Detecting enteropathogenic Campylobacters in chicken feces by PCR and comparing with culture method

امروزه کامپیلوباکترها از شایع ترین علل اسهال های باکتریایی در سرتاسر جهان محسوب می شوند. در جنس کامپیلوباکتر 18 گونه و تحت گونه وجود دارد که گونه های Campylobacter jejuni و Campylobacter coli مسبب اکثر عفونت های انسانی ناشی از کامپیلوباکترها می باشند و مهم ترین بیماری آنها نیز اسهال می باشد. بیماری در انسان متعاقب مصرف آب و مواد غذایی بویژه گوشت ماکیان رخ می دهد. تشخیص باکتریولوژیک این باکتری با دشواری زیادی از قبیل طولانی بودن مدت انکوباسیون و محدودیت تست های افتراقی همراه است. بنابراین استفاده از روش های دیگر تشخیصی امروزه مورد توجه قرار گرفته است. تعداد 116 نمونه رکتال سواپ از مرغ های موجود در مرغداری های شهر اصفهان گرفته شده و پس از 18-12 ساعت غنی سازی در محیط Campy-Thio، در محیط اختصاصی Campylobacter selective agar کشت شدند. برای شناسایی و تعیین گونه ها از رنگ آمیزی گرم و تست های اکسیداز، کاتالاز، هیدرولیز هیپورات و سنجش حساسیت به نالیدیسیک اسید و سفالوتین استفاده شد. جهت استخراج DNA از کیت های اختصاصی استفاده شد و با استفاده از پرایمرهای ویژه گونه های ژژونی و کلی اقدام به انجام PCR (Palymeras Chain Reaction) گردید. نتیجه کشت در 11 مورد مثبت بود (5/9) که از این تعداد 8 مورد مربوط به گونه ژژونی و 3 مورد مربوط به گونه کلی بود. نتیجه PCR نمـونه هـا در 27 مـورد مثـبت بود (2/23) که از این تعداد 18 مورد مربوط به گونه ژژونی و 9 مورد مربوط به گونه کلی بود. حساسیت و ویژگی روش PCR در مقایسه با روش کشت بترتیب 100 و 7/84 درصد بود. روش PCR مورد استفاده در این تحقیق قادر به شناسایی تمام نمونه های کشت مثبت بود و نتایج تعیین گونه با این روش تطابق کاملی با روش بیوشیمیایی داشت. بنابراین با توجه به دقت بالا و سرعت زیاد به نظر می رسد روش PCR جایگزین مناسبی جهت کشت در تشخیص کامپیلوباکترها در نمونه های مرغ ها باشد

Determining a pattern for antibiotic resistance in clinical isolations of pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is an opportunistic pathogen and one of the important factors of hospital infection. It causes many issues such as urinary tract infections, respiratory infection in cystic fibrosis patients, and wound infection in burn patients, septicemia and meningitis. Antibiotic resistance through various mechanisms is one of the challenges for the treatment of pseudomonad-caused infections. According to the inherent and acquired capacity of this bacterium in creating resistance against the antimicrobial factors, it is very important to identify a pattern for its antibiotic resistance. The aim of this study was to deliberate the frequency of pattern antibiotic resistance of pseudomonas aeruginosa strains. Methods: In this cross-sectional study, 200 pseudomonas aeruginosa isolations (from 86 males and 114 females) were collected from different samples such as urine, blood, wound, catheter and other samples from teaching hospitals in Zahedan City during nine-month period in 2017. After conducting biochemical tests and confirming bacterium type, based on Clinical Laboratory Standards Institute (CLSI), the antibiotic resistance of strains for 10 antibiotics was determined using disk diffusion method. In addition, the minimum inhibitory concentration of three antibiotics such as imipenem, piperacillin/tazobactam and ceftazidime were determined through E-test. The Chi-square test was used for statistical analysis through the SPSS software, version 16 (IBM SPSS, Armonk, NY, USA). Results: Out of 200 pseudomonas aeruginosa isolations (from 86 males and 114 females), the maximum resistance was related to ciprofloxacin (37%) and gentamicin (28.5%). The minimum resistance was related to piperacillin/tazobactam (6.5%) and ceftazidime (6%). The highest separated strain was from urine sample (54%), blood sample (23.5%) and wound sample (10.5%). Additionally all strains were sensitive to colistin. In this study, the percentage of multidrug-resistance (MDR) and extensively drug-resistant (XDR) strains were investigated, which were 13% and 5.5%, respectively. Conclusion: In this study, pseudomonas aeruginosa isolates had the lowest resistance to ceftazidime which this antibiotic could be the main treatment option. The high prevalence of MDR strains is a serious warning

Coexistence of Virulence Factors and Efflux Pump Genes in Clinical Isolates of Pseudomonas aeruginosa: Analysis of Biofilm-Forming Strains from Iran

Background. Biofilm formation and efflux pumps (EPs) correlation play a critical role in the pathogenicity and antibiotic resistance of Pseudomonas aeruginosa. In this study, biofilm formation and EP’s collaborative role in clinical isolates of P. aeruginosa infection were investigated. Methods. Eighty-six (86) P. aeruginosa isolates were collected from different clinical specimens and were confirmed using different biochemical tests. The formation of biofilm was investigated by using a crystal violet assay. Also, EP genes were identified by the PCR method. Results. Based on the results, gentamicin-resistant (n = 50, 66.29%) and ciprofloxacin-resistant (n = 61, 69.66%) strains were the most frequent and colistin (n = 1, 1.12%) and ceftazidime (n = 12, 7.86%) resistant strains were the least prevalent. Furthermore, 22 isolates (31.42%) were MDR, and 11 isolates (12.35%) were XDR strains. Also, 19 isolates (22.47%) were classified as strong biofilm, 29 isolates (21.34%) as moderate biofilm, and 3 (11.23%) isolates as weak biofilm producers. The distribution of the EP genes was as follows: mexA (n = 44, 34.83%), mexB (n = 33, 32.58%), oprM (n = 59, 29.21%), oprD (n = 61, 30.33%), tetA (n = 22, 25.58%), tetR (n = 19, 22.09%), and emrE (n = 21, 24.41%). However, there was a strong significant association between biofilm formation and EPs in P. aeruginosa. Conclusions. In this study, we suggested that the presence of a multidrug resistance efflux pump, MexEF-OprN, significantly reduced P. aeruginosa pathogenicity. In contrast, the presence of the MexAB-OprM and MexCD-OprJ pumps did not affect virulence

Comparison of Susceptibility Testing of E-test Strips with Cefoxitin and Oxacillin Disks in Identification of Methicillin-resistant Staphylococcus saprophyticus Strains

Background and Objectives: Over the years, Staphylococcus saprophyticus, in addition to becoming a serious pathogen, has become resistant to a wide range of antibiotics. Offering an inexpensive, rapid, and available method for identification of resistant bacteria can prevent the spread of infections caused by these bacteria. The aim of this study was to compare susceptibility testing of E-test strips with cefoxitin and oxacillin disks in identification of methicillin-resistant Staphylococcus saprophyticus strains in clinical samples. Methods: In this study, 590 isolates of Staphylococcus saprophyticus were isolated from different parts of treatment centers in Zahedan city. After confirmation of the genus of isolates, resistance to methicillin, was defined in 262 samples using cefoxitin 30 μg and oxacillin 1 μg antibiotic disks. To determine minimum inhibitory concentration, E-test strips and cefoxitin and oxacillin disks, were used. The mecA gene was identified by PCR and considered as gold standard. Results: Sensitivity of the test performed for cefoxitin and oxacillin antibiotics in the disk diffusion method, was 89.09% and 68.81%, respectively, and in the minimum inhibitory concentration, was 98.18% and 97.27%, respectively. Out of 262 samples, 110 were identified to be resistant to methicillin based on PCR assay. Conclusion: According to the findings of this study, oxacillin antibiotic disk showed acceptable sensitivity to determine methicillin-resistant Staphylococcus saprophyticus strains. Also, the methods based on the use of E-Test strips had the highest sensitivity and specificity

An Investigation of Toxic Shock Syndrome Toxin-1 Gene in Methicillin-Resistant Clinical Strains of Staphylococcus aureus using Multiplex PCR Method

Background and Objectives: Toxins produced by the bacteria are one of the most common cases, which can, together with other bacterial pathogens, cause or aggravate the disease. One of the diseases caused by bacterial toxins, is toxic shock syndrome. The tst gene encodes this toxin that can be easily transferred between different strains of Staphylococcus aureus. In this study, toxic shock syndrome toxin-1 gene was investigated in methicillin-resistant clinical strains of Staphylococcus aureus using multiplex PCR method. Methods: This study is a cross-sectional study, during a 9 month period, 470 samples were collected from patients hospitalized in different wards of treatment centers of Zahedan University of Medical Sciences in 2015. Phenotypic method was used for isolation and initial screening. Oxacillin and Cefoxitin discs were used. After isolation of resistant strains, femA and mecA genes and tst gene were investigated using phenotypic method and multiplex PCR method, respectively. Results: Of 170 clinical isolates of Staphylococcus aureus, 93 isolates were phenotypically methicillin-resistant, among which 89 isolates had mecA gene and 14 isolates had tst gene. Conclusion: The results indicated that the prevalence of methicillin-resistant strains and the strains carrying causative gene for TSST1, is high in Zahedan. Also, circulation of these isolates can lead to much more severe effects in individuals with weak immune system

An Investigation of the Prevalence of AmpC-producing Pseudomonas aeruginosa in Clinical Samples in Zahedan City, Iran

Background and Objectives: AmpC beta-lactamases are among cephalosporinases encoded on the chromosomes of many Enterobacteriaceae. In many bacteria, induction of AmpC enzymes can be made at a very high level by numerous mutations. In this study, the prevalence of chromosomal AmpC genes, was investigated in the isolates of Pseudomonas aeruginosa isolated from teaching hospitals in Zahedan city in 2015. Methods: In this descriptive cross-sectional study, 100 P. aeruginosa isolates were isolated from 391 clinical samples using biochemical and conventional methods. cefoxitin (30μg) disk diffusion method was used to isolate AmpC-producing strains, and multiplex PCR was used to identify chromosomal AmpC genes. ESBL containing strains was assessed using ceftazidime (30μg) and cefotaxime/clavulanic acid (30μg/10μg) disk diffusion tests. Data analysis was performed using χ2 test. Results: In primary phenotypic screening, out of 100 P. aeruginosa isolated, 88 isolates were ESBL producers and 20 isolates (20%) were AmpC beta-lactamase producers. Among 20 phenotypically identified AmpC producing isolates, 19 isolates (95%) had FOX gene, 7 isolates (35%) had EBC gene, 4 isolates (20%) had ACC gene, and 15 isolates isolates (75%) had DHA gene, which were detected by multiPlex PCR assay. Conclusion: The results of the present study indicated that the presence of AmpC leads to resistance of bacteria to many cephalosporins. Also, use of multiplex PCR yields the best results in the group identification of these genes

First report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-producing Klebsiella pneumoniae from Iran

Abstract: INTRODUCTION: Extended-spectrum beta-lactamases (ESBLs) are bacterial enzymes capable of hydrolyzing beta-lactams. The aim of this study was to describe the prevalence of TEM- and SHV-type ESBL-producing Klebsiella pneumoniae strains in Zahedan, Southeast Iran. METHODS: A total of 170 non-repetitive K. pneumoniae strains were collected from patients referred to three teaching hospitals of Zahedan. Antibiotic susceptibility testing was determined for 17 antibiotics using the Kirby-Bauer disc diffusion method. The frequency of ESBL-producing strains was calculated, and minimum inhibitory concentrations of ESBL-producing strains were determined for cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime. The presence of bla TEM and bla SHV genes was tested in all ESBL-producing strains using polymerase chain reaction and DNA sequencing. RESULTS: Among the 170 K. pneumoniae clinical isolates, 55 (32.4%) were ESBL producers; 92.7% (n=51) and 72.7% (n=40) of the isolates carried the bla SHV and bla TEM genes, respectively, and 67.3% (n=37) carried both genes. The sequencing results showed that all bla TEM types were bla TEM-1, except for two isolates that were bla TEM-104. The bla SHV types were bla SHV-1, bla SHV-11, bla SHV-12, bla SHV-99, bla SHV-108, and bla SHV-110. CONCLUSIONS: The percentage of bla TEM and bla SHV among ESBL-producing K. pneumoniae isolates from Zahedan is relatively high, indicating the need for further surveillance and consideration in antibiotic use. To the best of our knowledge, this is the first report of TEM-104-, SHV-99-, SHV-108-, and SHV-110-type ESBLs among clinical isolates of K. pneumoniae from Iran, and TEM-1, SHV-1, SHV-11, and SHV-12 appear to be the dominant ESBLs in this region