RAPD-PCR Fingerprinting Research Articles

Analysis of antimicrobial resistance and genetic diversity of Acinetobacter baumannii in a tertiary care hospital in Haikou City.

This study addresses the distribution and antimicrobial resistance of Acinetobacter baumannii (A. baumannii) in a medical facility in Haikou City, aiming to provide essential insights for enhancing in-hospital treatment and prevention strategies. We conducted a retrospective analysis of 513 A. baumannii isolates collected from a tertiary care hospital in Haikou between January 2018 and December 2020, focusing on their antimicrobial resistance patterns. Random Amplified Polymorphic DNA (RAPD) analysis was performed on 48 randomly selected A. baumannii strains. Using Gel-pro4.0 and NTSYSspc2.10 software, we constructed dendrograms to assess the genetic diversity of these strains. Our results indicate that males between 60 and 70 years old are particularly vulnerable to A. baumannii infections, which are most frequently detected in sputum samples, with a detection rate exceeding 70%. Alarmingly, over 50% of the isolates were identified as multi-drug resistant. The RAPD-PCR fingerprinting cluster analysis demonstrated substantial genetic diversity among the strains. Using primer OPA-02 at a 45% similarity coefficient, the strains were categorized into four groups (A-D), with group A being predominant (39 strains). high-prevalence areas like the Neurosurgery and Intensive Care Medicine Wards require enhanced surveillance and targeted interventions to manage Group C infections effectively. Additionally, the varied presence of other groups necessitates customized strategies to address the specific risks in each ward. Similarly, primer 270 at a 52% similarity coefficient classified the strains into five groups (E-I), with group E being most common (36 strains). The study highlights a concerning prevalence of antimicrobial resistance, particularly multi-drug resistance, among A. baumannii strains in the Haikou hospital. The significant genetic diversity, especially within groups A and E, underscores the need for tailored hospital treatment protocols and prevention measures. These findings contribute to the growing body of research on antimicrobial resistance, emphasizing the urgent need for effective management strategies in healthcare settings.

Cloning, sequencing, and characterizing of soil antibiotic active-producing Streptomyces species-specific DNA markers

Association of the antibiotic activity of the soil Streptomyces isolates to their genetic profiles analyzed through RAPD-PCR fingerprints prompted us here in this study to use the most common bands as specific markers to identify homologous proteins within these isolates by cloning, sequencing, and characterizing these markers. Six out of twelve DNA bands ranged between 600 and 1350 bp previously obtained by RAPD-PCR analysis were purified out of the RAPD gels, and then cloned into pGEM-T Easy vector system. Success of the cloning process was confirmed by digesting purified plasmids with EcoRI. The clones namely No. 54, 55, 20, 56, 57, and 58 were sequenced using the DNA BigDye Terminator Sequencing System utilizing the M13 primer. Results indicated that the size of the inserted sequences is 599, 566, 522, 870, 857, and 254 bp, in clones No. 54. 55, 20, 56, 57, and 58, respectively. Homologous proteins of the six cloned sequences generated by DNA blast software indicated that the highest score of protein homology was scored for clone No. 54 with 87 % homology to putative secreted pectate lyase [Streptomyces coelicolor A3(2)]. The other clones showed less homology with 77 % homology for the clones No. 55 and 56, 73 % homology for the clone No. 20, and 55 % homology for the clones No. 57 and 58. The association of homologous proteins to the reported RAPD pattern is confirmed here for the first time, and the resulting DNA cloned fragments deserve further molecular analysis.

Potential probiotic lactobacilli strains isolated from artisanal Mexican Cocido cheese: evidence-based biosafety and probiotic action-related traits on in vitro tests.

The biosafety of four potentially probiotic lactobacilli strains, isolated from artisanal Mexican Cocido cheese, was assessed through in vitro tests aimed to determine (1) the antibiotic susceptibility profile by broth microdilution, (2) the transferability of antibiotic resistance determinants by filter-mating, and (3) the phenotypic and genotypic stability during serial batch sub-culture (100-day period) by evaluating physiological and probiotic features and RAPD-PCR fingerprinting. Lactobacilli strains exhibited multidrug-resistance; however, resistance determinants were not transferred in the filter-mating assay. Significant (p < 0.05) differences were observed in bacterial morphology and some functional and technological properties when strains were serially sub-cultured over 50 generations (G50), compared to the initial cultures (G0). Conversely, the strains did not show mucinolytic and hemolytic activities either at G0 or after 100 generations (G100). Genetic polymorphism and genomic template instability on selected strains were detected, which suggest possible evolutionary arrangements that may occur when these bacteria are largely cultured. Our findings suggest that the assessed strains did not raise in vitro biosafety concerns; however, complementary studies are still needed to establish the safe potential applications in humans and animals.

A comparative study of the clonal diversity and virulence characteristics of uropathogenic Escherichia coli isolated from Australian and Turkish (Turkey) children and adults with urinary tract infections.

The virulence-associated gene (VAG) repertoire and clonal organization of uropathogenic Escherichia coli (UPEC) strains is influenced by host demographic, geographic locale, and the setting of urinary tract infection (UTI). Nevertheless, a direct comparison of these features among Australian and Turkish UPEC remains unexplored. Accordingly, this study investigated the clonal composition and virulence characteristics of a collection of UPEC isolated from Australian and Turkish UTI patients. A total of 715 UPEC strains isolated from Australian (n=361) and Turkish (n=354) children and adults with hospital (HA)- and community-acquired (CA)-UTIs were included in this study. Typing of the strains using RAPD-PCR and PhPlate fingerprinting grouped all strains into 25 clonal groups (CGs). CG representatives were phylogrouped and screened for the presence of 18 VAGs associated with extraintestinal pathogenic E. coli. Turkish UPEC strains were characterized by high clonal diversity and predominance of the phylogroup D, while few distinct clonal groups with phylogenetic group B2 backgrounds dominated among the Australian strains. Twelve identical CGs were shared between ≥1 patient group from either country. Australian strains, particularly those isolated from children with HA-UTI, showed higher virulence potential than their Turkish counterparts, carrying significantly more genes associated with adhesion, iron acquisition and capsule biosynthesis. This study identified identical CGs of UPEC causing HA- and CA-UTIs among Australian and Turkish UTI patients. These CGs frequently carried VAGs associated with adhesion, iron acquisition, immune evasion, and toxin production, which may contribute to their ability to disseminate internationally and to cause UTI.

Molecular characterization of integrons and Genotyping of Salmonella enterica isolated from diarrheal animals and humans

Resistance to antibacterial drugs is a major health problem because of its impact . It is important to study the prevalence of antibiotic resistance genes in order to understand.Twenty five Salmonella enterica isolated from human and animal diarrhea. Traditional culture methods, biochemical methods, and molecular methods were used to characterize the samples during December to September 2020 to 2021. Disk diffusion method was used to determine antimicrobial susceptibility patterns of isolates identified as MDR by the Clinical and Laboratory Standards Institute (CLSI). The PCR amplification was used to show presence of class 1, 2 and 3 integrons. Class 1 integrons and class3 were detected in 100% of the multidrug-resistant Salmonella isolates,whlie class2 detected among 40%. RAPD-PCR fingerprinting regarded successfully as a reliable, reproducible, accurate and sensitive discriminatory method to Salmonella enterica. The results of RAPD-PCR amplification of Salmonella enterica isolates revealed that 24 isolates (96.%) also showed about one to six amplification bands per isolate, except one isolates were untypeble. The most frequent band was a 1400bp band (72%), while the lowest frequent band was 400bp (8%) among an individual isolates.

Salegentibacter maritimus sp. nov., isolated from marine coastal sediment

Two bacterial strains were isolated from a marine sediment sample taken from Jingzi Wharf, Weihai, China. These two strains were characterized at the phenotypic, chemotaxonomic, and genomic level. The two strains possessed almost identical 16S rRNA gene sequences (99.9 %). However, RAPD-PCR fingerprint patterns discriminated that they were not from one clonal origin. The average nucleotide identity (ANI) value and the digital DNA-DNA hybridization (dDDH) value between the two strains were 98.3 % and 85.4 %, respectively, suggestingthat they belonged to the same species. On the basis of the result of phylogenetic analysis of the 16S rRNA gene sequences, the two strains belonged to the genus Salegentibacter and were closely related to S. holothuriorum KCTC 12371T (98.6 %) and S. salegens DSM 5424T (98.2–98.3 %). The ANI and dDDH clearly separated strains F63223T and F60176 from the the most related type strains with values below the thresholds for species. The genome sizes of strains F63223T and F60176 were approximate 3.89 and 3.59 Mbp, respectively. The strain F63223T had 3,335 predicted genes with DNA G + C content of 35.6 %. The major respiratory quinone was MK-6 and the major polar lipids were phosphatidylethanolamine and one unidentified lipid. According to the results of the phenotypic, chemotaxonomic characterization, phylogenetic classification and genome analysis, the two isolates could be considered to represent a novel species of the genus Salegentibacter, for which the name Salegentibacter maritimus sp. nov., is proposed, with F63223T (=MCCC 1H00433T = KCTC 82417T) as the type strain.

Phenotypic and molecular analysis of dominant occurring antibiotic active-producing Streptomyces soil flora in Northern Jordan.

This investigation aimed to determine the relatedness of dominant occurring soil Streptomyces spp. in Northern Jordan based on their RAPD-PCR fingerprints, and to compare RAPD technique with the conventional phenotypic characterization of Streptomyces isolates. Fifty-eight white and gray color-bearing aerial mycelia antibiotic active-producing Streptomyces soil isolates along with three reference strains were genetically analyzed by RAPD-PCR. Polymorphisms between the isolates showed 1 to 10 bands per isolate and ranged from 200 to 3200 bp in size. Results revealed one common band of ~600 bp shared by ~85% of the isolates, and the observation of bands specific to some reference strains and some soil isolates. When RAPD patterns were analyzed with the UPGMA, results revealed clustering the tested isolates into two equal main super clusters (50% each). Super cluster I appeared to be homogenous and include the three reference strains. However, super cluster II was heterogeneous and but not including any of the reference strains. The association of the antibiotic activity of the dominant white and gray aerial mycelium-bearing Streptomyces isolates to RAPD clustering is reported for the first time, and the RAPD-PCR fingerprints generated here deserve to be cloned, characterized and sequenced in future as Streptomyces species-specific DNA markers. The more random primers used in the analysis may add to RAPD technique a cost-effective, fast, precise result, and less labor work solution for analyzing the similarities and differences among the Streptomyces isolates.

Radio-protective role of interferon and fenugreek on γ- radiation induced DNA instability

The radio-protective role of fenugreek (fen) and IFN was studied against the damage effects induced by gamma irradiation in liver of albino rats. Male and female albino rats were paired. The produced generations were separated into 3 classes PC, PT& P-ir, representing untreated, fed with standard food mixed with 5% Fen seed powder (FSP) and exposed to whole body irradiation (WBI) respectively. Animals were allowed for mating to give F1. F1 was separated into 3 subgroups (ir-ir), (ir+FSP) and (ir-IFN), subjected to another dose of irradiation, fed with standard food mixed with FSP, injected with IFN respectively. All individuals were arranged for pairing until the production of F2. DNA assay was carried out using RAPD-PCR fingerprinting technique. Six arbitrary primers were used. They produced various numbers of fractions ranging between zero to 8 within each of the studied groups. Some specific fractions were picked out indicating polymorphic alleles. Quantitative mutations were observed within the percentage area of the generated bands. Highest and / or lowest similarity indices were observed between the studied groups indicating complementary to the used primer or pointing to some degree or to complete disturbance in the DNA sequencing as a result of the different treatments. In conclusion the remarked changes in DNA fingerprinting confirmed the potential transmission of radiation damage of genome to the progeny while each of fen and IFN ameliorated the harmful effects of irradiation.

Molecular characterization of lpxACD and pmrA/B two-component regulatory system in the colistin resistance Acinetobacter baumannii clinical isolates

Colistin is drug of choice for treatment of carbapenem-resistant Acinetobacter baumannii infections. Unfortunately, global increase in clinical outbreaks of colistin and carbapenem resistant A. baumannii infections is on the rise and cause public health concern. In the present study, a total of 187 A. baumannii recovered from specimens of 240 patients admitted to intensive care units (ICUs) of two hospitals in Kerman, Iran during 2017–2018. Among the isolates, we found four extensive drug-resistant (XDR) with Minimum Inhibitory Concentration (MIC) ≥4 μg/mL against colistin. The colistin-resistant (Col-R) isolates harbored blaOXA–51 and blaOXA-23 carbapenemase genes, exhibited resistance to all antibiotic classes except tigecycline and ampicillin-sulbactam. They belonged to clonal complex 2, a new MLST type 1752 and displayed identical RAPD-PCR fingerprints. Phylogenetic tree analysis suggested that, the Col-R A. baumannii emerged by endogenous mutations rather than acquisition of preexisting clones. Expressions of pmrCAB by quantitative real-time PCR (qRT-PCR) revealed 8 and 7 folds increased in the transcription levels of pmrB/C genes in strain 1 grown in presence of 16 μg/mL colistin (p ≤ 0.01). However, no change in the expression of the pmrA was observed. Furthermore, DNA sequencing of Col-R genes illustrated three nonsynonymous substitutions in the LpxA (N136 → K), LpxC (P293 → Q), and PmrB (V21 → F, S28 → R, I149 → F) in the strains 1 and 3, respectively showing MIC 32 μg/mL against colistin. Multiple amino acids alignments demonstrated several substitutions in N-terminal region of PmrB. In conclusion, the above results provide valuable insights into the mechanism of Col-R in A. baumannii and the expressions of relative genes.

Commonality of adherent-invasive Escherichia coli isolated from patients with extraintestinal infections, healthy individuals and the environment.

Adherent-invasive Escherichia coli (AIEC) has been implicated as a microbiological factor in inflammatory bowel disease (IBD) pathogenesis. These strains are defined by their ability to adhere to and invade intestinal epithelial cells, and to survive and replicate in macrophages. We postulated that AIEC strains may commonly inhabit the gut of healthy individuals (HI), cause extraintestinal infections, and be found in sewage treatment plants (STP) and surface waters (SW). A total of 808 E. coli strains isolated from HI; patients with community-acquired urinary tract infection (CA-UTI), septicaemia and urosepsis; STP; and SW, showing a diffuse adhesion pattern to Caco-2 cells were included in this study. Typing of the strains using a combination of RAPD-PCR and PhPlate fingerprinting grouped them into 48 common clones (CCs). Representatives of each CC were tested for the ability to invade Caco-2 cells, survive and replicate in macrophages, and for the presence of six virulence genes commonly found among AIEC strains. Twenty CCs were deemed AIEC based on their ability to survive and replicate in macrophages, while encoding htrA, dsbA and clbA genes. These CCs primarily originated from HI and CA-UTI patients but were also detected in secondary locations including STP and SW. Strains lacking intramacrophagic survival and replication abilities were regarded as diffusely adhering E. coli (DAEC). Certain clones of AIEC are common in the gut of HI whilst promoting CA-UTI. The survival and persistence of AIEC in STP and SW may have serious public health ramifications for individuals predisposed to IBD.

Involvement of the AcrAB Efflux Pump in Ciprofloxacin Resistance in Clinical Klebsiella Pneumoniae Isolates.

Increasing prevalence of multiple antibiotic resistance in Klebsiella pneumoniae strains confines the therapeutic options used to treat bacterial infections. We aimed in this study to investigate the role of AcrAB and qepA efflux pumps and AAC(6')-Ib-cr enzyme in ciprofloxacin resistance and to detect the RAPD-PCR fingerprint of K. pneumoniae isolates. A total of , 117 K. pneumoniae isolates were collected from hospitalized patients in three hospitals in Tehran, Iran, from August 2013 to March 2014. Antimicrobial susceptibility tests were performed by the disk diffusion method. Molecular identification and expression level of encoding quinolone resistance genes, acrA, acrB, qepA, and aac(6')-Ib-cr, were performed by PCR and real-- time PCR assays, respectively. All the K. pneumoniae isolates containing the mentioned genes were used simultaneously for RAPD-PCR typing. Colistin and carbapenems were the most efficient antibiotics against the clinical isolates of K. pneumoniae. PCR assay demonstrated that among the 117 isolates, 110 (94%) and 102 (87%) were positive for acrA and acrB gene and 5 (4%) and 100 (85%) isolates showed to have qepA and aac(6')-Ib-cr genes, respectively. Determination for AcrAB pump expression in 21% of strains demonstrated an increased expression, and the mean increase expression for acrB genes was 0.5-81. The results of RAPD-PCR reflected that in 95% CI, all isolates belonged to a clone. A high prevalence of genes encoding quinolone resistance in K. pneumoniae was detected in clinical samples. Therefore, the control of infection and prevention of drug-resistant bacteria spread need careful management of medication and identification of resistant isolates.

Evaluation of bacterial communities of Grana Padano cheese by DNA metabarcoding and DNA fingerprinting analysis

The composition of the bacterial community of Grana Padano (GP) cheese was evaluated by an amplicon-based metagenomic approach (DNA metabarcoding) and RAPD-PCR fingerprinting. One hundred eighteen cheeses, which included 118 dairies located in the production area of GP, were collected. Two hundred fifty-four OTUs were detected, of which 82 were further discriminated between dominant (32 OTUs; > 1% total reads) and subdominant (50 OTUs; between 0.1% and 1% total reads) taxa. Lactobacillus (L.) delbrueckii, Lacticaseibacillus (Lact.) rhamnosus, Lact. casei, Limosilactobacillus fermentum, Lactococcus (Lc.) raffinolactis, L. helveticus, Streptococcus thermophilus, and Lc. lactis were the major dominant taxa (‘core microbiota’). The origin of samples significantly impacted on both richness, evenness, and the relative abundance of bacterial species, with peculiar pattern distribution among the five GP production regions. A differential analysis allowed to find bacterial species significantly associated with specific region pairings. The analysis of pattern similarity among RAPD-PCR profiles highlighted the presence of a ‘core’ community banding pattern present in all the GP samples, which was strictly associated with the core microbiota highlighted by DNA metabarcoding. A trend to group samples according to the five production regions was also observed. This study widened our knowledge on the bacterial composition and ecology of Grana Padano cheese.

Unidirectional animal-to-human transmission of methicillin-resistant Staphylococcus aureus ST398 in pig farming; evidence from a surveillance study in southern Italy

BackgroundLivestock-associated methicillin-resistant Staphylococcus aureus (MRSA) belonging to clonal complex 398 is recognized as an occupational hazard for workers employed in intensive animal husbandry, especially in the swine-breeding chain. In this study, we compared the prevalence and epidemiological type of MRSA isolates from swine and farm workers in a large area of southern Italy.MethodsBetween January and March 2018, 88 workers from 32 farms where we had previously performed a survey for MRSA colonization of farmed pigs, were sampled by nasal swabbing. A follow-up investigation was conducted on seven workers 1 year after primary screening. MRSA isolates were characterized by MLST, spa and SCCmec typing, and tested for susceptibility to 15 antimicrobials. Epidemiological correlations between human and swine MRSA isolates were supported by Rep-MP3 and RAPD PCR fingerprinting, and whole-genome sequencing.ResultsThe overall colonization rate of MRSA in swine farm workers was 21.6%, being significantly higher in intensive farms and in workers with direct animal contact. All human MRSA isolates were multi-drug resistant, belonged to the ST398 livestock clade, and did not carry Panton-Valentine leukocidin and enterotoxin genes. Notably, 94.1% of human MRSA isolates belonged to the same epidemiological type as swine MRSA isolates from the corresponding farm. Persistent MRSA carriage was documented in some workers 1 year after primary sampling.ConclusionsWe report a high prevalence of MRSA among swine farm workers, with higher colonization rates associated with intensive breeding and animal exposure. Our findings suggest unidirectional animal-to-human transmission of LA-MRSA and denote the high zoonotic transmissibility of the ST398 livestock clade.

RAPD-PCR Fingerprinting for Some RhodotorulaSpecies Isolated from Natural Sources and their Antagonistic Potential Against Erwinia Carotovora and Erwinia Chrysanthem

Rhodotorula is a genus of yeasts, color classified in the phylum Basidiomycota, family Sporidiobolaceae, subfamily of Rhodotorulalodeae and class Microbotryomycetes.

ERIC- PCR Typing, RAPD-PCR Fingerprinting and Quorum Sensing Gene Analysis of Pseudomonas aeruginosa Isolated from Different Clinical Sources

Recently, Pseudomonas aeruginosa infections proportions have increased significantly. Molecular typing and virulence analysis are good techniques, which can lead us to know P. aeruginosa infections. P. aeruginosa isolates were identified by using molecular method (16S rDNA gene) via PCR technique for accurate identification. The highest percent 41.26% of P. aeruginosa bacteria was found in the burn infections followed by 28.57% in wound swabs, 17.46% in ear discharge and lowest percentage were obtained from sputum samples. All isolates classified into six groups (A-F) according to classes of antibiotics. Of the 63 bacterial isolates, 100% were resistant to carbencillin, whereas 31.74% were resistant to ticarcillin and all isolates susceptible to imipenem. In addition all of clinical isolates indicated multidrug resistant (MDR) patterns, the highest rate of MDR was observed with pattern C these isolates were able to resist (9-12) antibiotics. All isolates were typed genotypically by using two methods of amplification, ERIC and RAPD-PCR. The results of the ERIC-PCR typing of P. aeruginosa bacteria that 96.82% showed amplification bands ERIC-PCR also revealed 17 groups of genotypes (A-R) and 4 unique isolates. The results of RAPD-PCR fingerprint revealed 12 groups of genotypes (A-M) of 40–90% similarity according to coefficient values and 4 unique isolates, except 7.93% were untypeble. QS genes (lasI, lasR, rhlI, rhlR), screen showed all isolates 100% were positive for one or more QS genes, in the other hand 82.53% carrying lasI, lasR, rhlI, and rhlR, while the 15.87% carrying lasI, rhlI, and rhlR and 1.58% carrying lasI, lasR, and rhlR genes. ERIC genotyping significantly correlated resistance patterns but not with virulence control QS genes. RAPD genotyping significantly correlated with source of infection, resistance patterns and virulence control QS genes. These results can help initial diagnosis MDR P. aeruginosa outbreaks associated with specific genotyping patterns.

DETECTION OF DNA ALTERATIONS IN MUSCOVY DUCKS (CAIRINA MOSCHATA) NATURALLY INFECTED WITH HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS SUBTYPE H5N1

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 is circulating in Egypt since 2006, with escalating apprehension about its possibility to become more transmissible amongst humans. In this study, three serial outbreaks of HPAIV H5N1 in domestic Muscovy ducks in Sharkia Governorate, Egypt were investigated. Nervous signs with 62% mortality were observed in Muscovy ducklings. Gross examination revealed severely congested meningeal vessels, hemorrhages on the duodenum, pancreas, and coronary fat. Perivascular lymphocytic cuffing, gliosis and vacuolation of the neuropil were observed in the brain microscopically. Viral antigens were identified in the neurons and the glial cells of the cerebral cortex, submucosal Meissner's plexus neurons of the intestine and the hepatic Kupffer cells by immunohistochemistry. The HPAIV subtype H5N1 was isolated from different duck tissues in 66.7% of examined duck samples. Using RAPD-PCR fingerprinting, there were different patterns in the DNA of Muscovy ducks naturally infected with AIV (24, 48 and 72 hours post appearance of clinical signs) compared to uninfected birds. Differences in RAPD-PCR profiles between infected and uninfected ducks, and genomic instability percent (37.7%±1.76) pointed to the incidence of DNA alterations induced at 24 hours following the appearance of clinical signs. Further in vivo and in vitro experiments need to be done to determine the relative importance of these findings.

Characterization of clarithromycin heteroresistance among Helicobacter pylori strains isolated from the antrum and corpus of the stomach

Mixed infections and heteroresistance of Helicobacter pylori contribute to decreased efficacy of treatments. This study aimed to investigate frequency of clarithromycin heteroresistance and its link with mixed infections, medication history, and disease severity. A total of 40 pairs of H. pylori strains were isolated from the antrum and corpus of 97 patients. Susceptibility of the strains to clarithromycin was measured by agar dilution method. Site-specific mutations of 23S rRNA at A2143G, A2142G, and A2142C positions were analyzed by PCR and genomic relatedness of pairs of the strains was determined by random amplified polymorphic DNA (RAPD)-PCR. The results showed a prevalence of 35% (14/40) clarithromycin resistance. Diversity of the antrum and corpus isolates in resistance to clarithromycin was detected among 17.5% (7/40) of the patients. Similarly, diversity in MIC value was also detected in two patients infected with the sensitive strains. Significant difference in frequency of resistance was detected among patients with peptic ulcer disease (PUD) (MIC90 32μg/mL) and severe gastritis (MIC90 16μg/mL), compared with those who suffered from non-ulcer dyspepsia (NUD) (MIC90 8μg/mL) and chronic gastritis (MIC90 0.25μg/mL). MIC values showed 8-32 folds increased levels in the corpus. A2142G, A2143G, and A2142C mutations were detected in three, two, and two patients, respectively, but not observed in 46% of the resistant strains. RAPD-PCR fingerprints showed identical molecular patterns for the isolates of the corpus and antrum in each patient. In conclusion, microevolution of H. pylori strains during chronic infection, rather than mixed infection, and inappropriate medication appear to be main reasons of treatment failure in adults.

Genetic relationships and population structure of Malassezia pachydermatis strains isolated from dogs with otitis externa and healthy dogs

ABSTRACTMalassezia pachydermatis causes infections of the skin and mucous membranes, especially in animals. It is commonly accepted that symptom manifestation depends on the physiological status of the host (different metabolic, hormonal, and immunological disorders). However, it should be considered whether distinct strains of M. pachydermatis could have different pathogenic potential and maintain opposite relations with the host, such as commensalism or parasitism. The scope of this study was to explore the population structure, genetic diversity, and phylogenetic relationships of M. pachydermatis strains isolated from dogs with clinical symptoms of otitis externa and from healthy dogs in order to investigate their relationships and evolutionary history. For all tests, a group of 30 strains derived from dogs with otitis externa and 34 strains from healthy dogs were used. The level of genetic diversity was initially assessed by polymerase chain reaction (PCR)-based random amplification of polymorphic DNA (RAPD-PCR), whereas evolutionary history was assessed by comparison of the nucleotide sequences of the internal transcribed spacer ITS1 region of nuclear rDNA. RAPD-PCR fingerprinting revealed a high level of genetic polymorphism in both tested groups (85% of unique profiles), but clinical isolates usually grouped together with other strains from otitis externa cases. Sequencing analysis identified 17 distinct genotypes with 59 polymorphic sites within both populations; however, putatively virulent strains were more closely related, indicating a probable correlation between the genotype and the virulence potential. Therefore, the hypothesis that M. pachydermatis virulence depends solely on the host’s properties should be reconsidered including evolutionary and epidemiological data.

Polyphasic identification of Penicillia and Aspergilli isolated from Italian grana cheese

Penicillium and Aspergillus genera, both including mycotoxin producing species, were reported as associated to cheese and cheese working environment, but never studied in an extensive way in Italian grana cheese (Grana Padano and Parmigiano Reggiano). The aim of this work was to address the identification of Aspergilli and Penicillia associated to grana cheese in order to lay down the basis for risk assessment and safe processing for a high quality production. One hundred and four strains belonging to Aspergillus and Penicillium genera were obtained from cheese crust and from ripening room air (with the latter largely dominant), and identified following a polyphasic approach, strongly required for the identification at the species level. Morphological observation was used along with molecular techniques, RAPD-PCR fingerprinting and calmodulin gene sequencing (CaM), the former aimed to limit as much as possible the latter sequencing effort. Seventy four percent of the strains were assigned to Penicillium subgenus Penicillium, section Fasciculata. Main mycotoxin producing species identified were A. flavus, P. crustosum and P. verrucosum, while the dominant species in both air and cheese crust was P. solitum, which has never been so far reported as mycotoxigenic. Results obtained in this study confirmed that mycotoxin contamination is a possible issue to face during grana cheese making.

Evaluation of Genetic Analysis of ESCHERICHIA COLI Isolated from Two Different Environmental Sources: Sewage Water Verses Soiled Bedding Materials of Laboratory Rodents

ABSTRACT The present investigation details an assessment of genetic relationship of E. coli isolates collected from two different environmental sources viz. sewage water and soiled bedding materials of laboratory rodents. Five sewage water samples were collected from the industrial area of Lucknow city and 5 samples of soiled bedding materials of laboratory animals were collected from Animal facility at CSIR-IITR, Lucknow. For this study Random amplified polymorphic DNA markers (RAPD) was chosen as the molecular fingerprinting method. In this study, 10 RAPD primers were used to evaluate the genetic similarity of E. coli. isolates. The RAPD-PCR fingerprints were analyzed and data were scored as 1, 0 matrix. The generated data were fed on Popgene software for calculating genetic diversity and creating dendrogram. The genetic similarity of 85% was recorded from soiled bedding materials and only 71% in sewage water samples in E.coli samples. The dendrogram based generation of clustering of E. coli isolates show two major clusters. Within major cluster sub-cluster were also observed which indicating diversity within isolates of E. coli. The RAPD-PCR based fingerprinting provided a rapid means of discriminating E. coli isolates and considered a relevant tool for molecular typing.