Hyaluronic acid/silicon nanoparticle scaffold induces proliferation and differentiation of mouse spermatogonial stem cells transplanted to epididymal adipose tissue.

Spermatogonia stem cells (SSCs) are a unique cell population maintaining male spermatogenesis during life, through their potential for proliferation and differentiation. The application of silicon nanoparticles (SNs) and hyaluronic acid (HA) to induce the differentiation of SSCs seems promising. Herein, we investigate the effect of SN and HA scaffolds on the progression of SSCs spermatogenesis in mice. Initially SSCs were isolated from healthy immature mice and cultured on prepared scaffolds (HA, SN, and HA/SN) in a 3D culture system. Then viability of SSCs cultured on scaffolds was examined using MTT assay and Acridine Orange staining. Then SSCs cultured on scaffolds were transplanted into epididymal adipose tissue (EAT) in mature mice and the result was studied by H&E and IHC staining 8 weeks after transplantation. MTT and Acridine Orange analysis revealed that among three different scaffolds HA/SN based scaffold causes considerable toxicity on SSCs (P < 0.05) while H&E staining showed that culture of SSCs on HA, SN, and HA/SN scaffolds has a positive effect on the progression of SSCs spermatogenesis after transplantation into EAT. IHC staining identified TP1, TEKT1, and PLZF as crucial biomarkers in the spermatogenesis development of SSCs transplanted to EAT. According to the presence of these biomarkers in different experimental groups, we found the most spermatogenesis development in SSCs cultured on HA/SN scaffold (PLZF, P < 0.01) (TEKT1, P < 0.01) (TP1, P < 0.001). Our study showed that, although the cytotoxic effect of the HA/SN scaffold decreases the viability rate of SSCs; however, SSCs that survive on HA/SN scaffold showed more ability to progress in spermatogenesis after transplantation into EAT.

Agonist and antagonist NMDA receptor effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture.

In this work, agonist and antagonist N-methyl-D-aspartate (NMDA) receptor activation effect on cell fate during germ cell differentiation and regulate apoptotic process in 3D organ culture were studied. Afterwards, the effect of D-serine, retinoic acid (RA) and MK801 on spermatogenesis development was investigated. The animals were injected a single dose (40 mg/kg, intraperitoneal) of busulfan. After confirming the model, ten 5-day-old NMRI mice were used as spermatogonial stem cells (SSCs) transplantation donors. The SSCs were confirmed by detecting the promyelocytic leukaemia zinc finger (PLZF) protein. Then, tissue culture of the azoospermia model which had received SSCs was performed in various conditions (seven groups). The apoptosis markers levels of cells were significantly decreased in differentiation media containing RA and serine. In contrast, the expression of apoptotic markers including caspase 3, caspase 9 and Bax was increased in the presence of MK801. In conclusion, a new in vitro system capable of producing mature spermatozoa was developed that would be useful for investigating the medicinal effects of agents on the male reproductive system. Also, a comparison of spermatogenesis development in different media revealed that the presence of D-serine and RA (retinoic acid) in the culture medium has a positive effect on spermatogenesis.

Characterization of biofilm formation, antimicrobial resistance, and staphylococcal cassette chromosome mec analysis of methicillin resistant Staphylococcus hominis from blood cultures of children.

INTRODUCTION:: Methicillin resistant Staphylococcus hominis (MRSHo) has been recognized as an important human pathogen, particularly in immunocompromised patients. METHODS:: A total of 19 S. hominis isolates were collected from children at the Children's Medical Centre, Tehran, Iran, from March 2012 to February 2013. MRSHo susceptibility against 13 antimicrobial and 3 antiseptic agents was determined using disk diffusion (DAD) and minimum inhibitory concentration (MIC), respectively. All isolates were subjected to polymerase chain reaction (PCR) assay for 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs). Biofilm production of the isolates was determined using a colorimetric microtiter plate assay. RESULTS:: Of the 19 isolates, 16 were resistant to oxacillin and harbored mecA. High resistance was also observed against trimethoprim/sulfamethoxazole (81.2%). All MRSHo isolates were susceptible to the three disinfectants tested (Septicidine-PC, Septi turbo, and Sayacept-HP). In total, 15 (78.9%) isolates produced biofilms. Three isolates had SCCmec types (V and VIII), 13 were untypable (UT), and 5 had ACME type II. CONCLUSIONS:: The results indicate that MRSHo with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients such as children.

Characterization of biofilm formation, antimicrobial resistance, and staphylococcal cassette chromosome mec analysis of methicillin resistant Staphylococcus hominis from blood cultures of children

Abstract INTRODUCTION: Methicillin resistant Staphylococcus hominis (MRSHo) has been recognized as an important human pathogen, particularly in immunocompromised patients. METHODS: A total of 19 S. hominis isolates were collected from children at the Children's Medical Centre, Tehran, Iran, from March 2012 to February 2013. MRSHo susceptibility against 13 antimicrobial and 3 antiseptic agents was determined using disk diffusion (DAD) and minimum inhibitory concentration (MIC), respectively. All isolates were subjected to polymerase chain reaction (PCR) assay for 15 distinct resistance genes, staphylococcal cassette chromosome mec (SCCmec), and arginine catabolic mobile elements (ACMEs). Biofilm production of the isolates was determined using a colorimetric microtiter plate assay. RESULTS: Of the 19 isolates, 16 were resistant to oxacillin and harbored mecA. High resistance was also observed against trimethoprim/sulfamethoxazole (81.2%). All MRSHo isolates were susceptible to the three disinfectants tested (Septicidine-PC, Septi turbo, and Sayacept-HP). In total, 15 (78.9%) isolates produced biofilms. Three isolates had SCCmec types (V and VIII), 13 were untypable (UT), and 5 had ACME type II. CONCLUSIONS: The results indicate that MRSHo with high antibiotic resistance and unknown SCCmec might become a serious problem in the future for the treatment of patients such as children.

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.

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad.

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.

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.

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.