles as a new nonantibiotic therapeutic strategy have been widely used in recent years to treat bacterial infections. The present study aimed to investigate the antibacterial and antibiofilm effect of selenium nanoparticles (SeNPs) in vitro against V. cholerae O1 ATCC 14035 strain. SeNPs were prepared and characterized using ultraviolet-visible (UV-Vis) spectroscopy, DLS (dynamic light scattering), zeta potential measurement, and Fourier transform infrared (FTIR) analysis. The concentration of SeNPs was calculated by ICP (inductively coupled plasma) method. Also, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to assess the cytotoxic effect of SeNPs on Caco-2 cells. Antibacterial and antibiofilm activity of SeNPs was determined by broth microdilution and crystal violet assays, respectively. The average particle size of SeNPs was 71.1 nm with zeta potential -32.2 mV. The SEM images supported the uniform spherical morphology of the prepared nanoparticles. The antibiofilm effect of SeNPs was evident at concentrations of 50-200 µg/mL. This study results provided evidence that SeNPs are safe as an antibacterial and antibiofilm agent against V. cholerae O1 ATCC 14035 strain.

Investigating the effect of an identified mutation within a critical site of PAS domain of WalK protein in a vancomycin-intermediate resistant Staphylococcus aureus by computational approaches.

BACKGROUND: Vancomycin-intermediate resistant Staphylococcus aureus (VISA) is becoming a common cause of nosocomial infections worldwide. VISA isolates are developed by unclear molecular mechanisms via mutations in several genes, including walKR. Although studies have verified some of these mutations, there are a few studies that pay attention to the importance of molecular modelling of mutations. METHOD: For genomic and transcriptomic comparisons in a laboratory-derived VISA strain and its parental strain, Sanger sequencing and reverse transcriptase quantitative PCR (RT-qPCR) methods were used, respectively. After structural protein mapping of the detected mutation, mutation effects were analyzed using molecular computational approaches and crystal structures of related proteins. RESULTS: A mutation WalK-H364R was occurred in a functional zinc ion coordinating residue within the PAS domain in the VISA strain. WalK-H364R was predicted to destabilize protein and decrease WalK interactions with proteins and nucleic acids. The RT-qPCR method showed downregulation of walKR, WalKR-regulated autolysins, and agr locus. CONCLUSION: Overall, WalK-H364R mutation within a critical metal-coordinating site was presumably related to the VISA development. We assume that the WalK-H364R mutation resulted in deleterious effects on protein, which was verified by walKR gene expression changes.. Therefore, molecular modelling provides detailed insight into the molecular mechanism of VISA development, in particular, where allelic replacement experiments are not readily available.

Methicillin-resistant Staphylococcus aureus colonization of infectious and non-infectious skin and soft tissue lesions in patients in Tehran.

BACKGROUND: The most common clinical manifestations of Staphylococcus aureus strains in the community are skin and soft-tissue infections. S. aureus could colonize the body sites and complicate the pathogenesis of skin diseases. S. aureus colonization is a risk factor for severe conditions such as bone and joint infections, pneumonia, bacteremia, and endocarditis. This study aimed to investigate the prevalence of S. aureus strains in skin and soft tissue infections and other skin disorders in patients referring to dermatology clinics and to evaluate the antibiotic resistance pattern and molecular characteristics of S. aureus isolates. METHODS: Skin swabs were collected from the lesional sites in 234 outpatients referring to dermatology clinics in three hospitals in Tehran. Antibiotic susceptibility, biofilm formation, and hemolysis tests were performed for isolates. PCR was done for SCCmec typing, agr grouping, and virulence genes detecting. RESULTS: The prevalence of S. aureus strains among patients with skin and soft-tissue infections and other skin lesions was 44.77% (30/67) and 44.91% (75/167), respectively. Also, 59 (56.19%) isolates were MRSA, 35.57% were HA-MRSA, and 30.5% were CA-MRSA. The psmα gene was more prevalent (62.8%) among isolates, followed by hlaα (56.1%), tsst-1 (15.2%) eta (13.3%), etb (6.6%), and pvl (2.8%). The agr specificity groups I, II, III, and IV were identified in 49.5, 21.9, 11.4, and 14.2% of S. aureus isolates, respectively. Most (56%) S. aureus isolates produced a moderate biofilm, and 23.8% of them produced strong biofilms. α-hemolysin (46.6%), ß-hemolysin (25.7%), γ-hemolysin (19%), and both α and ß-hemolysin (5.7%) were also produced by isolates. CONCLUSION: The present study results indicated high colonization of skin lesions by HA-MRSA and CA-MRSA clones; MRSA strains were more resistant to antibiotics, contained various toxin genes, and were able to form biofilms. Therefore, they could play a vital role in the pathogenesis of various skin diseases; also, they could spread and cause infections in other body sites. Eradication and decolonization strategies could prevent recurrent infections and the spread of resistant strains and improve skin conditions.

Vibrio cholerae toxin coregulated pilus provokes inflammatory responses in Coculture model of Caco-2 and peripheral blood mononuclear cells (PBMC) leading to increased colonization.

The aim of this study was to assess the modulatory effect of TcpA in the expression of CEACAM1 adhesin molecule and IL-1, IL-8, and TNF-α pro-inflammatory cytokines in the Coculture model of Caco-2/PBMC (peripheral blood mononuclear cell) that can mimic the intestinal milieu. The TcpA gene from Vibrio cholerae ATCC14035 was cloned in pET-28a and transformed into Escherichia coli Bl-21. The recombinant TcpA-His6 protein was expressed and purified using Ni-column chromatography. The sequencing of transformed plasmid and Western blot analysis of purified protein confirmed the identity of rTcp. The cytotoxicity of different concentrations of recombinant protein for human colon carcinoma cell line (human colorectal adenocarcinoma cell [Caco-2 cell]) was assessed by MTT assay and showed viability of 92%, 82%, and 70%, for 10 µg/mL of TcpA after 24, 48, and 72 h, respectively. Co-cultures of Caco-2 and PBMCs were used to mimic the intestinal milieu and treated with different concentrations of rTcpA (1, 5, 10, and 50 µg/mL). Our data showed about 2.04-, 3.37-, 3.68-, and 42.7-fold increase in CEACAM1 gene expression, respectively, compared with the nontreated Caco-2/PBMC Coculture. Moreover, the expression of IL-1, IL-8, and TNF-α genes was significantly increased up to 15.75-, 7.04-, and 80.95-folds, respectively. In conclusion, V. cholerae TcpA induces statistically significant dose-dependent stimulatory effect on TNF-α, IL-,1, and IL-8 pro-inflammatory cytokines expression. Of these, TNF-α was much more affected which, consequently, elevated the CEACAM1 expression level in IECs. This suggests that TcpA protein is a critical effector as an inducer of increased adhesion potential of V. cholera as well as inflammatory responses of host intestinal tissue.

High prevalence of Campylobacter jejuni CC21 and CC257 clonal complexes in children with gastroenteritis in Tehran, Iran.

BACKGROUND: Campylobacter jejuni (C. jejuni) is a leading cause of acute gastroenteritis in human worldwide. The aim of study was to assess the distribution of sialylated lipooligosaccharide (LOS) classes and capsular genotypes in C. jejuni isolated from Iranian children with gastroenteritis. Furthermore, the level of dnaK gene expression in C. jejuni strains with selected capsular genotypes and LOS classes was intended. Moreover, a comprehensive study of C. jejuni MLST-genotypes and inclusive comparison with peer sequences worldwide was intended. METHODS: Twenty clinical C. jejuni strains were isolated from fecal specimens of 280 children aged 0-5 years, suspected of bacterial gastroenteritis, which admitted to 3 children hospitals from May to October, 2018. Distribution of sialylated LOS classes and specific capsular genotypes were investigated in C. jejuni of clinical origin. The expression of dnaK in C. jejuni strains was measured by Real-Time-PCR. MLST-genotyping was performed to investigate the clonal relationship of clinical C. jejuni strains and comparison with inclusive sequences worldwide. RESULTS: C. jejuni HS23/36c was the predominant genotype (45%), followed by HS2 (20%), and HS19 and HS4 (each 10%). A total of 80% of isolates were assigned to LOS class B and C. Higher expression level of dnaK gene was detected in strains with HS23/36c, HS2 and HS4 capsular genotypes and sialylated LOS classes B or C. MLST analysis showed that isolates were highly diverse and represented 6 different sequence types (STs) and 3 clonal complexes (CCs). CC21 and CC257 were the most dominant CCs (75%) among our C. jejuni strains. No new ST and no common ST with our neighbor countries was detected. CONCLUSIONS: The C. jejuni isolates with LOS class B or C, and capsular genotypes of HS23/36, HS2, HS4 and HS19 were dominant in population under study. The CC21 and CC257 were the largest CCs among our isolates. In overall picture, CC21 and CC353 complexes were the most frequently and widely distributed clonal complexes worldwide, although members of CC353 were not detected in our isolates. This provides a universal picture of movement of dominant Campylobacter strains worldwide.

Clonal diversity and genomic characterization of Panton-valentine Leukocidin (PVL)-positive Staphylococcus aureus in Tehran, Iran.

BACKGROUND: Some Staphylococcus aureus strains produce Panton-Valentine leukocidin (PVL), a bi-component pore-forming toxin, which causes leukocyte lysis and tissue necrosis. Currently, there is very limited information on the molecular epidemiology of PVL-encoding S. aureus strains in Iran. This study aimed to determine the molecular epidemiology and genetic background of PVL-positive S. aureus clinical strains isolated from Iranian patients. METHODS: A total of 28 PVL-positive S. aureus strains were detected from 600 S. aureus isolates between February 2015 and March 2018 from different hospitals in Tehran, Iran. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Molecular genotyping was performed using SCCmec and accessory gene regulator (agr) typing, PVL haplotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). RESULTS: The highest antibiotic resistance rate was found to be against erythromycin (57.1%), followed by ciprofloxacin (42.8%) and clindamycin (35.7%). Moreover, 19 (67.9%) out of 28 S. aureus isolates were identified as MRSA, including CA-MRSA (14/19, 73.7%) and HA-MRSA (5/19, 26.3%). SCCmec type IVa was detected as the predominant type (10/19, 52.6%), followed by type III (5/19, 26.3%) and type V (4/19, 21.1%). The agr type I was identified as the most common type (14/28, 50%), and H and R haplotype groups were observed at frequencies of 67.9 and 32.1%, respectively. Among H variants, the predominant variant was H2 (78/9%). The isolates encompassed 21 different sequence types (STs), including 16 new STs (ST5147 to ST5162). Based on eBURST analysis, the isolates were clustered into five CCs, including CC30, CC22, CC1, CC8, and CC5 (ST5160), and nine singletons. PFGE typing showed that 24 isolates were clustered into A (4 pulsotypes), B (9 pulsotypes), and C (11 pulsotypes) clusters. CONCLUSIONS: A high prevalence of PVL-positive CA-MRSA strains was detected in Iran. The majority of PVL-positive isolates were of H (mostly H2) variant, while R variant was harbored by 100% of PVL-positive MRSA strains. Also, CC8, CC22, and CC30 were identified as the dominant clones among PVL-encoding S. aureus strains. This study promotes a better understanding of the molecular epidemiology and evolution of PVL-positive S. aureus strains in Iran.

Significant contribution of the CmeABC Efflux pump in high-level resistance to ciprofloxacin and tetracycline in Campylobacter jejuni and Campylobacter coli clinical isolates.

BACKGROUND: Campylobacter resistance to antimicrobial agents is regarded as a major concern worldwide. The aim of this study was to investigate the expression of the CmeABC efflux pump and the RAPD-PCR pattern in drug-resistant Campylobacter isolates. METHODS: A total of 283 stool specimens were collected from children under the age of five with diarrhea. The minimum inhibitory concentration (MIC) of tetracycline and ciprofloxacin was determined by broth microdilution method and E-test, respectively. Detection of tetracycline and ciprofloxacin determinants was done by amplification of tetO gene and PCR-sequencing of the gyrA gene. The cmeABC transcriptional expression was analyzed by Real-time (RT)-PCR. Clonal correlation of resistant strains was determined by RAPD-PCR genotyping. RESULTS: Out of 283 fecal samples, 20 (7.02%) samples were positive for Campylobacter spp. Analysis of duplex PCR assay of the cadF gene showed that 737 and 461 bp amplicons were corresponding to Campylobacter jejuni and Campylobacter coli, respectively. All of the 17 phenotypically tetracycline-resistant Campylobacter isolates harbored the tetO gene. Also, four phenotypically ciprofloxacin-resistant Campylobacter isolates had a point mutation at codon 257 of the gyrA gene (ACA to ATA; Thr > Ile). High-level expression of the cmeA gene was observed in ciprofloxacin-resistant and high-level tetracycline-resistant Campylobacter isolates, suggesting a positive correlation between the cmeA gene expression level and tetracycline resistance level. Moreover, a statistically significant difference was observed in the cmeA gene expression between ciprofloxacin-resistant and ciprofloxacin-susceptible strains, which signifies the crucial contribution of the efflux pump in conferring multiple drug resistance phenotype among Campylobacter spp. RAPD analysis of Campylobacter isolates exhibited 16 different patterns. Simpsone`s diversity index of RAPD-PCR was calculated as 0.85, showing a high level of homogeneity among the population; however, no clear correlation was detected among tetracycline and/or ciprofloxacin resistant isolates. CONCLUSION: Significant contribution of the CmeABC efflux pump in conferring high-level resistance to tetracycline and ciprofloxacin was observed in C. jejuni and C. coli clinical isolates. The resistant phenotype is suggested to be mediated by CmeABC efflux pumps, the tetO gene, and point mutation of the gyrA gene. Genotyping revealed no clonal correlation among resistant strains, indicating distinct evolution of tetracycline and ciprofloxacin resistant genotypes among the isolates.

Lactobacillus acidophilus attenuates toxin production by Vibrio cholerae and shigella dysenteriae following intestinal epithelial cells infection.

AIMS: The main objective of the present study was to assess and compare the safety and inhibitory efficacy of Lactobacillus acidophilus against cholera toxin and shigatoxin production by measuring CTX-B and Stx1 expression level in Caco-2 cells exposed to Vibrio cholerae (as a non-invasive small intestine pathogens and Shigella dysenteriae (as an invasive colon pathogen). METHODS: Caco-2 cells were incubated with L. acidophilus 2 h before infection by V. cholerae and S. dysenteriae. Following RNA extraction and cDNA synthesis, relative toxins mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. L. acidophilus didn't show any cytotoxic effect on Caco-2 cells. RESULTS: L. acidophilus revealed a protective effect for Caco-2 cells against S. dysenteriae and V. cholera by 51% and 57%, respectively, which was determined by MTT assay and further confirmed by morphological examination. Pretreatment of Caco-2 cells with L. acidophilus prior to exposure to V. cholerae, attenuated the CTX-B expression in V. cholerae to about 1.76 folds. Expression of Stx1 by S. dysenteriae was also down-regulated to 1.6 folds following pretreatment of Caco-2 cells by L. acidophilus. No significant difference was observed in the attenuator role of L. acidophilus in toxin production by S. dysenteriae as a colon-invasive bacterium, compared with V. cholerae, the non-invasive pathogen of small intestine. CONCLUSIONS: The results of the present study suggest that L. acidophilus is safe with protective effect for human epithelial colorectal cells, and is effective enough to be applied as a supplementary treatment for attenuation of toxin production in acute infectious diarrhea caused by V. cholerae and S. dysenteriae.

The anti-adhesive and anti-invasive effects of recombinant azurin on the interaction between enteric pathogens (invasive/non-invasive) and Caco-2 cells.

Anti-adhesion therapy and anti-adhesin immunity are meant to diminish the interaction between pathogens and host tissues, either by prevention or by exclusion of bacterial adhesion and entrance to cells. Azurin is a scaffold protein possessing antiviral, antiparasitic, and anticancer activities. The purpose of the present study was to determine the effect of recombinant Azurin (rAzurin) on the adhesion and invasion capacity of invasive (Shigella sonnei, Shigella flexneri, Campylobacter jejuni) and non-invasive (Vibrio cholerae) enteric bacteria to cells. The non-toxic dose of rAzurin and the best MOI (Multiplicity of Infection) of bacterial species was assessed by MTT assay. Bacterial species were used at MOIs of 20:1 and Azurin was applied at the concentrations of 5 and 25 µg/mL and added to Caco-2 cells in competition and replacement assay to assess the anti-adhesion and anti-invasion properties of rAzurin. The protein caused significant decrease in the adhesion rate of S. sonnei, S. flexneri, C. jejuni, and V. cholerae strains to Caco-2 cells by 43, 39, 72, and 38% in competition and 45, 46, 75, and 48% in replacement assays, respectively. Also, S. sonnei, S. flexneri, and C. jejuni strains invasion rate was reduced to 50, 50, and 70% in anti-invasion assay, respectively. The inhibitory effect of Azurin against C. jejuni and V. cholerae strains adhesion was more significant (p < .001) compared to Shigella spp. (p < .05) which may be due to smaller size of the former bacteria. On the contrary, in invasion assay, rAzurin showed a greater inhibitory effect against Shigella spp. (p < .001) compared to C. jejuni (p < .05), which may probably be due to the interaction of rAzurin with several effectors or ligands, involved in Shigella invasion and internalization. The findings of the present study opens new insights of rAzurin as a new and potent candidate for reducing or probably preventing enteric bacterial attachment, invasion, and pathogenesis.

Modulatory effect of Vibrio cholerae toxin co-regulated pilus on mucins, toll-like receptors and NOD genes expression in co-culture model of Caco-2 and peripheral blood mononuclear cells (PBMC).

Vibrio cholerae, the causative agent of cholera, tend to colonize the small intestine as a Gram-negative pathogen. The intestinal mucus layer forms mucin physical barrier, consisted of high molecular weight proteins. Regarding the role of toxin-coregulated pilus (TCP) as one of the most important colonization factors of V. cholerae, this experimental study was designed to determine the role of TcpA in induction of mucin production and its regulatory effect on innate immunity molecules including toll like receptors (TLRs) and Nucleotide-binding oligomerization domain-containing proteins (NODs) using Caco2- PBMC co-cultures as an interactive model. The rTcpA protein of V. cholerae was expressed in BL21 Escherichia coli, purified using Ni-column chromatography and confirmed by western blotting. Nontoxic doses of rTcpA was determined on Caco-2 cell lines and different concentrations of rTcpA (1, 5, 10 and 50 µg/mL) showed a statistically significant effect on the expression of muc genes (MUC3 and MUC4) in a dose-dependent manner. This finding is supposed to facilitate physical adhesion and colonization of V. cholerae in intestinal lumen. The rTcpA moderately stimulated the expression of tlr4 and overexpressed tlr1, both of which are supposed to induce a mucosal protective response against bacterial infection. NOD2 was significantly increased which suggests that it may contribute in pro-inflammatory responses observed in cholera disease. No change in NOD1 expression was seen which might be attributed to the non-invasive nature of V. cholerae as an intestinal pathogen. In conclusion, the rTcpA protein of V. cholerae showed a statistically significant modulatory effect on the human gut epithelium gene expression which would help promising protection in prophylaxis applications.

Antibacterial and antibiofilm activity of bone marrow-derived human mesenchymal stem cells secretome against Vibrio cholerae.

The ability of V. cholerae to survive and spread in the aquatic environment combined with the scarcity of effective antimicrobial agents, especially those effective against multidrug-resistant strains highlights the need for alternative non-antibiotic approaches for the treatment of V. cholerae infections. The aim of this study was to specifically examine the potential direct effect of unstimulated MSC secretome on V. cholerae killing and biofilm formation as a representative of non-invasive enteric bacterial pathogen. The bmMSCs were characterized by the presence of CD44 and CD73 and the absence of CD45 and CD34 molecular markers. Moreover, self-regeneration and differentiation capacity of MSCs into adipocytes and osteogenic lineages was assessed by immunohistology (IHC) method. The antibacterial activity of unstimulated MSCs supernatant against V. cholerae in broth microdilution assay decreased the bacterial suspension from 108 CFU/ml to 107 CFU/ml and showed a significant antimicrobial activity in a dose-dependent manner at dilutions of 1:8 to 1:128 (P < 0.05). The role of MSC secretome without preconditioning in the prevention of biofilm formation was assessed through plate-crystal violet assay and showed high antibiofilm activity against V. cholerae also in dose-dependent manner. As antibacterial mechanisms of MSC secretome are different from conventional antibiotics, together with its antibiofilm activity, proposes its application as a novel therapeutic approach combatting multi-drug resistant pathogens with no fear of developing antimicrobial resistance.

Pulsed-field gel electrophoresis fingerprinting of Campylobacter jejuni and Campylobacter coli strains isolated from clinical specimens, Iran.

The aim of this study was to determine the clonal correlation of Campylobacter strains isolated from diarrheal children under 5 years of age in Iran using the PFGE method and to determine the antimicrobial susceptibility and virulence gene content of strains. Of 750 patients with bacterial diarrhea, 33 (4%) Campylobacter spp., including 31 C. jejuni (94%) and 2 C. coli (6%), were isolated during 18-month period in Tehran, Iran. Except for one strain, remaining Campylobacter strains were positive for the flaA gene. A complete set of cytolethal distending toxin (CDT) encoding genes (cdtABC) were detected in 52% of the C. jejuni strains, while the 2 C. coli isolates under study only harbored cdtA and cdtB of the CDT cluster. All strains were resistant to at least three antibiotic classes. Resistance to ampicillin among C. coli and C. jejuni strains was 100% and 84%, respectively, and 80% of all strains were susceptible to gentamicin. PFGE genotyping generated 19 pulsotypes with two major clusters, displaying the maximum and minimum similarity of 100% and 26%, respectively. The C. coli strains showed clearly distinct pulsotypes and each fell within separate clusters. A very homogeneous Campylobacter population was detected among Iranian patients with 33 % of strains showing identical banding patterns and no clear correlation was observed between antibiotic resistance profiles and PFGE patterns of the isolates.

A sensitive gold-nanorods-based nanobiosensor for specific detection of Campylobacter jejuni and Campylobacter coli.

BACKGROUND: Campylobacteriosis is a zoonotic infectious disease that can be mostly undiagnosed or unreported due to fastidious Campylobacter species. The aim of this study was to develop a simple, sensitive, and quick assay for the detection of Campylobacter spp. and taking advantage of the great sensitivity of gold nanorods (GNRs) to trace changes in the local environment and interparticle distance. METHODS: Characterized GNRs were modified by specific ssDNA probes of cadF gene. First, the biosensor was evaluated using recombinant plasmid (pTG19-T/cadF) and synthetic single-stranded 95 bp gene, followed by a collection of the extracted DNAs of the stool samples. The sensing strategy was compared by culture, PCR, and real-time PCR. RESULTS AND DISCUSSION: Analysis of 283 specimens showed successful detection of Campylobacter spp. in 44 cases (16%), which was comparable to culture (7%), PCR (15%), and real-time PCR (18%). In comparison with real-time PCR, the sensitivity of the biosensor was reported 88%, while the specificity test for all assays was the same (100%). However, it was not able to detect Campylobacter in 6 positive clinical samples, as compared to real-time PCR. The limit of detection was calculated to be the same for the biosensor and real-time PCR (102 copy number/mL). CONCLUSIONS: Taking high speed and simplicity of this assay into consideration, the plasmonic nanobiosensor could pave the way in designing a new generation of diagnostic kits for detection of C. jejuni and C. coli species in clinical laboratories.