دو ماهنامه میکروب شناسی پزشکی ایران
سال هفتم شماره 2 (تابستان 1392)

Pathogenic bacteria are important causes of disease in humans and agricultural products contamination. Side effects of chemical preservatives necessitate research on the use of a special blend of natural oils to prevent the growth of bacteria. The aim of this study was elucidating the antibacterial effect of the essential oil of Teucrium polium on human pathogenic bacteria in the Kerman province. In order to investigate the chemical composition and antibacterial activity of essential oil of Teucrium polium, the leaves with young branches of this plants which grows naturally in a village in Kerman province at full flowering stage in May 2010 were collected. The samples were cleaned and then dried in the shade. The plants essential oil was prepared by hydro-distillation method. Essential oil was analyzed by capillary gas chromatography (GC) using flame ionization (FID) and capillary gas chromatography coupled mass spectrometry (GC/MS). The antibacterial activity of the essential oil was determined against Gram negative and Gram positive bacteria by disc diffusion method. The total oil content of Teucrium polium plant was 0.75%. Twenty eight compounds were identified in the essential oil that included 99.75% of the total oil. The major components were α-pinene (12.52%), Linalool (10.63%) and Caryophyllene oxide (9.69%). For study of antimicrobial activity of the oil sample, the essential oil was tested against 9 bacteria by disc diffusion method. The antimicrobial effects of this essential oil was determined against three Gram positive bacteria Staphylococcus areous (PTCC 1431), Staphylococcus epidermidis (PTCC 1436), Streptococcus faecalis (PTCC 1237); as well as six Gram negative bacteria Pseudomonas aeroginosa (PTCC 11430), Shigella flexneri (PTCC 1716), Kellebsiella pneuomonae(PTCC=1053), Salmonella typhi (PTCC=1609), Serratia marcescens (PTCC 1187) and Escherichia coli (PTCC 1533). The antimicrobial effects of this essential oil on the Gram positive bacteria (Staphylococcus aureus and Staphylococcus epidermidis) and on all the Gram negative bacteria tested was much higher than those observed by tetracycline. The results showed the essential oil of Teucrium polium had strong anti-bacterial effects. The relatively high contents of α-pinene and Linalool in the essential oil may be the cause of its potential medicinal effects

Microwave radiation power of 180, 360 and 540 W was used. The bacterial strain used was Pseudomonas aeruginosa (ATCC 27853). Bacterial aerosols in the range of 107 and 106 of bacteria per ml of air, which was prepared using a nebulizer, was injected into the system. The results of this study showed that the number of bacteria remaining in the system decreased with increasing microwave power. There was absolutely no bacterial growth following 240 min of exposure to 540 watt radiation density for 107 bacteria in each ml of air current. Microwave radiation with high functionality can be used to remove bacterial air pollutions. They can help to control biological agents in hospitals and medical centers with good efficiency.

Listeria monocytogenes is a facultative intracellular pathogen that causes listeriosis which has extensive clinical manifestations. Infections with L. monocytogenes are a serious threat to immunocompromised persons. The aim of this study was to determine the frequency of L. monocytogenes strains recovered from clinical and non-clinical samples using phenotypic methods and confirmed by PCR. In this study, 617 specimens were analyzed. All specimens were cultured in the specific PALCAM agar. Colonies were initially identified by routine biochemical tests. Finally, PCR assays using primers specific for inlA gene were performed. In all, 46 (8.2%) L. monocytogenes isolates were recovered from 617 specimens. Fourteen (8.2%) strains, including 4 (7.5%), 2 (5.7%), 5 (14.2%) and 3 (8.5%) isolates were obtained from placental tissue, urine, vaginal and rectal swabs, respectively. In addition, 9 (7.4%) strains of L. monocytogenes which were isolated from 107 different dairy products originated from cheese 5 (7.1%), cream 2 (10%) and kashk 2 (11.7%), respectively. Among 11 (5.2%) strains isolated from 210 different meat products, 5 (5.5%), 4 (7.2%) and 2 (3%) strains belonged to sausage, meat and poultry extracts, respectively. Finally, 12 (9.2%) Listeria strains were recovered from 130 animal specimens that included 6 (10%), 4 (8%) and 2 (10%) strains from goat, sheep and cattle, respectively. Furthermore, all Listeria isolates (100%) were found to be carriers of inlA gene in PCR assay. The present study showed that the clinical and non-clinical specimens were contaminated with L. monocytogenes. So, it seems necessary to use a simple and standard technique such as PCR for rapid detection of this organism from various sources.

Uropathogenic Escherichia coli strains are the predominant causative organisms of urinary tract infections (UTIs). Aminoglycosides are clinically useful antibiotics with bactericidal activity against this bacterium. The most common mechanism for resistance to these antibiotics are mediated through production of aminoglycoside modifying enzymes (AMEs). The most common of these enzymes are Aminoglycoside Acetyltransferases (AACs). The epidemiology of the dominant type of these enzymes, AAC(3)-II, varies from region to region. The aim of this study was to determine the antimicrobial susceptibility pattern with a focus on aminoglycosides and the prevalence of aac(3)-IIa gene among clinical isolates of uropathogenic Escherichia coli obtained from Delfan, Lorestan, Iran. In this descriptive study, a total of 100 uropathogenic Escherichia coli isolates were collected from BoAli hospital in Delfan city, Lorestan, from July to November 2010. Antibiotic susceptibility patterns of the isolates were determined using disk diffusion method according to Clinical and Laboratory Standards Institute)CLSI) guidelines. Prevalence of aac(3)-IIa gene was determined by PCR and the relationship between resistance phenotypes to aminoglycosides and presence of aac(3)-IIa gene was evaluated. Among the 100 tested isolates, maximal resistance was seen to ampicillin (85%); whereas, no resistance to imipenem was found. Sixty percent of the isolates demonstrated resistance to at least one of the tested aminoglycosides. Resistance rate towards these agents were as followed: gentamicin 39%, kanamycin 26%, neomycin 31% and amikacin 1%. Forty–four isolates (44%) harbored the aac(3)-IIa gene. The maximal rate of gene presence (36 isolates, 92.3%) was detected in strains with gentamicin resistant phenotype (39 isolates, 39%). On the basis of our findings, use of antibiotics such as nitrofurantoin, amikacin or imipenem are recommended for empirical treatment of UTIs. In addition, locally widespread distribution of aac(3)-IIa gene will pose concerns about progressive resistance against potent aminoglycosides such as gentamicin, tobramycin and others in the future.

A lot of children suffer from urinary infections which can lead to pyelonephritis and cystitis. The main cause of these infections is uropathogenic strains of Escherichia coli. Serogroups and virulence factors of this bacterium play an important role in occurrence of infection’s symptoms. Unfortunately, due to the occurrence of antibiotic resistances, common treatments are ineffective and costly. The aim of this study was to study the distribution of virulence factors, serogroups and antibiotic resistance properties of uropathogenic Escherichia coli isolated from pediatrics with pyelonephritis and cystitis. In this study which was conducted during the year 2012 to 2013, 121 Escherichia coli strains were isolated from boys (51 samples) and girls (70 samples) with pyelonephritis and cystitis impatient in Baqiat-alah Hospital, Tehran. Antibiotic resistance of the isolates against commonly used antibiotics in the cases of urinary infections was investigated using disk diffusion method. Finally, PCR reactions using specific primers were done in order to detect virulence factors, serogroups and antibiotic resistance genes. Totally, 45.09% of boys and 55.71% of girls were positive for presence of uropathogenic Escherichia coli. The highest distribution of bacterium was seen in less than one year old boys (71.42%) and 5-12 years old girls (75%). Results showed that the distribution of Escherichia coli in children with pyelonephritis and cystitis were 55.55% and 44.89%, respectively. The most commonly detected serogroups in children with pyelonephritis were O1, O2, O4 and O7 while, the most commonly detected serogroups in children with cystitis were O1, O2, O6 and O16. The most frequent virulence factors in the cases of pyelonephritis were pap (100%), afa (97.5%), sfa (95%), hly (95%) and cnf-1 (92.5%) and their most frequent in the cases of cystitis were fim (100%), pap (72.7%), afa (63.6%) and hly (45.4%). The genes which encode antibiotic resistance against gentamicin (aac(3)-IV) (96.7%), beta-lactams (blaSHV and CITM) (90.3% and 88.7%, respectively), tetracycline (tetA) (82.2%), had the highest incidence in the bacterial isolates. Escherichia coli isolates had the highest antibiotic resistance against gentamicin (95.1%), ampicillin (91.9%), amikacin (85.4%) and ciprofloxacin (83.8%). Determination of the antibiotic resistance pattern in uropathogenic Escherichia coli isolated from children with urinary infections is important in each area or in hospital. Imipenem, due to its low antibiotic resistance, can be an effective drug for treatments of children with pyelonephritis and cystitis.

Different methods have been used for detection of shiga toxins; such as, cell culture, ELISA, and RFPLA. However, all of these methods suffer from high cost, time-consumption and relatively low sensitivity. In this study we used Multiplex PCR method for detection of genes encoding shiga toxins. In this study, 63 clinical samples were obtained from positive cultures of Shigella and E. coli O157, from Bahman 1391 until Ordibehesht 1392 in Mazandaran province. Initial confirmation of shiga toxins producing bacteria was performed by biochemical and serological methods. After DNA extraction, detection of stx1 and stx2 genes was accomplished by multiplex PCR. For confirmation of the PCR amplicon, DNA sequencing was used. Antibiotic sensitivity tests were performed by disk diffusion method. Among the positive strains, 13 strains contained stx2 genes, 4 strains contained Stx/Stx1 genes and 4 strains harbored both Stx/Stx1 and Stx2. The DNA extracted from other Gram-negative bacteria was not protected by the relevant parts of these toxins. Sequencing of the amplified fragments indicated the correct toxin sequences. The sensitivity for identification of Stx/Stx1 gene was 1.56 pg/ µl and for Stx2 was 1.08 pg/µl. The toxin positive strains were all sensitive to Cefixime, Gentamicin, Amikacin, Ceftriaxone, and Nitrofurantoin. This method is fast and accurate for detection of bacteria producing shiga toxin and can be used to identify different types of shiga toxin.

Lactococcus lactis (L. lactis) is one of the most important microorganisms used in dairy industry for production of fermented milk products. Bacteriophages which attack L. lactis are a serious threat to the dairy industry because of their negative effects on fermentation processes. Samples of raw milk were examined for the presence of lactococcal bacteriophages. Samples were centrifuged and then filtered through 0.45µm pore size filters. The filtrates were added to early-exponential cultures of Lactococcus lactis subspp. Lactis (PTCC 1336). Overlay method was used to detect the formation of plaques. After isolation and concentration of phages, serial dilutions of phage stock were used to determine titer of phage in concentrated sample. Electron Microscopy was used for observation and characterization of structural details of bacteriophages. Two phages were isolated; one of them had a hexagonal head of 45×30 nm in diameter and a flexible non-contractile tail of 70nm long which belonged to Siphoviridae. The other had a short tail and a hexagonal head of 53×60 nm in diameter which was a member of Podoviridae family. In this study, for the first time, two phages were isolated from milk. This does not reduce the significance of phage control in different stages of the production. The spread of the phages in the production plant can be very harmful.

Ice cream is a dairy product that is very popular during warm seasons. Ice cream can be contaminated with various microorganisms including pathogenic bacteria if hygienic procedures are not followed during preparation, distribution and preservation processes. This may put the health of people using ice cream at risk. Our study aimed to examine the bacterial contamination of traditional ice creams in Kermanshah city during 2008. During summer 2008, 80 samples of traditional ice creams were collected. The samples were examined according to the Iranian National Standard protocols for E. coli, Coliforms, Salmonella, Staphylococcus aureus and complete count of microorganisms. Overall, 62 (77.5%) samples had microbial contaminations more than the standard limit. Results showed 59 (73.75%) and 54 (67.5 %) of the samples contained a high number of microorganisms and coliform, respectively. Furthermore 30 (37.5%) and 23 (28.75%) of the samples were contaminated with E. coli and S. aureus, respectively. However, Salmonella spp. was not found in any of the ice cream samples. The traditional ice creams tested in Kermanshah were heavily contaminated with bacteria. It could be due to the inappropriate preparation and preservation procedures using unpasteurized milk and other materials. Contaminations may also be induced by personals. So it is recommended to apply the hygienic procedures for preparation and preservation of ice cream including the use of pasteurized milk and other materials.