Detection of Integrons and Staphylococcal Cassette Chromosome mec Types in Clinical Methicillin-resistant Coagulase Negative Staphylococci Strains

OBJECTIVES: Integrons are thought to play an important role in the spread of antibiotic resistance. This study investigates class 1 and 2 integron-positive methicillin-resistant coagulase-negative staphylococci strains isolated in Iran and characterizes their patterns of antimicrobial resistance. METHODS: Hundred clinical isolates of coagulase-negative staphylococci were characterized for integron content and staphylococcal cassette chromosome mec (SCCmec) type. RESULTS: Sixteen isolates carried class 1 (intI1) integrons and four isolates carried class 2 (intI2) integrons. One resistance gene array was identified among the class 1 integrons (aadA1 cassette). The distribution of SCCmec types in 50 methicillin-resistant coagulase-negative staphylococci strains showed that SCCmec types III and V dominated among the tested strains. CONCLUSION: This is the first report of methicillin-resistant coagulase-negative staphylococci strains that carry two mobile genetic elements, including class 1 and 2 integrons and SCCmec, in Iran.

Molecular characterisation of Klebsiella oxytoca strains isolated from patients with antibiotic-associated diarrhoea

Background and study aim: Colitis is a common complication after treatment with antibiotics such as beta-lactarns, quinolones, and aminoglycosides. Recently, Klebsiella oxytoca has been implicated in this type of diarrhoea. The prevalence and characterisations of K. oxytoca isolated from patients with antibiotic-associated diarrhoea were investigated. The K. oxytoca isolates were also tested for cytotoxin production

Patients and methods: This study was conducted from May 2011 to Dec 2013. Faecal samples were collected from hospitalised patients receiving antibiotic treatment. Initial cultivation was performed on specific media. The clinical isolates were confirmed by poIymerasc chain reaction [PCR] using the specific K. oxytoca po1ygalacturonase [pehX] gene. The double-disc diffusion test was used to detect extended-spectrum beta-lactamase [ESBL]-producing strains. Tracking of ESBL-encoding genes was performed via PCR. The organism was cultured on Hep-2 cell lines for cytotoxin production

Results: Out of 331 samples collected from patients, 40 were confirmed molecularly to be clinical isolates of K. oxytoca. Fourteen [35%] ESBL-producing strains were isolated using the double-disc diffusion method. Among the molecularly confirmed K. oxytoca isolated seven [17.5%] tested positive for the blaSHV gene, 12 [30%] for blaTEM, 10 [25%] for blaCTX-M, three [75%] for blaOXA, nine [22.5%] for blaCTX M-15, and seven [17.5%] for blaTEM-1. Five [12%] isolates showed cytotoxin activity below 30%, 12 [30%] strains showed moderate cytotoxin activity between 30% and 60%, and 23 [58%] strains showed cytotoxin activity >/=60%

Conclusions: the cytotoxin-producing K.oxytoca is found to be one of the causes of antibiotic-induced colitis. Discontinuing treatment and allowing normal intestinal flora to be established or prescribing appropriate medication after antibiogram can help patients with antibiotic-induced haemorrhagic colitis in a timely manner

Antibacterial properties of composite resins incorporating silver and zinc oxide nanoparticles on Streptococcus mutans and Lactobacillus

OBJECTIVES: Recurrent caries was partly ascribed to lack of antibacterial properties in composite resin. Silver and zinc nanoparticles are considered to be broad-spectrum antibacterial agents. The aim of the present study was to evaluate the antibacterial properties of composite resins containing 1% silver and zinc-oxide nanoparticles on Streptococcus mutans and Lactobacillus. MATERIALS AND METHODS: Ninety discoid tablets containing 0%, 1% nano-silver and 1% nano zinc-oxide particles were prepared from flowable composite resin (n = 30). The antibacterial properties of composite resin discs were evaluated by direct contact test. Diluted solutions of Streptococcus mutans (PTCC 1683) and Lactobacillus (PTCC 1643) were prepared. 0.01 mL of each bacterial species was separately placed on the discs. The discs were transferred to liquid culture media and were incubated at 37degrees C for 8 hr. 0.01 mL of each solution was cultured on blood agar and the colonies were counted. Data was analyzed with Kruskall-Wallis and Mann-Whitney U tests. RESULTS: Composites containing nano zinc-oxide particles or silver nanoparticles exhibited higher antibacterial activity against Streptococcus mutans and Lactobacillus compared to the control group (p < 0.05). The effect of zinc-oxide on Streptococcus mutans was significantly higher than that of silver (p < 0.05). There were no significant differences in the antibacterial activity against Lactobacillus between composites containing silver nanoparticles and those containing zinc-oxide nanoparticles. CONCLUSIONS: Composite resins containing silver or zinc-oxide nanoparticles exhibited antibacterial activity against Streptococcus mutans and Lactobacillus.

High prevalence of multidrug resistance and metallo-beta-lactamase [MbetaL] producing acinetobacter baumannii isolated from patients in ICU wards, Hamadan, Iran

Acinetobacter baumannii is gram- negative opportunistic coccobacilli, the most important agent in nosocomial infections with high mortality rate. Multidrug resistance in strains isolated from nosocomial infections, making it difficult to treat and sometimes impossible. The aim of the present study was to investigate antibiotic resistance in A. baumannii isolates from Iranian patients in Hamadan, west of Iran. In this cross sectional study 100 A. baumannii isolated from trachea, blood, urine, sputum and wound samples of patients bedridden in Intensive care unit [ICU] wards of three educational hospitals during June 2011 to October 2012 was included. Isolates confirmed at species level using biochemical tests and tracing bla[OXA-51] gene using Polymerase chain reaction [PCR] and preserved frozen at -70 °C until examination. Their susceptibility to 17 antibiotics was performed using Kirby-Bauer disc diffusion method. Determination of minimum inhibitory concentration and Metallo-beta-lactamase production was carried out using E-test method. Resistance rate of isolates were 94%, 85%, 84%, 97%, 95% and 98% against meropenem, imipenem, amikacin, ciprofloxacin, piperacillin/tazobactam and cefotaxime, respectively. No resistant isolate was observed against tigecycline and also no sensitive isolate seen against aztreonam and cefotaxime. Results of E-test illustrated that 99% of all isolates were Metallo-beta-lactamase [MbetaL] producing, which were resistance to imipenem; also 85% of them were resistance to meropenem. MIC50 and MIC90 of the isolates were >/= 256 and >/= 32 micro/ml for imipenem and meropenem, respectively. The antibiotic resistance against most of the antibiotics, especially carbapenems is very high in Hamadan region. In addition colistin sulfate and tigecycline were most effective antibiotics and to be used in A. baumannii infections

Determination of adhesion virulence factors of enteropathogenic escherichia coli [eaeA,bfpA] isolates from asymptomatic individuals compared to those with diarrhea

The aim of the this study was to investigate the prevalence of toxB, paa, Ipf and iha adhesion genes in enteropathogenic Escherichia coli [EPEC] isolates lacking in two important adhesion factors, the eaeA and bfpA genes. We examined a total of 70 serologically confirmed EPEC [eaeA, bfpA] isolates. DNA from the isolates was extracted by the phenol-chloroform method. toxB, paa, Ipf and iha adhesion genes in the EPEC isolates were detected by polymerase chain reaction. Data were analyzed by SPSS software and statistical analysis using the chi square test. P-values less than 0.05 were considered significant. PCR was positive for the toxB gene in 2 [2.85%], paa in 3 [4.28%], Ipf in 32 [45.71%] and iha in 15 [21.42%] of the 70 strains. Statistically, none of the toxB.paa, and Ipf genes were associated with diarrhea. However, the iha gene showed a weak significant relation to diarrhea [P=0.11]. The main mechanism of pathogenicity for EPEC is attachment and effacement. Therefore, EPEC [eaeA, bfpA] should have another adhesin factor, which should be investigated. EPEC strains [eaeA-, bfpA-] that possess the Ipf gene are common. Further investigations of the virulence properties of these strains are necessary in order to elucidate the role of these virulence factors in diarrhea among Iranian children

Prevalence and antibiotic resistance patterns of diarrheagenic Escherichia coli isolated from adolescents and adults in Hamedan, Western Iran

Pathogenic strains of Escherichia coli are a common cause of acute infectious diarrhea. The aim of this study was to investigate the frequency, virulence markers and antibiotic resistance patterns of diarrheagenic E. coli [DEC] isolated from adolescents and adults in Hamadan, west of Iran. A total of 187 stool samples were collected from adults with acute diarrhea. Stool culture was performed by conventional methods for enteropathogenic bacteria. Virulence factor genes for DEC were detected by polymerase chain reaction. Antimicrobial susceptibility was tested using the disk diffusion method. Among the 187 patients, 40 [21.4%] were positive for DEC. The most frequently identified DEC was enteropathogenic E. coli [47.5%], followed by enteroaggregative [20%], enterotoxigenic [17.5%] and shiga-toxin producing E. coli [15%]. No isolates of enteroinvasive E. coli were detected. All STEC strains were stx[+] / eaeA[-]. Out of the seven ETEC strains, five [71.4%] produced ST, one [14.3%] produced only LT and one [14.3%] of the isolates produced both ST and LT encoded by est and elt genes, respectively. Among the 40 DEC strains 27[67.5%] were multidrug resistant. DEC contribute to the burden of diarrhea in adults in Hamadan. Enteropathogenic E. coli was the most commonly identified DEC strain in the region studied

Ribotyping of EPEC isolates from diarrheal and asymptomatic patients in Iran

We intended to find out the diversity of EPEC isolates among asymptomatic or diarrheal children in Iran using ribotyping. Enteropathogenic Escherichia coli [EPEC] is responsible for gastroenteritis especially in young children. A total of 39 EPEC collected strains were serotyped and the presence of virulence genes as well as EAF plasmid among the strains was studied. Adherence assay was also performed. Clonal diversity of the isolates was investigated using ribotyping. Of 39 studied strains of E. coli, 6 serogroups of EPEC were represented. The presence of the stx gene was ascertained in 7 isolates and the eaeA, eaeB and bfpA genes were harbored by 5, 3 and 1 strains, respectively. Ribotyping yielded 9 different clusters. According to our results there was not a significant correlation between the results of serotyping and those of ribotyping. However, different serotypes of E. coli may belong to the same ribotype clusters and vice versa

Molecular cloning and expression of human Gamma Interferon[IFN-Lambda] Full cDNA in Chinese Hamster Ovary [CHO] Cells

IFN-gamma is mostly secreted by activated CD4+, CD8+ T cells and NK cells. This cytokine has immunomodulatory, anti-cancer and anti-microbial effects and is important for prophylaxis, diagnosis and treatment of chronic infections and cancers. The purpose of this study was to clone the full cDNA of human IFN-gamma and express it on CHO cell line. Lymphocytes from a healthy individual were isolated and activated by phytohaemagglutinin [PHA] in vitro. After 4 hours, total RNA extracted and first cDNA str and was synthesized. cDNA was amplified with primers containing EcoRI and NotI sites. The amplified fragment and the PcDNA3.1 vector were cut by EcoRI and NotI and ligated. The construct [pcDNA3.1-IFN-gamma] was transferred into E.coli [strain: DH5 alpha] using CaCl2 method and selected by plating on a medium containing ampicillin. The construct sequence was confirmed by PCR and sequence analysis. Construct expression was achieved by performing a calcium phosphate-mediated transfection into CHO cells and followed by selection of stable drug [G418] resistant clones by limiting dilution assay [LDA]. The IFN-gamma production by transected CHO cells was measured using ELISA technique. Out of 33 grown transformed bacterial colonies, only 6 had the entire sequences of the insert and one of them was used for the transfection experiment. Out of 768 wells, 5 clones produced more than 100 ng/ml/10[6] cells of IFN-gamma. Among the 5 clones, one with the maximum production of INF-gamma [143 ng/ml/10[6] cells] was selected and used for propagation