Random Amplified Polymorphic DNA Typing Research Articles

Molecular typing of clinical and non-clinical <i>Escherichia coli</i> from Rivers State, Nigeria using amplification-based RAPD and ERIC-PCR typing methods

Due to the diverse nature of Escherichia coli, typing of these organisms is essential. This can provide key epidemiological information not just on the strains in circulation and their relationship, but also on the development and spread of drug resistant clones. Affordable PCR-based typing techniques have resulted in an increase in typing studies particularly in resource limited settings. But information from Nigeria on strain typing is limited. This study was therefore aimed at typing clinical and non-clinical strains of E. coli from Nigeria using the PCR based Random Amplification of Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) typing methods. 
 E. coli was isolated from clinical and non-clinical sources using Eosin Methylene Blue agar. Characteristic green metallic sheen colonies associated with E. coli were purified, identities determined using standard biochemical tests and confirmed using molecular methods. Susceptibility testing was carried out using the modified Kirby-Bauer disc diffusion test and RAPD and ERIC-PCR typing carried out on 48 confirmed E. coli isolates as previously described. ERIC-PCR typing resulted in the generation of 38 unique patterns with a 0.94 diversity. Nine of these were singletons comprised of only a single isolate, while the remaining 29 patterns were grouped to 10 clusters. RAPD gave similar results of 37 patterns, 9 singletons, 0.94 diversity and 13 clusters. For ERIC-PCR, all isolates with identical patterns were from the same source. RAPD, however, had identical patterns present in isolates from different sources. 
 Using the ERIC-PCR typing method, this study was able to identify non-clinical isolates as distinct from the clinical E. coli isolates as evidenced by 100% identical ERIC-PCR profiles of isolates from the same source and a lack of 100% relatedness in isolates from different sources.

Capsular gene distribution and RAPD typing of Streptococcus agalactiae isolated from pregnant women

Streptococcus agalactiae has different virulence factors, from which the capsule has the most significant role in the pathogenesis of this organism. We aimed to investigate the distribution of more prevalent capsular genes among different Random Amplified Polymorphic DNA (RAPD) types of S. agalactiae isolated from pregnant women. A total of 106 isolates were collected from 420 vaginal and rectal swabs obtained from pregnant women. The specimens were transferred using Todd Hewitt Broth and were cultured on a blood agar containing antibiotics. The S. agalactiae isolates were identified by the standard microbiological and biochemical tests. The genomic DNAs of S. agalactiae isolates were extracted using an extraction kit. Then, the PCR method was used to detection of the capsular genes. Moreover, The RAPD PCR was used to genotyping of the isolates. The colonization rate of the pregnant women was 25.23%, and there was a statistically significant correlation between the weeks of gestation and the probability of colonization (p-value < 0.05). Also, 31 (29.24%) and 18 (16.98%) pregnant women had a history of abortion and membrane rupture, respectively. In addition, 20 (18.86%), 32 (30.18%), 4 (3.77%), and 6 (5.66%) isolates carried genes encoding capsular types Ia, Ib, III, and V, respectively. None isolates had the type II capsular gene, and other 44 isolates were non-typeable. Nine clones (clusters) of S. agalactiae were observed in the present study with 70% similarity, and 53 different types were identified among the isolates. Except for capsular types III and V that belonged to clones 3, 5, 7, and 9, other capsular types were detected in different RAPD types. We found that the capsular types Ib and Ia were predominant among pregnant women in this area, indicating their significance for vaccine designation. Also, our isolates showed a lower genotypic diversity in RAPD typing. This may be due to the same sources of most isolates.

RAPD Fingerprinting and Genetic Diversity of Salmonella Spp. Isolated from Broiler and Layer Flocks in Karbala, Iraq.

Salmonellosis in poultry is one of the most significant bacterial infections causing mortality, reduced production, and serious economic losses. This study aimed to study the molecular diversity among Salmonella isolates and investigate the epidemiological spread of these bacteria in broiler and layer chicken flocks in five different farms in Karbala, Iraq, using random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR). In total, 217 cloac a swabs were collected from the farms, out of which 129 and 88 swabs were taken from broiler and layer chickens. The samples were screened by PCR for S. enterica subsp. enterica using primers specific for the invA gene. Afterward, RAPD-PCR with uniplex or multiplex octamer primers was applied to genotype the isolates. The incidence rate of Salmonella infections in broilers and layers was estimated to be 27.9% and 12.5%. The uniplex primers P2 and P3, along with the multiplex primers yielded discriminatory patterns. Moreover, the RAPD typing showed a diverse range of banding patterns of Salmonella spp. Dendrograms created through GelJ software revealed various Salmonella genotypes in broilers and layers. The RAPD-PCR could be used as an accurate and fast tool to identify genetic relatedness among Salmonella spp. The obtained results would assist researchers in epidemiological studies and controlling salmonellosis in poultry fields.

Determination of incompatibility group plasmids and copy number of the bla NDM-1 gene in carbapenem-resistant Klebsiella pneumoniae strains recovered from different hospitals in Kerman, Iran.

Introduction. New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern.Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer.Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran.Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains.Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable.Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.

Randomly amplified polymorphic DNA analysis of Staphylococcus chromogenes isolated from bovine and bubaline mastitis in Karnataka.

Background and Aim:In recent times, non-aureus staphylococci (NAS) have emerged as the major organisms isolated from mastitis cases in dairy animals, with a predominance of Staphylococcus epidermidis and Staphylococcus chromogenes. As compared to Staphylococcus aureus, much less is known about the molecular types or the spatiotemporal epidemiology of these NAS species. In the present study, randomly amplified polymorphic DNA (RAPD) was employed to detect genetic polymorphisms, intraspecies diversity, and epidemiology of S. chromogenes strains (n=37) isolated from bovine and bubaline mastitis cases in the state of Karnataka.Materials and Methods:Thirty-seven S. chromogenes isolates (14 from bovines and 23 from bubaline) isolated from subclinical mastitis cases, from organized and unorganized sectors, were subjected to RAPD typing. Further, methicillin resistance was determined by cefoxitin disk diffusion method.Results:The amplified DNA fragments ranged from 150 to 3000 base pairs and yielded several RAPD profiles. Further analysis using Digital Image Correlation Engine correlation coefficient and UPGMA method showed that the 37 isolates could be classified into 12 distinct RAPD types (A to L) at 62% similarity (D=0.889). Four of the most predominant RAPD types, B, A, C, and E, in that order, and together, represented 65% of the isolates. High diversity was observed among the isolates both within farms and between geographic locations. Most of the isolates exhibited methicillin resistance. This is the first such report from India.Conclusion:In the absence of defined multilocus sequence type protocols or sufficient sequences available in the public domain, RAPD can be employed to determine genetic diversity of S. chromogenes isolates.

Prevalence and Mechanisms of Carbapenem Resistance Among Acinetobacter baumannii Clinical Isolates in Egypt.

The increasing number of carbapenem-resistant Acinetobacter baumannii clinical isolates is a major concern, which restricts therapeutic options for treatment of serious infections caused by this emerging pathogen. The aim of this work is to assess the antimicrobial resistance profile and identify the molecular mechanisms involved in carbapenem resistance in A. baumannii isolated from different clinical sources in Mansoura University Hospitals, Egypt. Antimicrobial susceptibility testing has shown that resistance to carbapenem has dramatically increased (98%) with concomitant elevated levels of resistance to quinolones, trimethoprim/sulfamethoxazole, and aminoglycosides. Polymyxin B and colistin are considered the last resort. Random amplified polymorphic DNA (RAPD) typing method revealed great diversity among A. baumannii isolates. Coexistence of diverse intrinsic and acquired carbapenem-hydrolyzing β-lactamases has been detected in the tested isolates: Ambler class A: blaKPC (56%) and blaGES (48%), and Ambler class B: blaNDM (30%), blaSIM (28%), blaVIM (20%), and blaIMP (10%). Most isolates (94%) carried blaOXA-23-like and blaOXA-51-like simultaneously. blaOXA-23-like was preceded by ISAba1 providing a potent promoter activity for its expression. Sequencing analysis revealed that ISAba1 has been also inserted in carbapenem resistance-associated outer membrane protein (OMP) (carO) gene in three isolates, two of which were clonal based on RAPD typing, leading to interruption of its expression as confirmed by SDS-PAGE analysis of OMP fractions. Carbapenem resistance genes are widely distributed among A. baumannii clinical isolates from different clinical sources. Therefore, enhanced infection control measures, effective barriers, and rational use of antimicrobials should be enforced in hospitals for minimizing the widespread resistance to carbapenems and all other antibiotics.

Corynebacterium striatum: an emerging respiratory pathogen.

Corynebacterium spp. are primarily considered normal flora and dismissed when isolated from clinical specimens. In recent years, Corynebacterium striatum has emerged as a multi-drug resistant human pathogen which can cause nosocomial outbreaks. The organism has infrequently been noted to cause respiratory infections. A retrospective study was conducted to identify the clinical and microbiological features of respiratory infection by Corynebacterium striatum. C. striatum isolates from clinical and surveillance samples were tested for susceptibility to antimicrobials and typed by Random Amplification of Polymorphic DNA (RAPD). Clinical data was obtained through a retrospective review of records. 15 isolates from clinical and surveillance samples of 11 hospitalised patients were included. The patients suffered from either an exacerbation of COPD (n = 9) or pneumonia (n = 2). The isolates were all multi-drug resistant. RAPD typing found no evidence of an outbreak/ transmission between patients. Corynebacterium spp. must be considered potential pathogens. Suspicious isolates should be identified to the species level since Corynebacterium striatum is often multi-drug resistant.

RAPD- and ERIC-Based Typing of Clinical and Environmental Pseudomonas aeruginosa Isolates.

Pseudomonas aeruginosa is a major cause of nosocomial infection in children and adults, resulting in significant morbidity and mortality due to its ability to acquire drug resistance. The ability of P. aeruginosa in the environment to cause infection in individuals has been reported previously; henceforth, surveillance of the emergence and transmission of P. aeruginosa strains among patients is important for infection control in a clinical setup. Various gene-typing methods have been used for epidemiological typing of P. aeruginosa isolates for the purpose of surveillance. In this work, the suitability and comparability of two typing methods, enterobacterial repetitive intergenic consensus (ERIC)-PCR and random amplification of polymorphic DNA (RAPD)-PCR fingerprinting, were studied to characterize P. aeruginosa strains isolated from clinical and environmental sources. Forty-four clinical and environmental bacterial isolates of P. aeruginosa were collected between October 2015 and January 2016. DNA extraction, ERIC-PCR and RAPD-PCR, agarose gel electrophoresis, and phylogenetic analyses were carried using the unweighted pair-group method with mean. RAPD typing revealed less clonality among clinical isolates, whereas the ERIC method showed greater similarity in comparison with RAPD. Environmental isolates, however, showed greater similarity using RAPD compared with ERIC typing. With only a few exceptions, most clinical isolates were distinct from environmental isolates, irrespective of the typing method. In conclusion, both the RAPD and ERIC typing methods proved to be good tools in understanding clonal diversity. The results also suggest that there is no relationship between clinical and environmental isolates. The absence of clonality among the clinical isolates may indicate that most P. aeruginosa infection cases could be endemic and not epidemic and that endemic infections may be due to nonclonal strains of P. aeruginosa.

Antibiotic resistance profile and random amplification typing of β-lactamase-producing Enterobacteriaceae from the local area of Al-Taif and nearby cities in Saudi Arabia

Background: The emergence of extended-spectrum ?-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae constitutes a serious problem because of the transfer of resistance genes from one organism to another. Objectives: To screen the antibiotic susceptibility of E. coli and K. pneumoniae from inpatients at King Abdul-Aziz Hospital, Al-Taif, Saudi Arabia and to detect common ESBL genes, and random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) genotyping of the Enterobacteriaceae. Methods: We analyzed 43 bacterial strains isolated from clinical samples of sporadic cases for their reactivity to different antibiotics. Strains that showed resistance by monoplex PCR were screened for ESBL TEM- and CTXM-1-, -2-, and -9-encoding genes. Random amplifications were used for typing the resistant strains. Results: The majority of the ESBL containing strains were sensitive to meropenem, amikacin, gentamicin, and tigecycline. PCR detection using ESBL gene-specific primers showed that 17 of 43 strains harbored genes for ESBL TEM, CTX-M-1, or CTX-M-2. RAPD typing revealed marked variation among the ESBL-producing E.coli in relation to each other. Conclusions: Considerably high incidence of ESBL-producing bacteria was present patients from the local area of Al-Taif and nearby cities in Saudi Arabia with TEM and CTX-M subtypes being the most commonly detected variants. There was evidence of a polymorphic genetic pattern among ESBL-producing bacteria. Keywords: Antibiotic resistance, E.coli, Klebsiella pneumoniae, RAPD typing, Saudi Arabia

Identification of bacterial agents causing mortality in postlarvae of giant freshwater prawn (Macrobrachium rosenbergii ) in south-west coastal districts of Bangladesh

Following an incidence of freshwater prawn Macrobrachium rosenbergii postlarvae (PL) mortality in hatcheries in summer 2012, samples from dead PL, rearing water and prawn feed from two south-west coastal districts of Bangladesh were collected to isolate, identify and characterize the agents causing PL mortality. Antibiogram profile of sixteen randomly selected bacteria, isolated from dead PL, that grew on TCBS, to 20 different antibiotics belonging to 12 major groups revealed that the drug resistance pattern varied from moderate (56% to the drugs: ciprofloxacin, cefotaxime, nitrofurantoin, kanamycin) to complete (to penicillin, ceftazidime and oxacillin) level. To identify the isolates, amplified rDNA restriction analysis (ARDRA) classified them in to four groups, and RAPD (randomly amplified polymorphic DNA) typing yielded nine different types of isolates within these four ARDRA groups. The 16S rDNA gene sequences identified that the groups were genotypically diverse belonging to the bacterial species: Enterobacter cloacae, Klebsiella pneumoniae, Exiguobacterium profundum and Enterococcus casseliflavus, respectively, that all demonstrated their killing potential to PLs in a simulated environment. The study therefore identified four different bacterial pathogens, one of which, Exiguobacterium profundum is reported for the first here in Bangladesh, that demand special consideration for disease management strategy.

English

Klebsiella pneumoniae is one of the major causes of nosocomial infection. Understanding the route and dynamics of dissemination in the outbreaks of infections relies on using accurate typing methods. This is achieved through molecular typing using random amplified polymorphic DNA (RAPD) analysis technique in correlation to the infection source and antibiotic resistance. In this study, a total of 300 clinical isolates were collected from different clinical sources among Mansoura University Hospitals, Dakahlia governorate, Egypt. Ninety six isolates were identified as K. pneumoniae. The antimicrobial susceptibility test showed high resistance to the majority of tested antimicrobials, especially to β-lactams. K. pneumoniae isolates were categorized into 24 different antimicrobial susceptibility patterns (A1-A24). Furthermore, RAPD analysis was applied as a molecular typing method using two individual primers, AP3 and OPA13. RAPD typing showed 51 distinct patterns (R1-R51) verified into 5 groups (A, B, C, D, and E). The most common patterns were D11, D18 and D19 included in groups B and C. On the other hand, more molecular variable isolates were distributed in groups D and E. Correlation between RAPD analysis and antibiotying established that specific RAPD pattern D11 was associated with multidrug resistant isolates. This confirms that cross acquisition can play an important role in the epidemiology of nosocomial colonization and infection with K. pneumoniae at Mansoura University Hospitals. In conclusion, this study emphasized the need for appropriate monitoring of K. pneumoniae infections, by using both traditional and molecular methods. RAPD proved to be effective technique in discriminating K. pneumoniae isolates. Key words: Klebsiella pneumoniae, random amplified polymorphic DNA (RAPD), antimicrobial susceptibility, typing, dendrogram.

Molecular Characterization of Klebsiella Isolates from Enteral Diets

To evaluate the genotyping of Klebsiella isolates from enteral diets in public hospitals, as high-resolution support within epidemiological surveillance for controlling nosocomial infections. Methods: Klebsiella isolates were obtained from enteral diets at two public hospitals in the state of Minas Gerais, Brazil. These isolates were identified and serotyped. Total DNA of Klebsiella was extracted and subjected to three genotyping methods, to evaluate polymorphism between the strains and genetic diversity. Results: Twenty-one isolates of Klebsiella were obtained from the hospital enteral formulae; fifteen were identified as K. pneumoniae and six as K. oxytoca. The results from random amplified polymorphic DNA (RAPD) and 16S-23S rDNA analysis revealed high polymorphism among the K. pneumoniae isolates and a low level of polymorphism among the K. oxytoca isolates. The 1420 base pairs of DNA fragments generated through amplification of the 16S rDNA region and digestion with eight restriction endonucleases resulted in identical restriction fragment length polymorphism (RFLP) patterns for all isolates. Conclusion: Our data demonstrate that RAPD and 16S-23S rDNA analyses provide more reliable estimates of the genetic diversity among Klebsiella isolates than does amplification of the 16S rDNA gene. Therefore, we recommend that RAPD typing should be the preliminary method for fast and inexpensive investigation and that 16S-23S rDNA typing should be a confirmatory method if needed.

Bacteriological Screening of Water in Mangalore, India

Introduction: Diarrhoea caused by contaminated water is among the most prevalent waterborne diseases in the developing countries like India. In the interest of public health, water supplies should be tested regularly to confirm their freedom from contamination. Objective: The objectives of the study were to screen different water sources for bacterial contamination, to know the antibiotic susceptibility of the common bacterial isolates and typing of the bacterial isolates by random amplification of polymorphic DNA (RAPD)technique. Place and Duration of the Study: Kasturba Medical College Hospital, Microbiology Laboratory, Mangalore, Karnataka, India between August 2007 and August 2009. Methodology: Water samples (n=324) were analyzed by standard microbiological techniques for bacterial contamination. Isolates were identified biochemically and antibiotic susceptibility testing was done by disc diffusion method. Escherichia coli isolates were typed by RAPD technique. Results: Among the water samples tested, 246 were excellent and 78 were contaminated. Contaminated samples showed the growth of commensal bacteria belonging to the family Enterobacteriaceae along with pathogens like Salmonella spp. and Vibrio spp. Many of the isolates were found to be sensitive and a few were found to be resistant to the antibiotics tested. RAPD typing showed genetic similarity and differences among the E. coli isolates from different water sources. Conclusion: Genetic similarity among isolates of E. coli indicates a common ancestral origin or a common source. Bacterial contamination of water samples with pathogens like, Salmonella spp. and Vibrio spp. as well as the faecal coliform is a concern, as water quality is an index of health and well - being of the society. Degree of contamination observed in this study suggests a need to be vigilant to monitor water quality, in order to prevent enteric diseases.

Random Amplified Polymorphic DNA (RAPD) Typing of Multidrug Resistant Enterococcus faecium Urinary Isolates from a Tertiary Care Centre, Northern India

Enterococci, though they are a part of commensal flora, are becoming increasingly important as nosocomial pathogens, due to their inherited and acquired resistances to several antimicrobial agents. In this context, Enterococcus faecium (E.faecium) requires a special mention due to its characteristic of Multidrug Resistance (MDR) and its ability to disseminate. This study was undertaken to phenotypically characterize and determine clonal relatedness amongst the indoor isolates of Enterococcus faecium (E. faecium) which were isolated from patients with urinary tract infections (UTIs). This study was carried out prospectively in a tertiary care university hospital and in Department of Microbiology, Varanasi, India. Urine samples were collected from patients who were admitted in different departments of the hospital with a clinical diagnosis of UTIs and they were processed for a period of one year. Enterococcal species were identified by doing extensive biochemical tests. Anti-microbial susceptibility testing was done by disc diffusion and agar dilution methods. Molecular typing of the isolates was done by Random Amplified Polymorphic DNA (RAPD) typing method. A total of 48 Enterococcal urinary isolates were identified in indoor patients, among which a majority (46, 95.83%) were E.faecium isolates. These isolates exhibited high resistance to fluoroquinolones (91.3%) and to ampicillin (60.86%) in particular. Two isolates were found to be resistant to vancomycin on screen agar. RAPD typing showed two major clusters, one of which had ten strains of 100% similarity, all of which were isolated from a common source. This study showed dissemination of multidrug resistant E. faecium isolates within the hospital. Being a quick and cost effective method, RAPD typing can be used to show clonal relatedness and to trace possible sources of organisms for epidemiological purposes.

Profile and multidrug resistance determinants of Chryseobacterium indologenes from seawater and marine fauna

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Kaštela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-β-lactamases (MBLs). The clonality of β-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla (IND)-type gene, including 2 isolates carrying bla (IND-2) and 21 carrying bla (IND-7), that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.

Molecular characterization and genotypic antimicrobial resistance analysis of Campylobacter jejuni and Campylobacter coli isolated from broiler flocks in northern Italy

Genetic variability and genotypic antimicrobial resistance (AMR) of Campylobacter jejuni and Campylobacter coli from commercial broiler farms were investigated in this study. Campylobacter isolates were genetically characterized by random amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) and flaA-SVR and flaB-SVR sequence-based typing. Eight RAPD types were identified in C. jejuni and three in C. coli, while 16 fla profiles were detected among all isolates. Further, 13 flaA-SVR and 13 flaB-SVR alleles were identified. Both typing methods detected a high level of genetic diversity, but fla-SVR typing showed a higher discriminatory power. Indeed, Simpson's index of fla typing (D=0.920) was higher than that of RAPD typing (D=0.814). Moreover, the association of flaA-SVR and flaB-SVR sequence analysis showed a higher discriminatory power compared with the sequence analysis of single loci. Isolates were also analysed by the mismatch amplification mutation assay PCR test and the detection of cmeB gene to determine the occurrence of genetic determinants of AMR to macrolides and fluoroquinolones and multidrug resistance. The A2074C and A2075G mutations in the 23S rRNA gene, the C257T mutation in the gyrA gene, and the cmeB gene were higher in C. coli (19.0%, 67.0%, 100.0% and 100.0%, respectively) than in C. jejuni (0.0%, 3.1%, 48.3% and 48.3%, respectively). This study showed a high degree of genetic diversity and a high prevalence of genetic determinants of macrolide resistance, fluoroquinolone resistance and multidrug resistance among C. jejuni and C. coli isolates from Italian commercial broiler farms.

Prevalence, antibiotic resistance and RAPD typing of Campylobacter species isolated from ducks, their rearing and processing environments in Penang, Malaysia

We report for the first time on the prevalence, antibiotic resistance and RAPD types of Campylobacter species in ducks and duck related environmental samples in Malaysia. Samples were examined by enrichment in Bolton Broth followed by plating onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA) and/or plating directly onto mCCDA. A total of 643 samples were screened, and the prevalence of Campylobacter spp. in samples from different sources ranged from 0% to 85%. The method of isolation had a significant (P<0.05) effect on the isolation rate. One hundred and sixteen Campylobacter isolates, comprising of 94 Campylobacter jejuni, 19 Campylobacter coli and three Campylobacter lari, were examined for their sensitivity to 13 antibiotics. Majority of the C. jejuni isolates were resistant to cephalothin (99%), tetracycline (96%), suphamethoxazole/trimethoprim (96%), and very few were resistant to gentamicin (5%), chloramphenicol (7%) and erythromycin (1%). All C. coli isolates were resistant to cephalothin, nalidixic acid, norfloxacin and tetracycline but susceptible to chloramphenicol, erythromycin and gentamicin. The three C. lari isolates were resistant to all the antibiotics tested except chloramphenicol and gentamicin (1/3 and 2/3 susceptible, respectively). Genetic diversity of Campylobacter isolates were determined using random amplification of polymorphic DNA (RAPD). C. jejuni and C. coli isolates belong to fifty-eight and twelve RAPD types, respectively.

Comparison of Haemophilus parasuis reference strains and field isolates by using random amplified polymorphic DNA and protein profiles

BackgroundHaemophilus parasuis is the causative agent of Glässer’s disease and is a pathogen of swine in high-health status herds. Reports on serotyping of field strains from outbreaks describe that approximately 30% of them are nontypeable and therefore cannot be traced. Molecular typing methods have been used as alternatives to serotyping. This study was done to compare random amplified polymorphic DNA (RAPD) profiles and whole cell protein (WCP) lysate profiles as methods for distinguishing H. parasuis reference strains and field isolates.ResultsThe DNA and WCP lysate profiles of 15 reference strains and 31 field isolates of H. parasuis were analyzed using the Dice and neighbor joining algorithms. The results revealed unique and reproducible DNA and protein profiles among the reference strains and field isolates studied. Simpson’s index of diversity showed significant discrimination between isolates when three 10mer primers were combined for the RAPD method and also when both the RAPD and WCP lysate typing methods were combined.ConclusionsThe RAPD profiles seen among the reference strains and field isolates did not appear to change over time which may reflect a lack of DNA mutations in the genes of the samples. The recent field isolates had different WCP lysate profiles than the reference strains, possibly because the number of passages of the type strains may affect their protein expression.

Classification of 17 newly isolated virulent bacteriophages of Pseudomonas aeruginosa

Seventeen virulent bacteriophages specific to Pseudomonas aeruginosa strains were isolated by screening various environmental samples. These isolated bacteriophages were grouped based on results obtained from restriction fragment analysis of phage genomes, random amplification of polymorphic DNA (RAPD) typing, morphology observations under transmission electron microscope, and host range analysis. All 17 bacteriophages are double-stranded DNA viruses and can be divided into 5 groups based on DNA restriction profiles. A set of 10-mer primers was used in RAPD typing of phages, and similar conclusions were obtained as for restriction fragment analysis. One phage was randomly selected from each of the 5 groups for morphology observations. Four of them had an icosahedral head with a long contractile tail, belonging to the Myoviridae family, and one phage had an icosahedral head with a short tail, thereby belonging to the Podoviridae family. Host range experiments were conducted on 7 laboratory strains and 12 clinical strains of P.aeruginosa. The results showed that 13 phages had the same infection profile, killing 8 out of 19 tested P.aeruginosa strains, and the remaining 4 phages had different and unique infection profiles. This study highlights the diversity of bacteriophages specific to P.aeruginosa in the environment.

Evaluation of randomly amplified polymorphic DNA (RAPD) for discrimination of Coxiella burnetii ruminant strains isolated in France

Coxiella burnetii is the causative agent of Q fever, a worldwide zoonosis. Ruminant species represent the main reservoir of the bacterium, as high levels of the bacteria are shed in birth products and other excreta. Human infection generally occurs after inhalation of contaminated aerosols [1]. The availability of methods to study the distribution and the spread of a given C. burnetii strain, from different geographical areas or hosts, would promote a better understanding of the epidemiology of this pathogen. Several genetic typing methods for C. burnetii have been evaluated and appeared to be useful tools for epidemiological and phylogenetic purposes. However, RAPD [2,3] has never been evaluated for C. burnetii typing. In this work, a RAPD protocol was used to evaluate the genetic diversity among C. burnetii ruminant strains in comparison to published MLVA data [4]. Ten French C. burnetii isolates obtained from goats, sheep, cows and the reference strain Nine Mile, were used for RAPD typing (Table 1). Whole DNA was extracted using the Qiagen Dneasy kit (Courtaboeuf, France), following the manufacturer’s protocol. To assess optimal RAPD conditions several parameters were tested, for instance using a range of MgCl2 concentrations (from 1.5 to 5.5 mM), or different brands of Taq polymerase. RAPD PCRs were performed using a set of four 10-mer primers (P4M, 5¢-AAGACGCCGT-3¢; PR5, 5¢-AGTCGTCCCC-3¢; PR10A, 5¢AGGGCCGTCT3¢; and PR12A, 5¢-CAGCTCACGA-3¢). Unless specified, the optimised PCR conditions were, for a final volume of 25 lL in dH2O: 1X Taq polymerase buffer (without MgCl2); 3 mM MgCl2; 0.2 mM each dNTPs; 1U of Taq polymerase; 0.5 lM 10-mer primer; and 10 ng of DNA (all the reagents were purchased from Invitrogen, Cergy Pontoise, France). Negative controls consisting of dH2O only were included in each run. Amplifications were performed in an Eppendorf Mastercycler (Le Pecq, France), programmed for an initial denaturation step of 5 min at 94 C, 45 cycles of 96 C for 30 s, 37 C for 30 s, 72 C for 90 s and a final extension step at 72 C for 5 min. The PCR products were separated on 0.8% agarose gel containing ethidium bromide, visualised and photographed under UV light. Gel analysis was performed using the Quantity One 1-D Analysis software from Bio-Rad (Marnes la Coquette, France). An RAPD type was defined after the combination of the patterns obtained with the four primers used in this study (Table 1). The reproducibility of the method was evaluated by repeating the same assay several times, by different manipulators, as well as by using independent DNA preparations. Briefly, RAPD profiles were stable when varying MgCl2 concentration. However, a higher yield of amplification, estimated by the intensity of the bands observed on the gels, was observed using 2.5–3.5 mM MgCl2. This yield decreased when using higher concentrations of MgCl2 (data not shown). A MgCl2 concentration of 3 mM was therefore chosen for subsequent analyses. Different RAPD patterns were observed when different brands of Taq polymerases were used (data not shown). The Hot start platinum Taq polymerase (Invitrogen) produced the best patterns in our hands and was chosen for all the experiments. After optimisation of our RAPD protocol, the use of each primer generated distinct polymorphisms allowing differentiation of the studied strains. Strains from neighbouring flocks, that were indistinguishable using MLVA analysis, could be discriminated using RAPD typing (Table 1). For instance, CbB2 and CbB5 are two strains obtained in 2001 from neighbouring flocks, but were isolated from cows displaying different clinical signs (Table 1). CbB2 was isolated from a case of metritis and CbB5 from an aborted cow. Corresponding author and reprint requests: Dr V. Duquesne, AFSSA, Sophia Antipolis, Unite de Pathologie des Petits Ruminants, 105 Route des Chappes, BP111, 06902 SophiaAntipolis, France E-mail. v.duquesne@afssa.fr