Investigation of Efflux-Mediated Tetracycline Resistance in Shigella Isolates Using the Inhibitor and Real Time Polymerase Chain Reaction Method.

BACKGROUND: Shigella spp. are gram negative bacteria, which are of global public health importance. The growing of multidrug-resistant Shigella isolates are a major problem around the world. METHODS: Overall, 50 isolates of Shigella spp. from children diarrheic stools were studied. The isolates were identified and confirmed using biochemical, serological and molecular methods (ipaH, wbgZ and rfc genes). Antimicrobial susceptibility test was done according to the Clinical and Laboratory Standards Institute (CLSI) guidelines against minocycline, tetracycline, doxycycline, ampicillin, streptomycin, trimethoprim-sulfamethoxazole, nalidixic acid, norfloxacin, ciprofloxacin and levofloxacin. Also, the role of efflux pump in defense of Shigella against tetracycline was investigated by Minimum Inhibitory Concentration (MIC) with and without an efflux pump inhibitor. Detection of tetA, tetB, tetC and tetD genes in Shigella was evaluated by conventional Polymerase Chain Reaction (PCR) and real time PCR. RESULTS: Molecular identification revealed a prevalence of 14% for Shigella flexneri and 86% for Shigella sonnei. Minimum Inhibitory Concentration (MIC) of 90% of resistant isolates was changed in the presence CCCP. Results of conventional PCR exhibited that 66% of isolates were positive for tetA, while according to real time PCR method, 90% of isolates carried tetA. Positive results for tetB were 12% and 18% by conventional and real time PCR methods, respectively. No positive results were detected for tetC and tetD. Also, tetB was detected only in S. flexneri while tetA was detected in both S. flexneri and S. sonnei. CONCLUSION: It seems that efflux-mediated tetracycline resistance to tetracycline in S. flexneri can be related to tetB, however resistance in S. sonnei can be related to the expression of tetA.

Multi-locus sequence type analysis of Shigellas pp. isolates from Tehran, Iran.

BACKGROUND AND OBJECTIVES: Strains of Shigella spp. can cause shigellosis, or bacillary dysentery. that is a public health problem worldwide. The aim of this study was to describe the population structure and genetic relatedness of multidrug resistant S. sonnei and S. flexneri isolated during a one year period from children with diarrhea in Tehran, Iran. MATERIALS AND METHODS: A total of 70 Shigella spp. were detected during the study period. Twenty MDR isolates of Shigella spp. were randomly selected and used in this study. Bacterial identification was performed by conventional biochemical and serological and confirmed by molecular method. After antimicrobial susceptibility testing, we used Multilocus sequence typing (MLST) for subtyping isolates. RESULTS: We found 14 Shigella sonnei and 6 Shigella flexneri isolates. Results of MLST showed five sequence types (ST) (145, 152, 241, 245, 1502) and BURST analysis revealed the largest number of single locus variant (SLV) and highest frequency (FREQ) for ST152. ST 152 with nine members was predicted as the founder by BURST. Frequency for ST 1502 and ST 245 was four isolates and the least frequency was seen for ST 241 and 145 with one and two members, respectively. ST 145 and ST 245 were described as singletons in BURST. All isolates with ST145 and ST245 were identified as Shigella flexneri. CONCLUSION: Annual Multi locus sequence typing of MDR Shigella would help us in better understanding of dominant species and comparing our results with the same studies in other countries especially our neighbor countries in source tracking purposes.

Prevalence of Panton-Valentine leucocidin and phenotypic and genotypic characterization of biofilm formation among Staphylococcus aureus strains isolated from children with adenoid hypertrophy.

Adenoids as a first line of host defense against respiratory microbes play an important role in majority of upper airway infectious and noninfectious illnesses. Bacterial pathogen can colonize on the adenoid tissue and probably act as a reservoir for them. To determine phenotypic and genotypic characterization of biofilm forming capacity of Staphylococcus aureus isolates from children with adenoid hypertrophy and prevalence of Panton-Valentine leukocidin (PVL) gene we collected 17 consecutive, clinically significant S. aureus isolates from children with adenoid hypertrophy undergoing adenoidectomy with one or more of the upper airway obstruction symptoms, nasal obstruction, mouth breathing, snoring, or sleep apnea. Biofilm formation was evaluated by colorimetric microtiter plate's assay. Gene encoding PVL and adhesion- or biofilm formation-encoding genes were targeted by polymerase chain reaction (PCR) assay. According to the results, all strains produced biofilm. Seven (41.2%) isolates produced strong biofilm whereas 7 (41.2%) isolates produced week and 3 (17.6%) isolates produced medium biofilm. Regarding the adhesion- or biofilm formation-encoding genes, 16 (94.1%) isolates were positive for the gene eno, 13(76.4%) for icaA, 13 (76.4%) for icaD, 10 (58.8%) for fib, 10 (58.8%) for fnbB, 4(23.5%) for can, and 1(5.8%) for fnbA. The high prevalence of genes encoding biofilms and adhesins and phenotypic ability to form a biofilm by S. aureus strains emphasizes the pathogenic character of strains isolated from children with adenoid hypertrophy.

Calcium Enriched Mixture and Mineral Trioxide Aggregate Activities against Enterococcus Faecalis in Presence of Dentin.

INTRODUCTION: The purpose of this in vitro study was to compare the antibacterial activity of Calcium Enriched Mixture (CEM) with ProRoot Mineral Trioxide Aggregate (MTA) against Enterococcus faecalis (E. faecalis) in the presence/absence of dentin powder. MATERIALS AND METHODS: Two series of freshly mixed (10, 50, and 100 mg), set crushed powder (10, 50, and 100 mg), and pieces of uncrushed set (50, 100 mg) of CEM and MTA were prepared (n = 32 groups). All samples were suspended in normal saline for direct exposure test against E. faecalis; in the second series, 50 mg of the dentin powder was also added to the solution. Dentin powder suspension and bacterial suspension served as negative and positive control groups, respectively (n = 2). The suspensions were incubated at room temperature for 1, 60, and 240 min; each group was tested five times and survival of the bacteria in test solutions was assessed by 10-fold serial dilutions and cultured on Brain Heart Infusion (BHI) plates. The plates were incubated at 37ºC. The mean values of log10 CFU were calculated and compared in all tested groups. The total number of tests added up to 510 times. RESULTS: In presence of dentin powder, freshly mixed powder from set materials, and pieces of uncrushed set materials of both tested cements killed > 95% of the bacterial cell in 1 min. Adding dentin powder caused an increase in antibacterial activity of freshly mixed powder from crushed set CEM and MTA but no acceleration in bacterial killing was observed, when dentin was mixed with set or uncrushed cements. Dentin powder alone reduced the number of viable bacteria in the 4-hour duration. There were no significant differences between different weights of freshly mixed, crushed set powder and uncrushed set of CEM cement and MTA at different times. CONCLUSION: Under the conditions of this in vitro study, CEM cement as well as MTA have antibacterial effects against E. faecalis. The addition of equal amounts of dentin powder to the suspension of CEM or MTA resulted in swifter elimination of bacteria.

Novel durable bio-photocatalyst purifiers, a non-heterogeneous mechanism: accelerated entrapped dye degradation into structural polysiloxane-shield nano-reactors.

This research has designed innovative Ag/TiO(2) polysiloxane-shield nano-reactors on the PET fabric to develop novel durable bio-photocatalyst purifiers. To create these very fine nano-reactors, oppositely surface charged multiple size nanoparticles have been applied accompanied with a crosslinkable amino-functionalized polysiloxane (XPs) emulsion. Investigation of photocatalytic dye decolorization efficiency revealed a non-heterogeneous mechanism including an accelerated degradation of entrapped dye molecules into the structural polysiloxane-shield nano-reactors. In fact, dye molecules can be adsorbed by both Ag and XPs due to their electrostatic interactions and/or even via forming a complex with them especially with silver NPs. The absorbed dye and active oxygen species generated by TiO(2) were entrapped by polysiloxane shelter and the presence of silver nanoparticles further attract the negative oxygen species closer to the adsorbed dye molecules. In this way, the dye molecules are in close contact with concentrated active oxygen species into the created nano-reactors. This provides an accelerated degradation of dye molecules. This non-heterogeneous mechanism has been detected on the sample containing all of the three components. Increasing the concentration of Ag and XPs accelerated the second step beginning with an enhanced rate. Further, the treated samples also showed an excellent antibacterial activity.

A high prevalence of mupirocin and macrolide resistance determinant among Staphylococcus aureus strains isolated from burnt patients.

Infections due to Staphylococcus aureus have become increasingly common among burn patients. The antibiotic resistance profile of S. aureus isolates and inducible resistance against clindamycin were investigated in this study. The presence of mecA gene, mupA gene and macrolide resistance genes were detected using PCR and multiplex-PCR. The resistance rate to methicillin, erythromycin and mupirocin were 58.5%, 58% and 40%, respectively. The prevalence of constitutive and inducible resistance among macrolide resistant isolates was 75% and 25%, respectively. Ninety five percent of the isolates were positive for one or more erm genes. The most common genes were ermA (75%), ermC (72%) and ermB (69%), respectively. The ermA gene predominated in the strains with the inducible phenotype, while ermC was more common in the isolates with the constitutive phenotype. The msrA gene was only found in one MRSA isolate with the constitutive phenotype. A total of 27 isolates (25%) carried the mupA gene. All the mupirocin resistant isolates and almost all the erythromycin resistant isolates were also resistant against methicillin which may indicate an outbreak of MRSA isolates with high-level mupirocin and erythromycin resistance in the burn unit assessed.

Time-kill study and synergistic activity of cell-wall inhibitor antibiotics in combination with gentamicin against Enterococcus faecalis and Enterococcus faecium.

The synergy between gentamicin and vancomycin, teicoplanin, ampicillin and linezolid was studied by time-kill method. Two clinical vancomycin resistant enterococci (VRE) and two vancomycin susceptible enterococci (VSE) isolates were used. Different concentrations of antibiotics were combined. Two VSE strains and the control strain exhibited synergism with the combination of gentamicin, vancomycin, teicoplanin, ampicillin and linezolid. Two VRE strains exhibited synergism with the combination of gentamicin and ampicillin. Synergy between gentamicin and vancomycin, teicoplanin and linezolid was not observed against these isolates. The VRE isolates were positive for vanA, aac (6')-Ie aph (2") and aph (3')-IIIa genes and their vancomycin, teicoplanin and gentamicin MICs were 512 µg/ml, 512 µg/ml and >4000 µg/ml, respectively. In order to treat serious enterococcal infections, further clinical evaluation is needed to examine the in vitro combined effects of gentamicin and vancomycin, teicoplanin and linezolid.

Molecular analysis and antimicrobial susceptibility of methicillin resistant Staphylococcus aureus in one of the hospitals of Tehran University of Medical Sciences: high prevalence of sequence type 239 (ST239) clone.

Methicillin-resistant Staphylococcus aureus (MRSA), particularly the multidrug-resistant clones, is an increasing worldwide problem. The average incidence rate of MRSA in Tehran was found to be over 40%. A total of 140 MRSA isolates obtained from patients attending a teaching hospital in Tehran, from May 2009 to December 2009, were included in this study. The antimicrobial susceptibility profile of MRSA isolates was determined by the agar disk diffusion method. Molecular analysis of MRSA strains was accomplished by Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST). Detection of mecA gene was used to confirm resistance to methicillin among the MRSA isolates. All the MRSA isolates were susceptible to chloramphenicol, teicoplanin, tigecycline and vancomycin. All MRSAisolates were resistant to oxacillin, whilst 139 strains showed resistance against ciprofloxacin, erythromycin, gentamicin, tetracycline and trimethoprim-sulfamethoxazole. PFGE analysis of all the 140 MRSA isolates produced five distinct pulsotypes designated as pulsotypes A-E. Most of the isolates (n=132) were clustered into pulsotype A. The most prevalent sequence type (ST) was ST 239 (pulsotype A) found in 82% (37/45) of the tested isolates. The second most prevalent type was ST 1238 (pulsotypes B, C and D) found in 15% (7/45) of the isolates. The remaining type, ST 8 (pulsotype E) was found in a single isolate. The results of this study indicated that the MRSA clone ST 239 was a major clone in the selected university hospital of Tehran and that it was widely spread among the different wards as well as all the age groups of patients.

A novel technique for producing durable multifunctional textiles using nanocomposite coating.

The treatment of textiles with Ag/TiO(2) nanoparticles causes a brownish color that limits the application of this otherwise good composite. In this paper, a novel method is introduced to overcome this problem. To this end, the effect of various concentrations of cross-linkable polysiloxane (XPs) and Ag mixed with XPs on TiO(2) treated fabrics has been investigated. The results reveal the performance of the method in the application of Ag and TiO(2) nanoparticles separately. In addition to the major effect of XPs on durability, the synergistic effect of applying XPs, especially Ag mixed with XPs, on TiO(2) has been confirmed. Unexpectedly, increasing the concentration of XPs not only did not limit the TiO(2) activity but allowed light absorption by the TiO(2) particles due to the low refractive index of XPs. Therefore, XPs treatment can be helpful for increasing the bioactivity and the general photo-catalytic activity of TiO(2). The results also showed that the hydrophilicity-hydrophobicity of treated substrate can be adjusted over a broad range by controlling the concentrations of these two nanoparticles and the XPs ratio. Other characteristics of treated fabrics such as antibacterial, self-cleaning, stain photo-degradability, UV protection, air permeability and washing durability were also investigated.

Phenotypic and genotypic evaluation of aminoglycoside resistance in clinical isolates of staphylococci in Tehran, Iran.

Aminoglycosides play an important role in the treatment of staphylococcal infections, despite the emerging widespread resistance among Staphylococcus. To determine the prevalence of aminoglycoside resistance and aminoglycoside modifying enzyme (AME) genes among infected patients at a teaching hospital in Tehran, Iran, we tested 585 Staphylococcus isolates, of which 322 were Staphylococcus aureus and 263 were coagulase-negative staphylococci, as determined by the disk diffusion method and multiplex PCR. The minimum inhibitory concentration of gentamicin for each isolate was determined by microbroth dilution. All methicillin-resistant staphylococci were mecA-positive by PCR. Of the 585 isolates, 27.6% were susceptible to gentamicin and kanamicin, 27.1% to tobramicin and amikacin, and 21.3% to netilmicin. The most prevalent AME genes included aac(6')-Ie-aph(2'') (93.7%) followed by aph(3')-IIIa (84.3%) and ant (4')-Ia (28.1%). More than 90% of aminoglycoside-resistant staphylococci contained at least one AME gene. The coexistence of two or three AME genes was detected in most gentamicin-resistant isolates. These results suggest an alarming rate of aminoglycoside resistance in this test location in Tehran, Iran. Continued surveillance at the genotypic and phenotypic levels, and adherence to well-designed antibiotic and infection-control policies are necessary to limit the spread of antimicrobial resistance.

Emergence of high-level vancomycin-resistant Staphylococcus aureus in the Imam Khomeini Hospital in Tehran.

OBJECTIVE: The objective of the study was to investigate the prevalence of vancomycin-resistant Staphylococcus aureus (VRSA). MATERIALS AND METHODS: Three hundred and fifty-six S. aureus isolates from the Imam Khomeini hospital in Tehran, Iran, were evaluated for methicillin and decreased vancomycin susceptibility by the microbroth dilution method. The mecA, vanAand vanB genes were targeted by polymerase chain reaction. RESULTS: Of the 356 isolates, 149 (41.85%) S. aureus strains were resistant to methicillin. Two strains of methicillin-resistant S. aureus were VRSA strains. One isolate, Teaching Hospital-1 (TEH-1), had a vancomycin minimum inhibitory concentration (MIC) of 64 microg/ml and was susceptible to teicoplanin while the other isolate (TEH-2) had a vancomycin and teicoplanin MIC of 512 and >256 microg/ml, respectively, and was positive for the vanA gene. CONCLUSION: This report shows that the emergence of VRSA in Iran warrants active microbiological surveillance and careful monitoring of vancomycin therapy.