Evaluation of Iranian Scientists Productions in Biotechnology and Applied Microbiology based on ISI through 2000 to 2008

In most countries, internationally indexed scientific publications provide the possibility for the scientometric experts to study their scientific progress. In Iran, the number of scientific productions indexed in ISI was 323 in 1993 and rose to 14832 in 2008 showing about 46 manifolds. The main aim of this research is to illustrate the dynamic structure of scientific productions of Iranian biotechnologists and applied microbiologists in WOS database during 2000–2008. The data was gathered through searching WOS database of ISI. The field of search was country (CU=IRAN). 681 scientific products were reported to be indexed in ISI. Iranians' international collaboration has been mainly with Canadian, Swedish and Australian coauthors. Compared to other Iranian universities, University of Tehran, Tarbiat Modares University and Tehran University of Medical Sciences have contributed mostly to ISI. Iranian biotechnologists’ and microbiologists’ intercontinental collaboration is generally in Biochemistry and Molecular Biology, Engineering and chemical and Biochemical research. There has been an increase in the fundamental research activities in Biotechnology and Applied Microbiology research projects which have triggered motivation for higher contribution to ISI

Antibacterial efficacy of lytic bacteriophages against antibiotic-resistant Klebsiella species

Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: <10 4 PFU/mL, medium: 10 4 -10 7 PFU/mL, and high: >10 7 PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method. KEYWORDS: bacteriophage, Klebsiella, antibiotic resistance Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1333 INTRODUCTION Bacteriophages (also called phages) are reported to be the most abundant organisms on earth Based on the replication type, phages are classified as either lytic or lysogenic. A lytic phage replicates in the bacterial host and destroys its host in a process, but a lysogenic phage inserts itself into the genome of its bacterial host and establishes a stable position in the infected bacterium After discovery, phages were the target of multiple research for the treatment of bacterial diseases, such as dysentery In spite of the great progress that has been made in the field of antimicrobial therapy, the appearance and spread of drug-resistant bacteria has caused a serious challenge in recent decades. As an example, the prevalence of resistant nosocomial infections is increasing at an alarming rate and their elimination is very difficult. This could be secondary to the wide use of antibiotics, as well as application of therapeutic measures that weaken the immune system and make subjects more susceptible to nosocomial infections. Phage therapy could be an effective alternative approach for the control of these infections, as several studies have shown their efficacy against both Gram-positive and Gram-negative bacteria The purpose of the present study was to isolate and enrich lytic bacteriophages against Klebsiella spp. and evaluate their antibacterial efficacy against antibiotic-resistant species. The impact of phage concentration, incubation duration, and method of culture (tube vs. plate) on the bactericidal effect was also investigated. MATERIALS AND METHODS Isolation of Klebsiella spp. Different samples, mainly from urine, vaginal smears, blood, wounds and their secretions, and burn lesions, were collected from patients referring to the Ghaem Hospital (Mashhad, Iran) during a course of about 1.5 years between November 2001 and March 2003. Samples were cultured on general (simple blood agar; supporting the growth of most microorganisms) as well as specific (MacConkey agar, desoxycholate agar, or eosin methylene blue agar; supporting the growth of Gram-negative bacteria) culture media. Culture media plates were incubated at 37°C for 24 h. To confirm the isolation of Klebsiella spp., Gram staining and multiple biochemical tests were performed, including glucose and lactose fermentation (Kligler iron agar medium), citrate utilization (Simmons citrate agar medium), urea (urea agar medium), hydrogen sulfide production, indole formation and motility (sulfide-indole-motility [SIM] agar medium; Kligler iron agar medium), and malonate utilization (malonate agar medium) tests. Determination of Klebsiella spp. Sensitivity to Antibiotics Mueller-Hinton agar medium was used to culture the appropriate bacteria. Colonies were first suspended in 5 mL of tripticase soy broth and kept at 37°C for several hours until the turbidity of the suspension changed, similar to that of barium sulfate solution in the 0.5 McFarland standard tube (the standard tube was shaken vigorously before usage). A sterile swab was stirred in the above suspension and the sample was cultured on Mueller-Hinton agar medium. Antibiotic disks were placed at a 15-mm distance from the Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1334 edge of the plate. Different disks were 24 mm from the center of each nearest disk. Following a 24-h incubation at 37°C, the growth inhibition zone was measured and compared with tables provided by the National Committee for Clinical Laboratory Standards (NCCLS). The results of sensitivity were reported as sensitive, resistant, or intermediate. Antibiotics that were evaluated included ampicillin, amoxicillin, amikacin, cephalexin, chloramphenicol, nitrofurantoin (for urine samples), gentamicin, kanamycin, nalidixic acid (for urine samples), rifampin, streptomycin, tetracycline, doxycycline, tobramycin, and sulfamethoxazole. Smooth agar containing glycerin was used to keep resistant Klebsiella colonies at -20°C as follows: four to five colonies were transferred to 20 mL of triple soy broth. After 4 h of incubation at 37°C, the tube containing tryptone soy broth was centrifuged at 2500 rpm. Then, 0.5 mL of the above-cultured bacteria was transferred to a Pyrex® test tube containing 3 mL of 3% Mueller-Hinton. Test tubes were incubated at 37°C for 4-6 h in order to accelerate bacterial growth. Following that, 0.5 mL of sterile glycerin was added to test tubes and tubes were transferred to -20°C. Isolation, Enrichment, Titration, and Bacteriophages Bacteriophages utilized in this study were isolated from wastewater of the septic tank in Ghaem Hospital that had been filter sterilized. To the aforementioned wastewater (45 mL), concentrated nutrient broth medium (5 mL) and 4-h antibiotic-resistant Klebsiella culture (5 mL) were added. Also added was 1% (v/w) MgSo 4 to provide optimum attachment of bacteriophage to bacteria. The mixture was then gently shaken and kept at 37°C for 24 h. Afterwards, chloroform was added (3 mL) and the mixture was shaken for 15 min. After being kept at room temperature for 2 h, the mixture was centrifuged (30 min, 3500 rpm) and the supernatant carefully isolated. For phage enrichment, the obtained supernatant was mixed with nutrient broth (10 mL) and 4-h Klebsiella culture (2 mL). The mixture was then processed as described above. Phage suspension was maintained in the nutrient broth at 4°C in a dark place using sterile and sealed glass containers. For the titration of phages, enriched samples were diluted by 10X in tubes containing 9 mL of tryptone broth. Then, 100 µL of each diluted sample was transferred to tubes containing 3 mL of soft agar. Afterwards, 4-h Klebsiella culture (1 mL) was added to each tube. Tubes were then shaken and their contents rapidly transferred to plates containing tryptone agar medium. The plates were incubated at 37°C for 24 h. Plates containing 30-300 plaques were used to calculate the number of phages in the primary solution using the following formula: Number of phages = Number of plaques × dilution titer × volume of media Evaluation of Antibacterial Activity The antibacterial effects of phages against antibiotic-resistant Klebsiella spp. were tested by the tube method and the double-layer agar plate method at two time points: after 1 h (for the tube method) and 24 h (for both tube and plate methods) of incubation at 37°C. In each method, three different concentrations of phages were tested: low (<10 4 PFU/mL), medium (10 4 -10 7 PFU/mL), and high (>10 7 PFU/ mL). According to the intensity of growth inhibition, the results were reported as +++ (75-100% reduction of bacteria compared to control), ++ (50-75% reduction of bacteria compared to control), + (25-50% reduction of bacteria compared to control), and -(<25% reduction of bacteria compared to control). Statistical Analysis All comparisons were performed using Fisher's exact test. A two-sided p value of <0.05 was considered to be statistically significant. Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1335 RESULTS Out of the total samples that were collected during the course of the study (a period of approximately 1.5 years), 72 antibiotic-resistant Klebsiella spp. were isolated. Most of these species were isolated from urine, wounds, and burn lesion samples Karamoddini et al.: Bacteriophages Against Resistant Klebsiella Species TheScientificWorldJOURNAL (2011) 11, 1332-1340 1336 In the tube method, different concentrations (low, medium, and high) of phages were evaluated for their inhibitory effect against the growth of isolated, resistant Klebsiella spp. after 1 and 24 h of incubation at 37°C. The results indicated that in both time points, all three assessed concentrations had antibacterial effects without even one strain being unaffected by phage treatment. There was a marked increase in the antibacterial effects after 24 h compared to 1 h of incubation, and this was observed for all three assessed phage concentrations. There was also a positive association between phage concentration and observed antibacterial effects at both assessed time points. This effect of concentration was found to be of high statistical significance when comparing the antibacterial effects of low concentration to those of medium (p < 0.001) and high (p < 0.001) concentrations. However, while there was a significant concentration effect at the 1-h incubation time point between medium and high phage concentrations (p < 0.001), no significant difference was observed after 24 h of incubation (p > 0.05) ( DISCUSSION The most obvious result to emerge from the present study was the promising antibacterial effects of phages against resistant Klebsiella spp. at all assessed (low, medium, and high) concentrations. The results also indicated that bactericidal effects of phages are augmented with increasing concentration and time of incubation. In addition, the double-layer agar plate method was associated with higher bactericidal effects compared to the tube method. Bactericidal effects of phages at low concentrations are due to their self-replication property. At low concentrations, the number of phages is exponentially increased in the presence of bacterial host 1337 Phages possess some unique properties that make them promising candidates for the treatment of bacterial infections. First, they need to bind to specific surface receptors in order to enter the bacteria and exert their effects. Hence, their bactericidal effects would be specific. Second, since eukaryotic cells lack phage receptors, phage preparations appear be harmless to human, animal, and plant cells Several reports have demonstrated the efficacy of phages in the treatment of infectious diseases caused by Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Vibrio vulnificus, and Salmonella spp., and also Gram-positive bacteria, such as Enterococcus faecium and Staphylococcus aureus 1338 In spite of the positive findings on the therapeutic efficacy of phages, this strategy has not been introduced into routine clinical practice for the treatment of bacterial infections. This stems from several reasons, the most important of which are the advent and widespread use of antibiotics in the Western world as well as the inconsistency and unsuccessful results of early trials. The main reasons for the inconsistent findings of the early trials are (1) inadequate scientific methodology that was used; (2) not heeding the prerequisites for phage therapy, such as lack of complete knowledge on phage biology, including lysogeny phenomenon (which might have led to the employment of a wrong phage); (3) lack of placebo control and robust trial design; (4) not identifying pure phage strains; (5) not meeting safety requirements for phage preparations, such as endotoxin removal; (6) not confirming adequate phage viability in the employed preparations; and (7) rapid clearance of phages from the body. The modern generation of phage research has attempted to overcome these shortcomings and promising results have been obtained. However, there is still much work to be done in order to extrapolate positive in vitro findings into more complicated in vivo experiments In recent years, there have been relatively few studies on the efficacy of phage therapy against Klebsiella infection, particularly resistant Klebsiella spp. The promising results of this investigation add to the existing body of literature about the potential efficacy of phage therapy. As Klebsiella spp. are among the most important causes of noscomial infections As a limitation of the current study, it must be mentioned that the 24-h bacterial cultures were not tested for bacteriophage resistance. Furthermore, it would be helpful to evaluate the bactericidal efficacy of phage preparations in more detailed time points. To sum, the results of this research support the idea that phages are effective bactericidal agents that could serve as potential alternatives for antibiotics in the treatment of resistant bacterial infections. In addition, the present findings provide evidence with respect to the impact of concentration, incubation duration, and method of culture on the bactericidal effects of phages. ACKNOWLEDGMENT

Antibacterial Efficacy of Lytic Bacteriophages against Antibiotic-Resistant Klebsiella

Bacterial resistance to antibiotics is a leading and highly prevalent problem in the treatment of infectious diseases. Bacteriophages (phages) appear to be effective and safe alternatives for the treatment of resistant infections because of their specificity for bacterial species and lack of infectivity in eukaryotic cells. The present study aimed to isolate bacteriophages against Klebsiella spp. and evaluate their efficacy against antibiotic-resistant species. Seventy-two antibiotic-resistant Klebsiella spp. were isolated from samples of patients who referred to the Ghaem Hospital (Mashhad, Iran). Lytic bacteriophages against Klebsiella spp. were isolated from wastewater of the septic tank of the same hospital. Bactericidal activity of phages against resistant Klebsiella spp. was tested in both liquid (tube method; after 1 and 24 h of incubation) and solid (double-layer agar plate method; after 24 h of incubation) phases. In each method, three different concentrations of bacteriophages (low: 107 PFU/mL) were used. Bacteriophages showed promising bactericidal activity at all assessed concentrations, regardless of the test method and duration of incubation. Overall, bactericidal effects were augmented at higher concentrations. In the tube method, higher activity was observed after 24 h of incubation compared to the 1-h incubation. The bactericidal effects were also higher in the tube method compared to the double-layer agar plate method after 24 h of incubation. The findings of the present study suggest that bacteriophages possess effective bactericidal activity against resistant Klebsiella spp. These bactericidal activities are influenced by phage concentration, duration of incubation, and test method

Burden of Bites by Dogs and Other Animals in Los Angeles County, California, 2009-2011

OBJECTIVES: The objective of this study was to analyze all animal-to-human bite reports during a 3-year period from a regional surveillance database. Results helped to inform local efforts to reduce and prevent animal-to-human bites. METHODS: We reviewed all cases of animal-to-human bites occurring from 2009 through 2011 that were reported to the Los Angeles County Department of Public Health’s Animal Bites Database. We collected data on the bite victim’s date of birth, age, and address; bite circumstances (ie, date, time, location, how bite occurred); anatomic site and treatment of bite; type of reporting facility; and breed and management of biting animal. RESULTS: From 2009 through 2011, 26   169 animal-to-human bites were reported, of which 23   103 (88%) were dog bites. Most animal-to-human bites (n = 7673, 29%) occurred between 4 pm and 8 pm and peaked during the month of July (n = 2663, 10%). Most animal-to-human bites occurred outdoors (n = 8772, 34%) and while victims engaged in recreational activities (n = 4353, 17%). The hands were the most common injury site (n = 9130, 35%), and only 1% of animal bites (n = 267) resulted in hospitalization. A total of 4115 bite victims (16%) received tetanus vaccinations. Of all animal-to-human bite cases, medical organizations reported 13   451 (51%), and animal control agencies reported 10   682 (41%). CONCLUSIONS: Animal-to-human bites can often lead to medical complications. Surveillance is essential in helping to identify, manage, and reduce these highly preventable injuries and direct public health actions and policies on animal bite risk and prevention