Acinetobacter baumannii clonal lineages I and II harboring different carbapenem-hydrolyzing-beta-lactamase genes are widespread among hospitalized burn patients in Tehran

The aim of this study was to analyze antimicrobial resistance patterns and their encoding genes and genotypic diversity of Acinetobacter baumannii isolated from burn patients in Tehran, Iran. The presence of extended-spectrum beta-lactamase- and bla[OXA]-encoding genes among 37 multidrug resistant [MDR] A. baumannii strains isolated from patients hospitalized in a teaching hospital in Tehran was evaluated. Susceptibility to 7 antibiotics was tested by disk agar diffusion and to polymyxin B and colistin was tested by E-test, according to CLSI guidelines. All isolates were then analyzed by PCR for the presence of bla[IMP],bla[VIM], bla[SIM] bla[OXA-23], bla[OXA-24], and bla[OXA-58]-like carbapenemase genes, and bla[OXA-51] like, bla[TEM],bla[SHV], bla[PER], bla[VEB], and bla[GIM] genes. Genotyping of A. baumannii strains was performed by repetitive sequence-based [REP]-PCR and cluster analysis of REP-PCR profiles. A. baumannii isolates were assigned to international clones by multiplex PCR sequence group analysis. Twenty-five A. baumannii isolates were classified as MDR, and 12 were classified as extensively drug resistant. All isolates were susceptible to colistin and polymyxin B. Eighty-one percent of the isolates was resistant to imipenem or meropenem and harbored at least one or both of the bla[OXA-23]-like or bla[OXA-24]-like carbapenemase genes. Co-existence of different resistance genes was found among carbapenem-resistant isolates. Multiplex PCR sequence group analysis most commonly assigned A. baumannii isolates to international clones I [18/37; 48.6%] and II [18/37; 48.6%]. An alarming increase in resistance to carbapenems and the spread of bla[OXA-23]-like and/or bla[OXA-24]-like carbapenemase genes was observed among A. baumannii strains belonging to clonal lineages I and II, isolated from burn patients in Tehran

Detection of the Efflux-Mediated Erythromycin Resistance Transposon in Streptococcus pneumoniae

BACKGROUND: The present analysis focuses on phenotypic and genotypic characterizations of efflux-mediated erythromycin resistance in Streptococcus pneumoniae due to an increase in macrolide resistance in S. pneumoniae worldwide. METHODS: We investigated the prevalence of efflux-mediated erythromycin resistance and its relevant genetic elements from 186 specimens of S. pneumonia isolated from clinical and normal flora from Tehran, Iran. The presence of erythromycin resistance genes was tested by PCR with two sets of primers, specific for erm(B) and mef(A/E), and their genetic elements with tetM, xis, and int genes. Isolates were typed with the BOX PCR method and tested for resistance to six antibiotics. RESULTS: Antibiotic susceptibility tests revealed that 100% and 47% isolates were resistant to tetracycline and erythromycin, respectively. The erythromycin and clindamycin double-disc diffusion test for macrolide-lincosamide-streptograminB (MLSB) resistance phenotype showed 74 (84%) isolates with the constitutive MLSB phenotype and the remaining with the M phenotype. BOX PCR demonstrated the presence of 7 types in pneumococci with the M phenotype. Fourteen (16%) isolates with the M phenotype harbored mef(A/E), tetM, xis, and int genes. CONCLUSIONS: The present results suggest dissemination of polyclonal groups of S. pneumoniae with the M phenotype carrying resistance genes attributed to transposon 2009.

Correlation of Ciprofloxacin Resistance with the AdeABC Efflux System in Acinetobacter baumannii Clinical Isolates

BACKGROUND: Acinetobacter baumannii is one of the most important pathogens capable of colonization in burn patients, leading to drug-resistant wound infections. This study evaluated the distribution of the AdeABC efflux system genes and their relationship to ciprofloxacin resistance in A. baumannii isolates collected from burn patients. METHODS: A total of 68 A. baumannii clinical strains were isolated from patients hospitalized in Motahari Burns Center in Tehran, Iran. Ciprofloxacin susceptibility was tested by the disk diffusion and agar dilution methods. PCR amplification of the adeRS-adeB drug efflux genes was performed for all resistant and susceptible isolates. To assess the role of the drug efflux pump in ciprofloxacin susceptibility, carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as an efflux pump inhibitor (EPI). RESULTS: Approximately 95.6% of the Acinetobacter isolates were resistant to ciprofloxacin, with minimum inhibitory concentration (MIC) values ranging from 4 to > or =128 microg/mL. The susceptibility of 86.1% of the resistant isolates increased by factors of 2 to 64 in the presence of CCCP. All resistant isolates were positive for the adeRS-adeB genes, and 73.2% of them had mutations in the AdeRS regulatory system. CONCLUSIONS: The results showed that AdeABC genes are common in A. baumannii, which might be associated with ciprofloxacin non-susceptibility, as indicated by the observed linkage to the presence of the genes essential for the activity of the AdeABC, several single mutations occurring in the adeRS regulatory system, and an increase of ciprofloxacin susceptibility in the presence of a CCCP EPI.

effect of reduced bacterial dilution on human amniotic membrane antibacterial activity, in vitro

The human amniotic membrane is the inner most layer of placenta and has antimicrobial effect, due to the presence of human beta-defensins and elafins. The purpose of this study is to investigate the effect of dilution reduction of 0.5 McFarland prepared from standard bacterial strains of Salmonella enterica BAA-708, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, Klebsiella pneumoniae ATCC7881, and Enterococcus faecalis ATCC29212 on antibacterial effect of human amniotic membranes in vitro. Amniotic membranes were obtained from the bank of organ transplantation in Imam Khomeini hospital, of women with elective cesarean section whose HIV, HBV, HCV and VDRL serological tests were negative. They were cut to 1.5x1.5 cm pieces. Then 0.5 McFarland suspensions of 1.5x10[8], 0.5x10[7] and 1.5x10[6] dilutions were prepared from bacteria which then were spread on Mueller Hinton medium agar and a piece of membrane was put in the center of each plate. After 24 hours incubation at 37[degree]C, the results were observed. In 0.5 McFarland standard dilution an inhibition zone was created in three standard strains of Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica unlike the other two strains. There was no change in the above results with two other dilutions and inhibition zone of sensitive strains was not created. Dilution reduction of microbial strains does not affect the antibacterial impact of amniotic membrane and dilution reduction does not yield to a false positive response and the conversion of resistant to sensitive strains

Evaluation of phenotypic methods for detection of klebsiella pneumoniae carbapenemase-producing K. pneumoniae in Tehran

One of the main mechanisms of resistance to carbapenems is potential of Klebsiella pneumoniae to produce K. pneumoniae Carbapenemase [KPC]. KPC is an important type of Carbapenemase, which can hydrolyze carbapenems and other beta-lactam antibiotics. Modified Hodge Test [MHT] and use of boronic acid as a KPC inhibitor are two types of phenotypic methods, which are used for detection of carbanemase-producing bacteria. Specificity of these two phenotypic tests for identification of KPC was assessed in this study. Forty-four K. pneumoniae strains were isolated from wound infections of burn patients. All isolates were identified with specific biochemical tests. Carbapenem-resistant K. pneumoniae isolates were identified by disc diffusion method and analyzed with cut off-points of CLSI 2011 guideline. For detection of KPC-producing strains, carbapenem-resistant isolates were examined with two different phenotypic [i.e. MHT and Boronic acid] methods. Subsequently, strains with positive phenotypic methods were examined by PCR as a molecular method. Twenty-eight [64%] out of 44 isolates were resistant to carbapenem according to CLSI breakpoints and 16 [36%] were susceptible. MHT was positive in all of carbapenem-resistant isolates but none of them have had the synergism effect between meropenem and boronic acid. Also, all isolates were negative for presence of KPC genes on gel electrophoresis. According to results MHT has not enough specificity for detection of KPC

immunohistochemistry and toluidine blue roles for helicobacter pylori detection in patients with gastritis

Background: helicobacter pylori, which is associated with many upper gastrointestinal diseases, is found in half of the population of the world. Several special stains and immunohistochemistry stain for H. pylori are available. The need for and usefulness of immunohistochemical [IHC] technique has been debated for years. Toluidine blue is a simple stain for microbiological studies and is easily available in laboratories. Therefore, this study was conducted to compare hematoxylin and eosin [HandE], Giemsa and toluidine blue staining with immunehistochemistry for detection of H. pylori in patients with gastritis and also to correlate the results of these staining methods with pathological grading

Methods: we reviewed 54 consecutive gastric biopsy specimens stained by HandE and Giemsa as well as by toluidine blue and immunohistochemistry stains for H. pylori

Results: H. pylori was positively identified by IHC in 43 [79.63%] patients, while positive samples were found in 18 [33.33%], 24 [44.44%] and 33 [61.11%] patients using HandE, Giemsa and toluidine blue staining methods. Our results showed that classical histological staining methods are not sensitive enough to identify low numbers or coccoid forms of organism, while toluidine blue and immunohistochemistry play an important role in detection of H. pylori infection

Conclusion: toluidine blue has been proved to be much more reliable than HandE and Giemsa in detection of H. pylori. In addition, in post treatment biopsies and in biopsies with unexplained chronic active gastritis without histological evidence of H. pylori should have immunohistochemistry done to detect possible low density or coccoid form of organisms. Iran. Biomed. J. 17 [1]: 36-41, 2013

Investigation of class I, II and III integrons among acinetobacter strains isolated from ventilator-associated pneumonia patients in intensive care unit of Rasoul Akram Hospital in Tehran, Iran

Multi-drug resistant strains of Acinetobacter spp. have created therapeutic problems worldwide. The objective of this study was to detect integrons in Acinetobacter spp. isolates from Ventilator-Associated Pneumonia patients using PCR method. A total 51 Bronchoalveolar lavage samples were obtained from patients in ICU and examined for Acinetobacter spp. infection by biochemical and PCR methods using blaOXA51-like primers. Antimicrobial susceptibility testing was performed using disk diffusion and MIC methods. Among 51 patients with VAP [62.7% males, 35.2% females, mean age 53 year], 50 [98%] were positive, with a high prevalence of gram-negative bacteria, mainly Acinetobacter spp. [70%], from which A. baumani was detected in 34 [68%] and A. lwoffii in 1 [2%] of isolates. More than 90% of isolates were resistant to imipenem, piperacillin+tazobactam, third generation cephalosporins and gentamicin, while the most effective antibiotic was colistin [100%]. The correlation coefficient between disk diffusion and MIC was 0.808 [p = 0.001]. Three Acinetobacter isolates [8%] harbored integrase /gene but none of isolates contained Class II or III integrons. The results showed that colistin was an effective antibiotic and can be used for treatment of patients in ICU. Due to the high number of MDR isolates lacking Integrons it can be concluded that although class I integrons are important among clinical isolates of A. baumannii, they have no significant role in dissemination of antibiotic resistance genes in Rasoul Akram Hospital in Tehran, Iran. The presence of IntI in A. Iwoffii may be related to transfer of integron to A. baumannii which can be considered as an important threat for hospitalized patients

[ effect of amniotic membrane transplantation in healing of primary stages of pseudomonas keratitis]

Human amniotic membrane [HAM] forms the inner wall of the membranous sac that surrounds and protects the embryo during gestation. The main advantages of amniotic membrane transplantation [AMT] in the treatment of bacterial keratitis are its epithelial bandage properties. Previous studies have documented the presence of some antimicrobial proteins and peptides in amniotic fluid such as lactoferrin, lysozyme, bactericidal or permeability increasing protein, calprotectin [MRP8/14 protein complex], LL37, and neutrophil defensins [Human Neutrophil Peptides, HNP 1-3]. Furthermore, the amniotic membrane does not express HLA-A, B, C or DR surface antigens, which may help avoid rejection after its transplantation. Thus, it can be used as a biological immune barrier. The purpose of this study was to evaluate the effectiveness of the amniotic membrane's healing properties in rabbits with pseudomonas keratitis. By using an animal model, 14 rabbits were divided into two groups of controls and cases. A syringe was used to inoculate the corneal stroma of the animals by Pseudomonas aeruginosa ATCC27853. After 20 hours pseudomonas keratitis was created and amniotic membrane was transplanted to the cornea of the case group. The infiltration size were observed on the first, third and seventh days after the experiment. Corneal perforation was seen in the controls [P<0.001] but amniotic membrane prevented perforation in the case group [P=0.02]. Transplantation of amniotic membrane in the primary stages of pseudomonas keratitis treatment remarkably prevents corneal perforation and it can be used to control the disease process

Effect of amniotic membrane combined with ciprofloxacin in curing the primary stages of pseudomonal keratitis

Keratitis caused by Pseudomonas aeruginosa is often resulted in severe corneal ulcers and perforation, which leads to losses of vision. Human amniotic membrane [HAM] forms the inner wall of the membranous sac which surrounds and protects the embryo during gestation. The purpose of this study was to evaluate the effectiveness of the amniotic membrane's healing in rabbits with pseudomonas keratitis. In total 14 rabbits divided in 2 groups of: 1 as Control and 2 as experimental amniotic membrane combined with ciprofloxacin. A 0.05 ml suspension of Pseudomonas aeruginosa ATCC 27853 was injected into rabbit's corneal stroma, with no interference in control group. In the second group, the amniotic membrane in pieces of 1.5 x 1.5 cm transplanted to the entire corneal surface by eight interrupted 10.0 nylon sutures. In the first day ciprofloxacin drop was injected to the second group every 30 minutes and through second to seventh days every 2 hours. The results of perforation in cornea and the amount of infiltration were registered. The results showed that amniotic membrane transplantation [AMT] + ciprofloxacin group had 0% perforation and the control group 85.6%. Average infiltrations were 5 mm in AMT + ciprofloxacin groups and 23.75 mm in control. The use of amniotic membrane with ciprofloxacin was effective in prevention of cornea perforation and controlling the process of pseudomonal keratitis remission. The improvement of inflammation rapidly happened in ciprofloxacin + AMT group.

Identification of KPC-producing pseudomonas aeruginosa and acinetobacter baumannii in a burned infant: a case report

The objective of this study was to determine the phenotypic characteristics of KPC-producing Pseudomonas aeruginosa and Acinetobacter baumannii isolates. A case report study was performed at a tertiary burn care centre in Tehran, Iran. Nine isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from a hospitalized case were isolated. The identity of isolates was confirmed and their antibiotic susceptibility testing was performed. Eight out of nine Pseudomonas aeruginosa and Acinetobacter baumannii isolates were resistant to Imipenem. Three out of 8 imipenem resistant isolates were also positive for KPC test. Findings of this study highlight the importance of implementation of an effective infection control strategy in order to prevent and reduce the emergence and spread of gram negative Carbapenemaseproducing organisms in Iran.

[ prevalence of nosocomial infections caused by Enterobacter cloacae and antibiotic resistant patterns in samples isolated from patients in two hospitals in Tehran]

The increasing use of beta-lactam antibiotics in clinics for the treatment of different bacterial infections since early 1980s has led to increased rates of resistant bacteria isolated from patients. One of the problems in the treatment of nosocomial infections is related to resistant bacteria such as Enterobacter cloacae due to cross resistance through extended-spectrum beta-lactamase production. The aim of this study was to determine the prevalence of extended-spectrum beta-lactamase producing E. cloacae from different clinical specimens collected from hospitalized patients. In the present study, 101 E. cloacae confirmed by standard specific microbiologic tests were collected from different specimens in Milad and Motahri hospitals in Tehran, Iran during February 2010 and September 2011. Antibiotic susceptibility tests were conducted according to the process recommended by the Clinical and Laboratory Standards Institute for 13 antibiotics of choice. Extended-spectrum beta-lactamase producing strains were screened for by combined disk method as a phenotypic diagnostic test. From a total of 101 E. cloacae, 33 [33%] were shown to produce extended-spectrum beta-lactamase by phenotypic tests; 5% of the bacteria were resistant to imipenem too. This study clearly showed the high prevalence of resistance to broad-spectrum beta-lactam antibiotics in the isolated E. cloacae among which 5% were multi drug resistant. All the isolated E. cloacae were susceptible to Colistin. These results can be alarming for physicians treating resistant E. cloacae infections, especially extended-spectrum beta-lactamase producing species

[ prevalence of extended-spectrum beta-lactamases and CTX-M-1 producing escherichia coli in urine samples collected at Tabriz city hospitals]

Numerous use of Beta Lactame in treatment of bacterial infections resulted in increments of drug resistance of such bacteria. One of difficulties in treatment of hospital infections is Extended Spectrum Beta Lactamase [ESBL] among isolated clinical strains of E.coli. Since some of ESBL strains shows double reaction in drug sensitivity test at in vitro and in vivo condition, therefore it makes difficulties in selection of right treatment. In the last years, CTX-M enzymes have become the most prevalent ESBLs in worldwide. The prevalence of ESBL types largely remains unknown in many parts of the Iran. This study was made to find the prevalence of ESBLproducing E.coli and molecular detection of CTX-M-1 in Tabriz. In the present study, 400 urine samples collected between November 2009 and April 2010, from Tabriz Hospitals were studied. Out of the 400 samples, 188 E.coli isolates were detected by standard biochemical tests. Susceptibility to antimicrobial agents was tested to 10 antibiotics by the disk agar diffusion [DAD] method. ESBL production was screened by phenotypic test that included both separate and combined disk agar diffusion techniques. The screened isolates were investigated by PCR assay to detect CTX-M-1 gene. From the total 188 E.coli isolates, 82 [43.6%] were shown to produce ESBLs by phenotypic test. During the PCR method on the 82 isolates, 69 [84.1%] were confirmed as CTX-M-1 producing group. The present study showed that CTX-M-producing isolates were increasing among E.coli strains and indicated the need for adequate susceptibility tests in laboratories for choosing the appropriate antibiotics for treatment

randomized controlled trial of 5-day regimen of azithromycin and a 10-day regimen of co-amoxiclav for treatment of acute sinusitis

Acute sinusitis constitutes a significant portion of health service utilization globally both in- and outpatient as well as emergency department visits, with 83% resulting in a prescription for an antibiotic. This study compared the efficacy of a 5-day regimen of azithromycin [a macrolid antibiotic] with a 10-day regimen of coamoxiclav [combination of an aminopenicillin with a betalactamase inhibitor] for the treatment of acute sinusitis. A total of 76 subjects with acute sinusitis were randomly assigned in two groups, azithromycin [n=40] and co-amoxiclav [n=36]. One group received azithromycin, 500mg in the first day and 250mg for 4 days and the other group received co-amoxiclav 625mg, 3 times a day for 10 days. Patients were visited 4 times during the study [baseline, phone call, end of treatment, end of study] and regression/progression of their symptoms and their response to the treatment was evaluated. There was no significant difference between the two groups' demographic and clinical presentations. Duration of regression of the symptoms in the azithromycin group was significantly shorter than the co-amoxiclav group [7.6 days versus 10.6, p=0.03]. Clinical success rate at end of the study was 80% for azithromycin and 66.7% for co-amoxiclav [p=0.025]. Clinical success rates among females in both groups seemed to be higher than males, but this difference was not statistically significant [p=0.13]. Results revealed that azithromycin regimen is more efficient, has less side effects, and required shorter treatment period. Patients were able to tolerate the medications better with a higher compliance and less economic cost than co-amoxiclav regimen

[Evaluation of presence of the bla-SHV and bla-AmpC [CITM, FOX] beta-lactamase genes in clinical isolates of Escherichia coli]

Resistance to beta-lactam antibiotics along with clinical isolates is frequently resulting production of beta-lactamase enzymes. In recent years, the production of extended spectrum beta-lactamases [ESBLs] and AmpC beta-lactamase among clinical isolate especially Escherichia coli is greatly increased. On the other hand, beta lactamase genes have several subfamilies, and designing universal primers could be valuable to detect all of them. Therefore, the aim of this study was to survey prevalence of phenotypic ESBLs and detection of SHV and AmpC [CITM, FOX]-type beta-lactamase genes by using universal primers through PCR. A total of 500 clinical samples were collected from hospitals of Tehran and 200 E.coli isolates were detected by standard biochemical tests. Subsequently, these isolates were screened for beta-lactamase production by Disk diffusion method and combined disk. Resistant isolates were evaluated for molecular assessing of SHV, CITM and FOX genes by using PCR. Among entire of 200 E.coli, 128 [64%] isolates were selected via phenotypic tests for detection of bla-SHV and bla-AmpC [CITM, FOX] genes via PCR. With 95% confidence, 7[5.5%] and 13[10.2%] E.coli harbor bla-SHV and bla-CITM, respectively. Fox gene was not detected in any samples. Results were showed that complete detection of beta-lactamase enzymes is essential for resistant control and the appropriate prescription of beta-lactam drugs. So using molecular assay with phenotypic test is important

Characterization of Pseudomonas aeruginosa in burn patients using PCR- restriction fragment length polymorphism and random amplified polymorphic DNA analysis

One of the major opportunistic pathogens in patients with burn injuries is Pseudomonas aeruginosa, which causes severe infections in burned patients. The objective of the study was to examine the molecular epidemiology of P. aeruginosa colonization in the burn unit of Shahid Motahari Hospital in Tehran, Iran. Restriction fragment length polymorphism [RFLP] and random amplified polymorphic DNA [RAPD] analysis were employed to study 127 clinical and two environmental P. aeruginosa isolates collected from January to June 2008. In RFLP, the PCR products of 16S rRNA gene were digested with restriction enzyme Alu I, Hae III, and Rsa I, and the fragments generated were analyzed by agarose electrophoresis. Molecular typing by RFLP did show no discriminatory power for P. aeruginosa isolates, but RAPD-PCR revealed eight different genotypes; RAPD 1 to RAPD8 in clinical and environmental isolates. RAPD1 was the major genotype in clinical [n=64, 50.4%] and environmental isolates [n=l, 50%]. The findings suggest that RAPD might have a superior typeabil-ity and discriminatory power over RFLP to study P. aeruginusa. Moreover, they highlight the need for further attention to the control of infection sources in Burn Units to prevent the transmission of the bacterium

Comparison of 3-day and 7-day ciprofloxacin regimen for the treatment of uncomplicated urinary tract infection in women: a randomized double-blind clinical trial

Urinary tract infections [UTIs] are among the most commonly bacterial infections in clinical practice. Almost half of all women experience at least one urinary tract infection in their lifetime. This study compared efficacy and safety of 3-day and 7-day ciprofloxacin regimen for the treatment of uncomplicated urinary tract infection in women. A total of 76 patients were randomly assigned to two treatment groups. One group received ciprofloxacin, 250 mg twice a day for 3 days [n=39] and the other group received ciprofloxacin 250 mg twice a day for 7 days [n=37]. Subjects were visited and assessed three times during the study period [baseline, end of treatment, and test for cure]. Clinical and bacteriological responses to the treatment were compared between the two groups. There was no significant difference between the two groups in terms of age distribution and clinical signs/symptoms during the baseline visit. There was no significant difference between clinical or bacteriological responses between the two groups. Three-day regimen of ciprofloxacin showed high microbiological eradication rate for E. coli [66.7%] which was similar to the eradication rate observed for 7-day regimen [64.8%]. No statistically significant difference was found in adverse effects between the groups, except for nausea [p=0.041]. A 3-day ciprofloxacin regimen appeared to be safe and effective for the treatment of UTI in women. Therefore, shorter therapy duration with ciprofloxacin can potentially improve patient compliance and decrease costs